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The biogeochemistry of tetrapyrrole pigments, emphasizing chlorophyll


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The biogeochemistry of tetrapyrrole pigments, emphasizing chlorophyll
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xxii, 638 p. : ill. ; 29 cm.
Louda, J. William
University of South Florida
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Tampa, Florida
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Tetrapyrroles   ( lcsh )
Chlorophyll   ( lcsh )
Dissertations, Academic -- Marine Science -- Doctoral -- USF   ( fts )


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Thesis (Ph.D.)--University of South Florida, 1993. Includes bibliographical references (leaves 464-514).

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usfldc doi - F51-00184
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THE BIOGEOCHEMISTRY OF TETRAPYRROLE PIGMENTS, EMPHASIZING CHLOROPHYLL. by 0. WILLIAM LOUDA A dissertation submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida August 1993 Co-Major Professor: William M.Sackett, Ph.D. Co-Major Professor: Earl W.Baker, Ph.D.


Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Ph.D. Dissertation This is to certify t hat the Ph.D. D issertation of J.William Louda with a major in Marine Science has been approved by the Examining Committee on 27 July 1993 as satisfactory for the dissertation requirement for the Ph .D. degree. Examining Committee: .,....tvh,....a tJ"o......,. ""'"D-. Co-Major Professor: E W .Baker,Ph D E.S. Van V leet,Ph.D.

J.William Louda 1993 All Right s Reserved


DEDICATION I wish to dedicate this work to my family,noting the unfeigned support of my Mother-Jeanne,who passed on but eight months ago, and my Father-Joe;and to my best friend-my wife/my life-Debbie. JWL: 8/93


ACKNOWLEDGMENTS A study of this breadth could not have been possible without certain assistances and inputs from many. Following, in no particular order, are those which I wish to thank for their help during my studies on the biogeochemistry chlorophyll Dr.Susan E.Palmer, as my predecessor in Dr.E.W.Baker's lab, is thanked for initial training on the mass spectrometer and with the 'wet' chemistry of tetrapyrroles. Since, then collaborative efforts on coal porphyrins (Palmer al. ,1982) and the hydrous pyrolysis studies reported here are also acknowledged. Dr.Patrick A.Hatcher is thanked for samples from and ancillaru data on Mangrove Lake, Bermuda. Dr.Bernd R.T.Simoneit is thanked for near surface sediment samples from the DSDP/IPOD site survey cruise in the Gulf of California and a sample of dihydrophytol acetate. Mr.Art Olander of American Gilsonite is thanked for fresh 'mine-face' samples of Gilsonite from three separate veins. Mr.Edward W.McKnight, a former F.A.U. employee,is thanked for assistance in design and the fabrication of an inert gas chamber for the loading of TLC plates(See Fig.lS). Dr.Mary J.Leenheer, formerly of Citeies Service Corporation, is greatfully acknowledged for samples of and data on the Bakken Formation in the Western Canadian Basin. Dr.Zvi Safer, formerly of Cities Service Corporation, is thanked for the performance of hydrous pyrolysis studies. Miss Debra Murphy, a former student in the Chemistry Department and lab assistant in Dr.E.W.Baker's lab,is thanked for her translation of the original German texts of Fischer and Orth(1937) and Fischer and Stern(1940).


Mr.Dom Underwood of Ace Glass Corporation(Vineland, N.J.) is thanked for his recommendation of and assistance with the Michel-Miller LPHPLC system. Dr.Peter S.Clezy of New South Wales is heartily thanked for his most generous gifts of nickel and vanadyl 'monobenzoetioporphyrins'(cmpds. [CXVII] and [CXVIII],Appendix A). Dr.Jon DeLeeuw is thanked for a gift of dihydrophytol. Drs.Pamela P.Zelmer and Eugene H.Man of the University of Miami are thanked for their interest and a gift of mesopyropheophorbide-a(cmpd. [ X],Appendix A). Dr.Ron S.Oremland is thanked for samples from and data on Big Soda Lake, Nevada. Dr.John F.McKay is thanked for samples of Anvil Points Green River oil shale and for the supercritical methanolwater study on same. The late Dr.Peter S.Given is remembered greatfully for many productive talks and for his unflagging insistence that bacterial cytochromes must play an important role in the generation of coal(etio) porphyrins. Drs.Robert B.Gagosian and John W.Farrington of Woods' Hole Oceanographic Institute are thanked for sedimenttrap and bottom sediment samples from the Peru upwelling system. Dr.Wilson L.Orr of Mobil Research and Development is thanked for samples, support, fruitful discussions and his input on the importance of weak C-S bonds in the early stages of catagenesis. The following University of South Florida faculty are heartily thanked for their assistance and especially,their patience, during the course of these studies: Drs. William M.Sackett, Edward S.VanVleet, Pamela Hallock-Muller and Gabriel S.Vargo. Finally, I wish to offer my sincerest gratitude to my mentor, colleage and friend-Dr.Earl W.Baker.


LIST OF TABLES LIST OF FIGURES TABLE OF CONTENTS LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE ABSTRACT CHAPTER 1. INTRODUCTION Tetrapyrrole nomenclature Tetrapyrrole biosynthesis Potential geoporphyrin precursors Geologic metalloporphyrins Geoporphyrin heterogeneity The controversial DPEP-to-ETIO crossover Purposes and goals of these studies CHAPTER 2. MATERIALS AND METHODS General laboratory procedures Standards Solvents Extractions Separation techniques Liquid/liquid separations Analytical derivatizations Metals and metalloporphyrin formation CHAPTER 3. RESULTS AND DISCUSSION I: ANALYTICAL EXPERIMENTATION Initial assay of bitumen Separation-isolation studies Electronic absorption spectroscopy and chromophore identification Mass spectrometric analyses iii vii xix xxi 1 9 19 21 35 39 47 51 52 52 53 53 54 56 61 67 73 77 7o 83 148 218


TABLE OF CONTENTS (cont'd). CHAPTER 4. RESULTS AND DISCUSSION II. GEOCHEMICAL INVESTIGATIONS 279 Chlorophyll alteration prior to incorporation into sediment 282 Sediment trap studies 294 CHAPTER 5. CONCLUSIONS Analytical experimentation Geochemical investigations REFERENCES APPENDIX Appendix A 443 448 464 515


Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9. Table 10. Table 11. Table 12. LIST OF TABLES Metals reported in geologic metalloporphyrins. Geoporphyrins for which unambiguous assignment of structure has been reported. Preparation of various weight percentages of aqueous hydrochloric acid for usage in the determination of the HCl-number of tetrapyrrole pigments. Representative pigments eluting in various fractions during chromatography over cellulose. Individual step and cumulative percent yields obtained during the purification of nickel porphyrins from petroleum crudes and oil shales. Recovery of vanadyl porphyrins during chromatographic analysis;individual step and cumulative yield data. Isotopically corrected,averaged and normalized low voltage mass spectra for chromatographic fractions of the 'main' vanadyl porphyrins from Boscan asphaltenes. Mass spectral indices derived for chromatographic fractions of vanadyl porphyrins from Boscan asphaltene. Retention behavior of free-base,nickel and vanadyl porphyrins during reverse phase LPHPLC. Electronic absorption spectra of free-base and metallo-dihydroporphyrins and free-base porphyrins. Electronic absorption spectra of metalloporphyrins. Binary mixtures of tetrapyrrole pigments and their band separation used in the study of second derivative electronic absorption spectroscopy. iii 36 45 64 86 98 109 122 124 145 165 168 190


Table 13. Quantitation of dihydroporphyrins;estimation of 205 purpurin-7 or purpurin-18 in the presence of pheo phorbide-a,as calculated from synthetic known mixtures. Table 14. Estimation o f absolute and relative amounts 210 of vanadyl benzoporphyrins in mixtures with vanadyl alkylporphyrins. Table 15. Estimation of the accuracy of calculating nickel 213 a nd vanadyl porphyrins in the presence of non porphyrin background. Table 16. Se lected standard pigments,their extinction 217 coefficients,mo lecular weights and carbon numbers as used herein for the quantitaion of geolog ical tetrapyrroles. Table 17. FORTRAN p r ogram for the generatio n of nominal 222 mass tables for monobenzo-,tetrahydrobenzo and alkyl analogs of DPEP and ETIO series porphyrins Table 18. Nominal mass table for C26 to C80 free-base 223 porphyrins Table 19. Nominal mass table for C26 to C80 nickel 224 porphyrins Table 20. Nominal mass table for C26 to C80 copper 225 porphyrins. Table 21. Nominal mass table for C26 to C80 zinc 226 porphyrins Table 22. Nominal mass t ab l e for C26 to C80 va nadyl 227 porphyrins Table 23. Indices derivable from tetrapyrrole mass spectra. 228 Table 24. High ene rgy electron impact mass spectrum of 239 va n ady l deoxophy lloerythroetioporphyrin recorded at probe a n d source temperatures of 290C. Table 25. (M+1) and (M+2)mass spectral peak enhancement 251 due to isotopic contributions,as calculated f o r C20 through C50 a lkyl porphyrins Table 26. Mass spectra survey of M/M+1/M+2 relatio nships 252 for several free-base,vanadyl and monoisotopic nickel porphyrins iv


Table 27. Changes in the overt isotopic pattern of nickel porphyrins due to carbon number increases. Table 28. Calculation of the 'expected' mass spectrometric pattern fo r an hypothetical 1:1 mixture of nickel DPEP and nickel ETIO at carbon number thirty. Table 29. Observed versus calculated distribution of isotopic peaks in the mass spectra of C32 and C36 nickel porphyrins Table 30. Estimation of vanadyl benzoporphyrins in mixtures with vanadyl alkylporphyrins. Table 31. Tetrapyrrole pigment composition of the diatom Synedra sp. in fresh-viable and dark stored-dead states. Table 32. Floating sediment trap data. Table 33. Tetrapyrrole pigment yield data for surface to near-surface sediments collected beneath or near the Peruvian upwelling system at about 15 South Table 34. Tetrapyrrole pigment yield data for samples fro m DSDP/IPOD Legs 56,57,58,60,61,63,64,65,66 and 71. Table 35. Preliminary classification of chlorophyll derivatives isolated from surface to near-surface sediments in the Peruvian upwelling zone at about 15 South. Table 36. Preliminary classification of chlorophyll derivatives isolated from various deep sea sediments Table 37. Geochemical data for brackish-to-marine sapropel from Mangrove Lake,Bermuda. Table 38. Electronic absorption spectra of selected tetrapyrrole isolates from marine sediments. Table 39. Relationship of 7,8-dihydroporphyrins to free-base porphyrins as found in deep sea sediments Table 40. Geochemical data for nine shales and a juvenile petroleum from a test well drilled in coastal California. v 255 258 262 270 290 298 313 320 340 341 350 360 390 400


Table 41. Table 42. Table 43. Table 44. Table 45. Table 46. Table 47. Table 48. Table 49. Mass spectrometric indices determined fro the nickel 402 and vanadyl geoporphyrins isolated from nine shales and a juvenile petroleum recovered from a test well 'Wildcat A' in coastal California. Geochemical dataincluding metalloporphyrin 408 concentrations, for Mississippian aged shales of the Bakken formation. Mass spectral characterization of nickel and 409 vanadyl porphyrins isolated from shales of the Bakken formation. Bulk petroleum pararneters,elernental and 412 porphyrinic metals and metal ratios for petroleum crudes from the Monerey Forrnation(Miocene) and the Big Horn Basin(Paleozoic). Concentration and mass spectrometric characteri-418 zation of nickel porphyrins in twenty-six petroleum samples. Concentration and mass spectrometric characteri-419 zation of vanadyl porphyrins in twenty-six petroleum samples. Comparison of extractable organic matter and nickel 427 porphyrins derived from Anvil Points Green River oil shale by exhaustive soxhlet extraction and by treatment with supercritical methanol-water. Yield and mass spectrometric data for the nickel 430 and vanadyl porphyrins isolated from marine sediments and shales following hydrous pyrolysis. Percentages of benzoporphyrins in vanadyl porphyrin 440 arrays isolated from shales and petroleum crudes of anoxic marine origins. vi


Figure 1. Figure 2. Figure 3. Figure 4. Figure 5 Figure 6. Figure 7. Figure 8 Figure 9. LIST OF FIGURES Structural relationships of chlorophyll-a /vanadyl DPEP and 'heme'/vanadyl etioporphyrin-III. Tetrapyrrole nuclei. Generalized structure of "pheopigments" to illustrate nomenclature. Structures of archtypical compounds mentioned in text. Generalized phorbide structure illustrating several "oxy-deoxo" derivatives as defined in text. Compounds involved in the biosynthesis of porphyrin and chlorophyll structures. Structures of algal and higher plant chlorophylls. The structures of the bacteriochlorophylls -a and -b. Structures of bacteriochlorophylls-c and -d. Figure 10. Heme type pigments. Figure 11. Structure relationships in the p rotoporphyrin-IX series. 3 11 14 16 18 20 23 26 28 30 32 Figure 12. Structures of unusual biologic 33 tetrapyrroles. Figure 13. Structures of geoporphyrins for which 44 unequivocal identification is reported in the literature. Figure 14. Reactions potentially involved in 50 tetrapyrrole geochemistry. Figure 15. Diagrammatic sketch of an inert gas chamber 60 for loading of oxygen sensitive pigments onto thin-layer chromatographic plates. vii


Figure 16. Electronic absorption spectra of total 62 Figure 17. Figure 18. extracts in which free-base porphyrins co-occur with (a)dihydroporphyrins or (b) metalloporphyrins. Electronic absorption spectra of (a)the total extract of spinach(Spinacea oleracea) (b)the hypophases of five successive extracts of 'a' with 90% methanol, and (c) the resultant ethereal epiphase following liquid/liquid partition. Electronic absorption spectra of the reaction products formed by the treatment of authentic pheophorbide-a free acid with diazomethane in the presence of alkali. Figure 19. The addition of diazomethane to porphyrin exocyclic double bonds(after Fuhrhop,1978) Figure 20. Electronic absorption spectra of various total extracts of marine sediments in sequential stages of tetrapyrrole evolution. 66 70 72 79 Figure 21. Analytical scheme for the isolation and 89 purification of free-base porphyrins from sedimentary bitumen. Figure 22. Electronic absorption spectra repre-90 sentative of the various stages during the isolation and purification of geologic free-base porphyrins. Figure 23. Composite flow chart representing the 94 various methods used for the isolation and purification of nickel or nickel plus copper porphyrins from marine sediment extracts. Figure 24. Low-pressure high-performance liquid-96 chromatography(LPHPLC)separations of nickel or nickel plus copper porphyrins. Figure 25. Mass spectral distributions obtained for 99 aliquotes of a nickel porphyrin array which were exposed to various acidic media. Figure 26. Mass spectra distributions for gel perm-102 eation fractions collected during the purification of nickel porphyrins. viii


Figure 27. Flow chart for the separation and purification of nickel and vanadyl porphyrins from geologic bitumen. Figure 28. Electronic absorption spectrum of vanadyl porphyrins as typically isolated from petroleum crudes and marine sediments. Figure 29. Figure 30. Figure 31. Figure 32. Figure 33. Figure 34. Figure 35. Figure 36. Chromatographic flow-chart for a study of the 'early-eluting' or 'non-polar' vanadyl porphyrins present in maturing oil shales and petroleum crudes. Electronic absorption spectra of (a) a benzo-porphyrin enriched and (b) the main fraction of vanadyl porphyrins from Boscan asphaltenes. Electronic absor?tion spectra of fractions Al-1(a) and Al-2(b) obtained from chromatography of a benzoporphyrin enriched vanadyl porphyrin isolate over alumina. Electronic absorption spectra of the early(a) and late(b) eluting vanadyl benzoporphyrins obtained during the LPHPLC of fraction BEE/Al-2 over methanol deactivated silica. Mass spectra histogram of the vanadyl benzoporphyrins isolated from the early eluting portion of the pigments in Boscan asphaltene. Quantitative and qualitative analysis of the elution of vanadyl porphyrins from columns of alumina. Mass spectra of Boscan asphaltene vanadyl porphyrins as separated into ten fractions by chromatography over alumina. Comparison of mass spectra obtained on repooled vanadyl porphyrin chromatographic fractions(a) and that obtained by the weighted reconstitution of same (b)using the individual mass spectra for each fraction. ix 105 108 112 114 116 117 118 121 123 127


Figure 37. Figure 38. Figure 39. Figure 40. Figure 41. Figure 42. Figure 43. Figure 44. Figure 45. Figure 46. Schematic of the low-pressure highperformance liquid-chromatographic (LPHPLC)system used in the present studies. Chromatogram obtained during a test of LPHPLC system using B-carotene,pheophytin-a and to simulate the range of non-polar' pigments obtained as fraction #1 from chromatography over cellulose. LPHPLC Chromatograms for the cellulose fraction-1 pigments obtained from acidified extracts of fresh viable axenic cultures of (a)the diatom Synedra sp. and ( b )the unicellular green alga Closterium sp .. LPHPLC Chromatograms of the cellulose fraction-1 pigments from extracts of marine sediments recovered at 15.0(a) and 180.6(b) meters sub-bottom i n the Guaymas Basin. LPHPLC Chromatograms of porphyrin/dihydroporphyrin test mixtures using 'normal phase'silica gel. Reversed phase LPHPLC chromatograms obtained during the development of a system with which t o separate pheophorbide-a and pyro-pheophorbide-a. Reverse phase LPHPL C chromatographic trial with vanady l porphyrins from DSDP sample 41-368-63-2. phase LPHPLC chromatogram o f the 6 Cu-porphyrins derived from the i n vitr o treatment of petroleum nickel pg3phyrins with methane sulfonic acid and C u sulfate. Main electronic absortion spectral types of concern in tetrapyrrole geochemistry. Electronic absorption spectra o f six main free-base p orphyrin t ypes. X 130 135 136 138 140 143 147 149 152 155


Figure 47. Figure 48. Figure 49. Figure 50. Figure 51. Figure 52. Figure 53. Figure 54. Figure 55. Figure 56. Figure 57. Electronic absorption spectra of selected 'chlorins'. Comparison of the electronic absorption spectra of the free-base and metalloforms of (a)chlorin-p6 TME and (b) mesopyropheophorbide-a ME. Analytical schemefor the in vitro manipulation of tetrapyrrOle chromophores using sodium borohydride and copper sulfate. Comparison of the electronic absorption spectra of pheophorbide-a,copper pheophorbide-a and their 'oxy-deoxo' derivatives. Comparison of the lectronic absorption spectra of purpurin-7 TME,copper purpurin -7 TME and their 'oxy-deoxo'derivatives. Comparison of the electronic absorption spectra of phylloerythrin ME,copper phylloerythrin and their 'oxy-deoxo' derivatives. Comparison of the electronic absorption spectra of nickel phylloerythrin ME and nickel 9-oxy-deoxo phylloerythrin ME. Comparison of the electronic absorption spectra of pyrrorhodin-XV, 'oxy-deoxo' pyrrorhodin-XV and their copper derivatives. Electronic absorption band shapes resulting from the overlap of closely spaced primary absorptions(redrawn from Rao,1967). The electronic absorption spectra of test mixtures containing pheophorbide-a and either purpurin-7 TME or purpurin-18 ME. The zero order and second derivative electronic absorption spectrum of a geochlorin fraction containing pheophorbide-a and purpurin-18 pigments. xi 159 164 174 176 178 180 183 185 188 191 193


Figure 58. Electronic absorption and second 195 derivative spectra of test mixtures containing nickel deophylloerythro-porphyrin ME and either nickel phylloerythrin ME,nickel 'monobenzoetioporphyrin' or vanady l etioporphyrin. Figure 59. Second derivative electroni c absorption 198 spectra of various test mixtures made with authentic vanadyl.etioporphyrin-III and vanadyl 'monobenzoetioporphyrin'. Figure 60. Delineation of non-pigment background 204 in electronic absorption spectra using 'best-fit' French curve methods. Figure 61. Visible absorption spectra for test 209 mixtures of vanadyl etioporphyrin-III plus vanadyl 'monobenzoetioporphyrin' in concentrations ranging from 1 to 32 molar percent. Figure 62. Quantitation of nickel porphyrins using 212 electronic absorption spectra contaminated by non-porphyrin background. Figure 63. Quantitation of vanadyl porphyrins using 215 electronic absorption spectra contaminated by non-porphyrin background. Figure 64. Generalized structures of geoporphyrin 219 mass spectral series. Figure 65. Averaged,isotopically corrected and 230 normalized low voltage mass spectrum of a vanadyl geoporphyrin array. Figure 66. Changes in the mass spectra of free-base, 233 nickel and vanadyl geoporphyrins during the volatilization step of DIP-EI-MS. Figure 67. Electronic absorption spectrum of the 235 tetrahydrofuran soluble extract of the residue on the ion source exit slit following the MS analyses of nickel and vanadyl geoporphyrin isolates. xii


Figure 68. Figure 69. Figure 70. Figure 71. Figure 72. Figure 73. Figure 74. Figure 75. Figure 76. Figure 77. Figure 78. Figure 79. Uncorrected real-time high voltage mass spectrum of vanadyl deoxophylloerthroetioporphyrin. High gain expansion of the high voltage mass spectrum of VO-DPEP. Changes in the mass spectrum of vanadyl etioporphyrin-III as ionization voltage increases. Changes in the mass spectrum of vanadyl deoxophylloerythroetioporphyrin as ionization voltage increases. Uncorrected real-time high energy mass spectrum of the vanadyl geoporphyrin array isolated from Boscan asphaltene. Alteration of the mass spectra of a vanadyl geoporphyrin array due to changes in the ionization voltage. Changes in the apparent relationships of M/M+l/M+2 peaks in nickel porphyrins due to increases in the carbon number of the ligand. Scan to scan variability in the relationship of M+2 and M+4 to the molecular ion(M) of nickel octaethylporphyrin during DIP-El-MS. Scan to scan variability in the relationship of the M+2 peak of nickel etioporphyrin-I during EI-MS. Selective volatization of vanadyl porphyrins during DIP-EI-MS;changes in VO-ETIO to VO-DPEP ratio at C-32. Selective volatization of ETIO over DPEP series porphyrins during DIP-EI-MS. Volatization profile obtained during the DIP-El-MS of a l:l(molar) test mixture of vanadyl etioporphyrin-III and vanadyl octaethylporphyrin. xiii 237 241 243 244 246 248 256 259 260 264 267 268


Figure 80. Comparison of the volatilization of vanadyl etioporphyrin-III and vanadyl 'monobenzoetioporphyrin'. Figure 81. Selected scans from the DIP-EI-MS of a 16 molar percent mixture of vanadyl 'monobenzoetioporphyrin' with 84 molar percent vanadyl etioporphyrin-III. Figure 82. Suggested overall profile for the DIP-EI mass spectrometric analysis of geeporphyrins. Figure 83. Repeatability of geoporphyrin mass spectral analyses. Figure 84. Electronic absorption spectrum(a) and second derivative(b) of pigments extracted from the green alga(Chlorophyta)Closterium sp. and the electronic absorption spectra\c) of the isolated chlorophylls-a and -b. Figure 85. Electronic absorption spectrum(a) and its second derivative(b) for the total pigment extract of the red alga Acro-chaetium sp.. ------Figure 86. Electronic absorption spectra of the total pigment extracts of fresh/viable and two-month post-mortum 'dead' diatoms (Bacilliariophycea:Synedra sp.). Figure 87. Electronic absorption spectrum of the polar fraction isolated from an extract of 2-month dead diatoms(Synedra sp.). Figure 88. World map with sample locations delineated. Figure 89. Site map for sampling locations off the coast of Peru near 15S. Figure 90. The dehydration/cyclization of pheophorbides-a to 132 ,173-cyclopheophorbide-a enol. Figure 91. Structural relationships between the chlorophylls-b and -c3 and 7-nor-DPEP. xiv 272 273 275 277 287 288 292 293 295 297 302 306


Figure 92. Figure 93. Figure 94. Figure 95. Figur e 96. Figure 97. Figure 98. Figure 99. Figure 100. Dissolved oxygen transect of the waters o f f Peru at about 15S(Redr awn from He nrichs and Farrington,1984 and data i n Gagosian al.,1980). Downhole trends in the Pigment Yield Index(PYI)for three sites sampled beneath the Peruvian upwelling system at abou t 15S. P l o t s of tetrapyrrole pigment y ield versus sub-bottom depth for various DSDP/IPOD sites. Summary of pigment _yiel d t rends with depth in the water column and under lyin g sediment s for a marine anoxic environment. Cyclization a n d rearrangement of the isocyclic ring in chl o rophyll-s(after Callot et al. ,1990;0campo et al.,1984). ----P otential alteration o f b a c teriopheoduring earl y diagenesis and expected' DPEP seies porphyrins. Suggested mechanism for the removal of the 9-keto moiety from chlorophylla derivatives. Comparison of the average low-voltage mass spectra for the coincident 7,8-dihydro-DPEP(a) and DPEP(b)series porphyrins isolated from an oil shale in the Sisquoc formation. Electronic absorption spectrum(a) a n d second derivative(b)for t h e monocarboxylic acid dihydroporphyrin s isolated from DSDP/IPOD sample 63-4713 2 Figure 101. E lectronic absorption spectra of purpurin-18 a n d chlorin-p6 isolated from DSDP/IPOD sample 64-481A2 2 Figure 102. Electronic absorption spectra of 132 1 73-cyclopheophorbidea enol and the dioxy-dideoxo derivative obtained via reduction with sodium borohydride. XV 314 317 330 336 345 354 363 365 368 370 371


_Figure 103. The dehydration-cyclization of pheophorbides to yield cyclopheophorbide -a enols. Figure 104. Electronic absorption spectrum of 132 ,173-cyclomesopheophorbide-a enol (tentative). ----Figure 105. Electronic absorption spectra of rhodins isolated from DSDP/IPOD sites 57-438(a) and 63-471(b). Figure 106. The cyclizations of mesoporphyrin-IX and pyrroporphyrins-XV to yield isomeric pseudo-DPEP compounds('CAP') with a cyclopropane moiety. Figure 107. Electronic absorption spectrum of 'CHLORIN-660' ,suggested as being a macromolecular complex. Figure 108. Downhole profiles for the aromatization reaction. Figure 109. Structural comparison of a 7,8-dihydroporphyrin and a free-base porphyrin in order to illustrate the 'aromatization' reaction. Figure 110. Electronic absorption spectrum of mixed rhodoporphyrins isolated from DSDP/IPOD sample 64-479-29-4. Figure 111. Suggested overall diagenetic trend for the aromatization and chelation reactions,incorporating the observed equilibrium reaction at the aromatization step. Figure 112. Downhole plot for the chelation(Ni) reaction as observed for DSDP/IPOD site 63-467 in the San Miguel Gap, California borderlands(Louda and Baker,1981). Figure 113. Electronic absorption(a) and mass(b) spectra of decarboxylated nickel 'phylloerythrins' isolated from a Quaternary diatomaceous ooze recovered in the Guaymas Basin,Gulf of California. xvi 373 375 377 380 382 385 386 387 389 393 396


Figure 114. Figure 115 Figure 11G. Figure 117. Figure 118. Figure 119. Figure 120. Figure 121. Figure 122. Figure 123. The evolution of vanadyl porphyrins in oil shales and petroleums of marine origin,as traced with mass spectral histograms of DPEP and ETIO series pigments(from Baker and Louda,1986a). Averaged low voltage mass spectra of the vanadyl porphyrins isolated from a downhole sequence of shales and an incalated juvenile petroleum in a wildcat test well,onshore California. Postulated incorporation of a chlorophyll derivative into a geopolymeric state. Low voltage mass spectra of the nickel and vanadyl porphyrins isolated from Mississippian aged oil shales of the Bakken formation. Plots of percent DPEP versus vitrinite reflectance(R0). Plot of nickel and vanadyl porphyrin concentrations weight percent sulfur for twenty-five petroleum crudes. Cross-plot of vanadyl porphyrin concentration and percent DPEP for a series of maturing oil shales and twentythree petroleum crudes. Plot of percent DPEP versus the alkylation index for vanadyl porphyrins isolated from shales and petroleum crudes. Mass spectra of the nickel porphyrins isolated from Anvil Points Green River Oil shale by (a) soxhlet extraction or (b)treatment with supercritical methanol and water. Mass spectral distributions of metalloporphyrins isolated from DSDP/IPOD sample 63-467-63-2. xvii 399 403 405 407 411 415 420 422 428 431


Figure 124. Figure 125. Figure 126. Figure 127. Figure 128. Figure 129. Figure 130. Figure 131. Figure 132. Low voltage mass spectra of the nickel porphyrins isolated from Bakken shale number 6362(R = 0.65) prior to and following pyrolysis. Low voltage mass spectra of the vanadyl porphyrins isolated from Bakken shale number 6362(R0= 0.65) prior to and following hydrous pyrolysis. Comparisons of the mass spectra and RP-HPLC fingerprints of three vanadyl geoporphyrin isolates. Suggested overall tetrapyrrole yield trends for sediments deposited in anoxic or oxic conditions. Structural comparison of the pigments involved in. the oxidative ruP.ture of the isocyclic ring in a chlorophyll derivative(pheophorbide-a),producing purpurin-18 and chlorin-p6. Proposed routes(partial)for the anoxic /reductive diagenesis of chlorophyll derivatives ending in the production of DPEP and 'DiDPEP' porphyrins. The cyclization of pheophorbide-a and mesopheophorbide-a and the participation of the enols in the production of DiDPEP'compounds. Formation of cyclopropane-porphyrins from mesoporphyrin-IX. Suggested overall geochemical transformations of chlorophyll and heme structures. xviii 433 435 447 451 453 455 457 459 460


LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE alt. incl. v/v w/v T t h d m em nm Km U V/VIS M.S. HPLC LPHPLC RP-LPHPL C cc G P C EI used in parenthetical notes, 'alternatively' used in parenthetical no tes, 'including volume t o vo lume measure weight t o volume measure temperature time hour(s) day(s) meter(s) centimeter(s) n anome ter(s) kilomete r(s) ultraviolet/visible spectroscopy(ia.350900nm) mass s pectrometr y or mass spectrum high performance liquid chromatog raph y l o w pressure HPLC reverse phase LPHPL C column chromatography gel permeation chromatography e lectro n impact DIP El-MS direct insertio n probe El-M S m/z OD o<-HEDA mass to charge ratio,replaces antiquated m/e oxydeoxo alpha-hydroxyeth y l desacetyl PDP propyl despropio DSDP/IPOD D eep Sea Drilling Project,International Phase of Ocean 64-4812 -2) are given as Leg-Site-Core-Secti on wavelength xix


THE BIOGEOCHEMISTRY OF TETRAPYRROLE PIGMENTS, EMPHASIZING CHLOROPHYLL. by J. WILLIAM LOUDA An Abstract Of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida August 1993 Co-Major Professor: William M .Sackett, Ph .D. Cq-Major Professor: Earl W.Baker, Ph D XX


Electronic absorption spectra recorded on native geepigments and on in vitro derivatives,obtained with combinations of copper insertion and borohydride reduction reactions, allowed a sensitive 'chromophore identification' scheme to be developed.Quantitation of the Ni and VO geoporphyrins,as well as the of each,was tested and precisions at the level of 100% found. An overall methodology for obtaining repeatable lowvoltage mass spectra on geoporphyrin arrays is presented. Repeatabilities for the various mass spectral derived indices were; % DPEP(%), X(%), A.I.(S%) and% BENZ(%).Quantitation of the metallobenzoporphyrins was much better(%) with UV/VIS techniques. Tetrapyrrole geochemistry was investigated at all stages of organic evolution.Thus,viable and senescent/dead uni-algal cultures,sediment trap material,surface(<1m)sediments,deep ocean long cores(DSDP),oil shales and petroleum crudes were investigated. Results indicate that the chlorophylls can serve as sources for a variety of geoporphyrins.Early in diagenesis two competing reactions dictate further 'fossilization'. First is the loss of the carbomethoxy group.This produces pyropheophorbides which can either lead to the true DPEP series,via a sequence of defunctionalization reactions,or, via intramolecular cyclization(dehydration),to certain 1 32 173-cyclopheophorbide enols.The latter,following defunctionalization,give rise to DiDPEP and/or DPEP-type pigments with a 7-membered exocyclic ring.Second,chlorophyll nuclei have undergone oxidative scission of the isocyclic ring can,through purpurins and chlorins,theoretically yield C28-C30 E7IO-series porphyrins.Bacteriochlorophyll-a was found to be a dominant pigment in several anoxic sediments and is suggested as an important and highly specific pre-xxi


cursor for certain geoporphyrins(l3-methyl-desethyl-DPEP). Heme type compounds(viz.cytochromes) appear to not only iield C 32 ETIO pigments but also,through derived by the cyclization(dehydration) of propionic acid groups,a series of DPEP-like compounds with 6-membered exocyclic rings. It was concluded from the studies reported both here and in the literature that, tetrapyrrole pigments evolve in at least five separate assemblages,the first four of which funnel into the last.These are; phytyl esters,steryl esters, carboxylic acids, geopolymers(bound) and alkyl pigments. Abstract approved: Co-major and Co-major Professor;E.W.Baker,Ph D Graduate Student Advisors Department of Marine Science Date of Approval xxii


CHAPTER 1 INTRODUCTION 1 Natural gas, coal and petroleum accumulations represent the three main energy yielding hydrocarbon sources available to mankind. Alternative yet interrelated 'fossil fuels' also e xist. These are condensates, peats/brown coals and oil shales, the less mature forms of each of the above (see Breger, 1963; Brownlow, 1979; Hunt, 1979; Tissot and Welte, 1978; Van Krevelen, 1961: and lead references in each). The vast majority of usable natural gas originates during metagenesis, and leads to the essentially methathetic reaction products of methane ('natural gas') and 'carbonized' ('pyrobitumen,' coke, graphite etc. ) forms of carbon, in both the terrestrial (peat-coal) and aquatic (sediment-sha l e-petroleum) organic fossilization scenarios. The ultimate biological origin of coal was determined rather straightforwardly, as cellular structure is retained during the earlier phases of coalification (Given, 1984; Tissot and Welte, 1978; Van Krevelen, 1984). Observational and intuitive evidence for biological sources (precursor Qrganic matter (OM)) of petroleum existed by the late nineteenth century (lead references in Hunt, 1979; Tissot and Welte, 1978). Ultimate proof required identification of chemicals clearly having origins in the biosphere. The isolation and identification of pigments from petroleum, shales, coals and allied bituminous matter (Treibs 1934a-b, 1935a-b, 1936) provided this proof


2 In the last of these papers Treibs postulated a reaction scheme to explain the transformation of 'biotic chlorophyll into 4eoxoQhyllogryth roetioQorphyrin (DPEP), as depicted in Figure 1. This first explanation of 'organic diagenesis', for the most part, established the field of organic geochemistry. Thought on the generation and accumulation of fossil bitumens (petroleum) has evolved from 'the compaction and accumulation of non aqueous soluble biochemicals in a geological phase separation' (Trask and Wu, 1930) to present theories encompassing the thermal/thermocatalytic conversion of geopolymers (kerogen), themselves ultimately derived from biochemicals, into the melange of organics we know as bitumen and, when abundant, petroleum (Hunt, 1979; Meinschein, 1959; Philippi, 1977; Tissot and Welte, 1978). Inherent to the ultimate goals of the present work is the premise that biochemical derivatives accompany the major organic constituents (kerogen) of sediments in such a way as to be clearly linked to their ultimate biotic origins. These remnant compounds have come to be collectively known as 'biological markers' or 'bi omarkers.' The concept of a 'biomarker' was first implied by Treibs (1936) when he forwarded a plausible scheme for the conversion of biologic chloro phyll-a into geologic DPEP and heme-porphyrins into etioporphyrin-III (Fig. 1). Subsequently, the concept of 'biomarkers' grew and began to evolve Articles addressing the concept and utility of biomarkers include: "Biochemical Fossils" (Fox, 1944), "Biochemical Limnology" (Vallentyne, 1954, 1955), "Paleobiochemistry" (Abelson, 1956), "Chemical Fossils" (Eglinton and Calvin, 1967) and, most recently, a multi authored book entitled "Biological Markers" (Johns, 1986). The last


CHLOROPHYLL a c=o O=c / \ HO OH 'HEME-TYPE' (e o_.Fe(lll)protoporphyri n-IX) VO DPEP VO ETI0-111 Figure 1. Structural relationships of chlorophyll-a/vanadyl DPEP and 'heme'/vanadyl etioporphyrin-III. 3


4 reference also contains a chapter entitled "Porphyrins in the Geologic Record" (Baker and Louda, 1986a), into which much of the present study has been incorporated Tetrapyrrole pigments serve as biomarkers in several ways. (1) They link fossil organic matter to their ultimate sources in the biosphere (Treibs 1934a-b, 1935a-b, 1936). (2) The presence of tetrapyrrole carbo x yl i c acids in crude o i l allowed initial upper-limit estimates to be placed upon the thermal history (g.g. <300C) involved in petroleum generation (Dunning and Moore, 1957; Dunning et gl., 1960; Treibs, 1940, 1948). (3) The advent of geoporphyrin analysis with mass spectrometry (Baker, 1966; Baker et gl., 1967), supplemented recently by HPLC (Eglinton et gl., 1980; Hajibrahim et gl., 1978, 1981; Quirke et gl., 1979, 1982; Sundararam, 1985) and GC-GC/MS (Eglinton et gl., 1984; Gallegos et gl., 1983; Hein et gl., 1985; Roberts and Scammells, 1983) techniques, lead to the utilization of the DPEP-to-ETIO ratio* (D/E) as a marker for the thermal history of sedimentary bitumen and petroleum crudes (Baker, 1969; Baker and Louda 1983, 1986a-b; Baker and Palmer, 1978; Baker et gl., 1977, 1978a, 1987; Barwise, 1982; Barwise and Park, 1983; Burkova et gJ., 1980a; Didyk et gj., 1975a-b; Morandi and Jensen, 1966; Mackenzie et gJ., 1980). Seemingly, so valid are trends in D/E, or the newly proposed (Barwise, 1982; Barwise and Park, 1983) and adopted (Baker and Louda, 1986a; Baker et gl., 1987; Louda, this study) % DPEP measure, that industry "places much emphasis" upon *DPEP, named after deoxophylloerythroetioporphyrin (XXXVIII), and ETIO, named after etioporphyrin-III (LII), represent mass spectral series with nominal masses of 476n and 478n, where n is an integer (Baker 1966; Baker and Palmer, 1978; gl., 1967). Currently, alternate or pseudo-DPEP structures are known and are discussed elsewhere in text.


5 same (J. M. Moldowan, P. Sundararaman; pers. communs. 1984). An alternate geoporphyrin parameter affected by thermal history is the weighted average mass (X) or average carbon number (C). In addition to applications with sedimentary bitumens or petroleum crudes (Baker and Louda, 1983, 1986a; Baker and Palmer, 1978), X appears to provide a potential for the ranking and sub-ranking of humic coals (Bonnett et gl., 1984; Palmer, 1979; Palmer et gJ., 1982). (4) Perhaps the oldest and most widely implemented utilization of tetrapyrrole pigments as 'biomarkers,' is the measurement of chlorophyll and derivatives (''pheo-pigments") as indicators of phytoplankton standing crop and/or water column productivity (Bunt, 1975; Creitz and Richards, 1955; Farmer et gJ., 1982; Garside and Riley, 1969; Glooschenko and Blanton, 1977; Hall and Moll, 1975; Hallegraeff, 1981; Harvey, 1934; Jones, 1977; Kiefer and SooHoo, 1982; Kreps and Verbinskaya, 1930; Marra et gl., 1982; Parsons, 1963; Parsons and Seki, 1970; Parsons and Strickland, 1963; Parsons et gl., 1977; Rai, 1973; Redalje, 1983; Richards and Thompson, 1952; Riemann, 1978; Riley and Chester, 1971; Ryther and Yentsch, 1957; Seely et gJ., 1972; Sellner, 1981; Steele, 1974; Zscheile and Harris, 1943). So important is the 'biomarker' utility of chlorophyll and derivatives in the assessment of primary productivity that the United Nations has published analytical guidelines (UNESCO, 1966) and spectral methods have been developed and implemented for its analysis in the world ocean from airborne (Hoge and Swift, 1985) and earth orbit (Gordon et gJ., 1980, 1982) reconnais ances (5) Chlorophyll derivatives are widely used as paleolimnological 'biomarkers.'


Except in surface sediments in or near (dysphotic) the photic zone, chlorophyll per se is rarely the form in which the tetrapyrrole remnants of photoautotrophs enter sediments. Chlorophyll derivatives in the natural environment have been called "pheo-pigments" (UNESCO, 1966; Yentsch, 1965), hlorophyll .Qecomposition .o.roducts" (i.g. SCDP: Vallentyne 1954, 1955, 1960), or simply "chlorophyll derivatives" (Orr and Grady, 1957; Orr et gl., 1958}, as used herein (cf. Baker and Louda, 1983, 1986a; Louda and Baker, 1986}. These derivatives enter sediments, or form rapidly on geologic time scales, 6 and are thus the compounds fossilized or recycled. Chlorophyll derivative paleolimnology rests on the premise that highly productive waters deliver concomitantly large amounts of organic matter including, inter alia, chlorophyll derivatives to underlying sediments. Conversely, sediments beneath waters of low productivity may receive lower amounts of autochthonous* OM, thus of sparse chlorophyll derivative content, but can still be organic-rich via inputs of allochthonous* OM. Concurrent with the input of chlorophyll derivative-rich or -poor OM are the general patterns of paleoenvironment leading to, in essence, anoxic or oxic deposition (cf. Brownlow, 1979; Demaison and Moore, 1980; Didyk et gl., 1975a; Glenn and Arthur, 1985; Hunt, 1979; Tissot and Welte, 1978}. This is, of course, vastly oversimplified but does follow the known trends of elevated chlorophyll-derivative fossiliza-tion in anoxic sediments versus lowered contents in oxic settings (Baker and Louda, 1980a-c, 1981a-b, 1982, 1983, 1986a-b; Brown et gl., *Autochthonous, in the present treatise, is meant to imply OM derived from overlying water columns and sedimentary biota while allochthonous is used to describe primarily terrestrial OM which is redeposited


7 1977; Daley et gl., 1977; Gorham, 1960; Gray and Kemp, 1970; Kemp and Lewis, 1968; Koyama et gl., 1968; Kozminski, 1938; Louda and Baker, 1981, 1986; Louda et gl., 1980; Orr et gl., 1958; Phinney, 1946; Sanger and Crowl, 1979; Vallentyne, 1954; Vallentyne and Craston, 1957). A precise methodology for the analysis of chlorophyll-derivatives in geochemical settings has been slow to emerge. Most studies have used gross estimates of total chlorophyll derivatives, determined on crude extracts, and related this yield to wet sediment weight. Vallentyne (1955) defined a "sedimentary chlorophyll unit" ("SCU"*) and suggested eventual normalization to "grams ignitable matter" to negate mineral matter dilution effects. Except for Orr et gl. (1958), no further attempts to standardize tetrapyrrole pigment yield data occurred until DSDP and !POD programs provided long-cores of oceanic sediment, inherently of variant deposition and water content (viz. compaction) and forced re-evaluation. Louda et gl. (1980) formulated a Qigment yield index or PYI. As first defined, PYI was the yield of tetrapyrrole pigments in pg/g-sediment: wet weight divided by the organic carbon (C0r9 ) content of the sediment in percent dry weight. The obvious error in this method, not allowing for compaction dewatering processes, forced a rapid change to dry PYI PYidry = pg tetrapyrrole pigment/g-sediment: dry wt. divided by corg in %, dry wt.: Baker and Louda, 1980a). This organic-carbon-based constituentyield index has also been extended to the study of carotenoids *According to Vallentyne (1955) 1 SCU equals the amount of pigment yielding 0.100 AU (absorption unit) in 10 ml of 90% acetone (aq.) when measured over a 1 em light path at 667 nm. Employing known milli-molar extinction coefficients Fuhrhop and Smith, 1975) this can be shown to be the same as 15.6 or 11. 2 pg of pheophytin-a or pheophorbide-a, respectively.


8 (tetraterpenoids: Baker and Louda, 1982} and the PAH perylene (Baker and Louda, 1982; Louda and Baker, 1984}. However, determination of the tetrapyrrole PYI required further refinement. Thus, separation into individual classes of pigments, determination of individual yields and summation of those yields were included (Louda and Baker, 1981}. Tetrapyrrole geochemistry has been the subject of periodic review The premier review of tetrapyrrole geochemistry and, in essence, of organic diagenesis is that of Alfred Treibs (1936), which yielded a plausible reaction scheme for the diagenesis of chlorophyll-a and the generation of vanadyl deoxophylloerythroetioporphyrin (Figure 1). Treibs also postulated the conversion of heme-type pigments into VOetioporphyrin III (Figure 1). Following Treibs' (1936) initial report of tetrapyrrole geochemistry, it took more than twenty years before enough additional data and/or interest prompted the next major synopsis Primarily with data emerging from the petroleum industry, due to economic interest in porphyrins as carriers of the industrial catalyst poisons nickel and vanadium (Beach and Shewmaker, 1957; Constantinides et gl., 1959; Dunning et gl., 1960; Erdman and Harju, 1963; Erdman et gl., 1956a-b, 1957; Groennings, 1953; Overberger and Danishefsky, 1952; Skinner, 1952}, Dunning and Moore published a review entitled ''Porphyrin research and origin of petroleum." Since this review, synopses chronicling the progress of tetrapyrrole geochemistry have appeared at intervals of from 2-6 years. The past twentyfive years have witnessed reviews published by Dunning (1963}, Hodgson al. {1967), Baker (1969b}, Hodgson (1973), Baker and Palmer (1978}, and Baker and Louda (1986a). The last two articles above included information on tetrapyrrole diagenesis studies in the long sediment


9 cores (g.g. >500-1000 m) obtained from the Deep Sea Drilling Project (DSDP) and the International Phase of Ocean Drilling (!POD). Review of organic geochemical studies, including tetrapyrrole pigments, on DSDP and !POD samples have also been published (Baker and Louda, 1980c; Eglinton et gJ., 1983). Six topics related to geologic tetrapyrroles will be reviewed as a foundation for further discussion. These six topics are: tetrapyrrole nomenclature; tetrapyrrole biosynthesis; potential geoporphyrin precursors (biologic tetrapyrroles); geologic metalloporphyrins, the metals; geoporphyrin heterogeneity; and DPEP-to-ETIO ratios. Tetrapyrrole Nomenclature This section provides the 'working vocabulary' of the tetrapyrrole geochemist/biological oceanographer and the precise nomenclature of the organic chemist. Selected structures are also cross-referenced as to common names and both the Fischer (Fischer and Orth, 1937; Fischer and Stern, 1940) and revised (Bonnett, 1978; IUPAC-IUB, 1978) nomenclatures (see Appendix A). The physicochemical manifestations of the tetrapyrrole pigments reside in the type of nucleus, chelated metal, nature of peripheral substituents and association with other organics and Orth, 1937; Fischer and Stern, 1940; Gouterman, 1978; Lehninger, 1975; Okunuki et gj., 1968; Rimington and Kennedy, 1962). Variations of chelated metals and peripheral substitution will, for the most part, be deferred until the section on 'potential geoporphyrin precursors'. This section, then, covers only the various tetrapyrrole nuclei and


certain selected structures of oceanographic/limnological/geochemical importance. 10 Compounds comprised of 4 pyrrole subunits are collectively referred to as tetrapyrroles. Primarily these include the macrocyclic porphyrin-and linear bilin-type tetrapyrroles. A selected assortment of tetrapyrrole nuclei is given in Figure 2. In the present treastise a modified 'Fischer' (Fischer and Orth, 1937; Fischer and Stern, 1940; cf. Willstater and Stoll, 1913) nomenclature, including many 'common names,' will be used. However, "provisional recommendations" for a "revised nomenclature" (Bonnett, 1978; IUPAC-IUB, 1978) will be mentioned. Most structures can also be cross referenced in text by bracketed Roman enumeration [N] to both the 'Fischer' and 'revised' names (Appendix A). Porphyrin [XLVIII], considered as the parent structure, is drawn twice in Figure 2 to give both the 'Fischer' (Fig. 2a) and 'revised' (Fig 2b) methods for atom identification. In the 'Fischer' system, Bpyrrole carbons are numbered 1-8 and methine-bridge carbons (i.g. mesopositions) are labelled a-6. This contrasts with the 'revised' nomenclature in which all ring carbons, starting with an a-pyrrole position, are labelled consecutively 1-20 Thus, in the 'revised' format, Bpyrrole positions are 2,3,7,8,12,13,17,18 and methine bridge atoms are 5,10,15,20. To the organic geochemist, porphyrin [XLVIII] can be considered as the lowest molecular weight (i.g. C20) member of the ETIO-series. Other structures given in Figure 2 are compared to porphyrin [XLVIII] in the 'Fischer' system (Fig. 2a) Chlorin (Fig. 2c) can be considered as 7,8-dihydro-porphyrin and is the parental nucleus for a wide variety of products [g.g.


a) b) d) e) g) h) c) f) i) 0 0 Figure 2. Tetrapyrrole nuclei. a) porphin("Fisher nomenclature", Fisher and Orth,1937), b) porphyrin("Revised nomenclature", IUPAC-IUB,1978), c) chlorin, d) bacteriochlorin, e) phorbide(alt. phorbin), f) bacteriophorbide, g) porphyrinogen, h) corrole, i) bilane ..... .....


12 XXIII-XXXI] derived from the oxidative chemical degradation of chlorophyll-a [I] (Fischer and Stern, 1940). This includes rhodins and purpurins, which contain 'rhodofying' (shifting absorption to the red: bathochromic) auxochromes such as carboxylic acids, aldehydes and/or ketones in conjugation with the chlorin nucleus.* Bacteriochlorins (Fig. 2d) are 3,4,7,8-tetrahydro-porphyrins and yield analogous products to the chlorins, upon the oxidative degradation of bacteriochlorophyll-a [Mg-XXI]. Phorbide (Fig. 2e) can be considered as 6,y-cycloethano-chlorin or 7,8-dihydro-6,y-cycloethano-porphyrin. Phorbide(s) may also be called phorbin(s) (Seely, 1966). The characteristic sub-structure of phor bides {Fig. 2e: see [I-XVIII]) and bacterio-phorbides {3,4-dihydro phorbides: see [XXI-XXII]) is the 6,y-ethano moiety. This sub-structure is also referred to as the 'isocyclic' or 'cycloethano' ring and consists of carbons number 9 and 10 in the 'Fischer' system. Each ring in the sub-structure of tetrapyrrole pigments is designated as A-D (or I-IV) with ring E (or V) being the so-called isocyclic ring {Fig. 2e). The isocyclic ring (ring E) of phorbide, when substituted by 9-keto and 10-methoxy carbonyl (methyl formate) moieties, is also referred to as the 'carbocyclic ring.' This struc-ture {pheophorbide) is part of chlorophylls-a [I], -b [XIV], -d and bacteriochlorophylls-a [Mg-XXI] and -b. 'Chlorophylls-c' [XIX] contains the carbocyclic ring given above but its nucleus is that of a 7,8-didehydro-phorbide. That is, it is a QhgQ-porphyrin. *source references for structures include: Aronoff, 1966; Fischer and Orth, 1937; Fischer and Stern, 1940; Holt, 1966; Inhoffen and Jager, 1965; Jackson eta}., 1968; Scheer and Inhoffen, 1978; Seely, 1966; Smith, 1975).


13 Phorbides with only 9-keto substitution of the isocyclic ring, yielding a 'cyclopentanone' subunit, are considered QYCQ-pheophorbides. Pyro-pheophorbide nuclei are the basic structure of the Chlorobium chlorophylls and related compounds which are also called bacteriochlorophylls -c, -d and (see Brockman, 1976; Holt and Hughes, 1961; Holt et gl., 1966; Hughes and Holt, 1962; Kenner et gl., 1976; Mathewson et gl., 1963; Smith et gl., 1980a-b). Phorbide (Fig. 2e) structure has been deta i led and emphasized above as compounds with the type of nucleus which can potentially yield DPEP-based (7,8-didehydro-phorbide) compounds (see [XXXII-XL]). Structures given as Figure 2g-i are for porphyrinogen, corrole (or corrin) and bilane, respectively. These differ from porphyrin [XLVIII] via reduction of all methine bridges, ring contraction due to 'loss' of a methine bridge carbon and the oxidative cleavage of macrocyclic structure at a meso-position, in that order. Descriptive terms and modifiers commonly found in the oceano graphic, geochemical and chemical literature,* as interpreted by the author, are presented below. Figure 3 is the generalized structure of a 'pheo-pigment', to serve as a reference 'Chlorophyll(s)'**: Photosynthetically active pigment present in oxygenic and anox ygenic photoautotrophs. E xact structure(s) present is a phenomenon of species-specificity. Contains Mg(II) "Routine' *Examples of general reference sources for these 'definitions' include the following: Aronoff, 1953; Dunning, 1963; Fischer and Stern, 1940; Holt, 1965; Richards and Thompson, 1952; Seely, 1966; Strickland and Parsons, 1968; Vallentyne, 1960; Willstater and Stoll, 1913; Yentsch, 1965; Yentsch and Menzel, 1963; Yentsch and Ryther, 1959. ** Chloro-phyll from the Greek: Kloros =green, phyllon = leaf. Pheo = brown.


CH3 I CH2 I a "meso" compound +2H M R2 CH2 I R1-o_..c,b E M=methyl E =ethyl a "meso" position '0 -"isocyclic ring" -....__________ 9-keto( 'oxo') ( if 9-H2 =desoxo) Figure 3. Generalized structure of "pheopigments" to i llustrate nomenclature. ......


15 qeterminations of "chlorophylls" via spectrophotometric or fluorescence technique i nclude 'chlorophy l l i des' as well. 'Pheophytin(s)' : 'Chlorophyll(s) which have had Mg(II) removed, essent ially the 'free-base' form o f chlorophyll(s). 'Pheophorbide(s)': 'Chlorophyll(s)' derivatives formed by the loss of both Mg(II) and the esteri fying isoprenoid alcohol (viz. phytol/chlorophylls-a, -b, -d(?) and bacteriochlorophylls-a, b; farnesol/Chlorobium chlorophylls-650, -660, bacteriochlorophyll-e). 'Chlorophyllide(s)': 'Chlorophyll(s)' which have lost the esterifying isoprenoid alcohol (see 'Pheophorbide(s)'). In th i s light, chlorophyll(s)-c are 'chlorophyllides', as is detailed i n Appendix A. 'Pheopigments' (phaeopigments) : 'Chlorophyll(s)' derivatives in the water column and sediments, including primarily 'pheophyt in(s)' and 'pheophorbide(s),' which are not determined by spectrophotometric or flu orescent techniques spec ific for 'ch lorophyll(s).' Determi nation of 'pheopigments' using these methodologies rests upon spectrometric d etermination of pigments in e x t racts befo r e (i. g native) and ing mild acidification which elicit s the loss of Mg(II) from c h l o r ophyll(s).' 'Chlorin(s)': Unfortunately, a term which has ambiguity evolved into its usage. In the broadest, and most common, usage (1) a compound (viz. tetrapyrropyrrole) which e xhibits an electr onic spectrum in which the visible band order is I>IV,III,II (see Results I). Also, yet still vague, (2) a 7,8-dihydroporphyrin Most specifically, and the usage implied herein, (3) a 7,8 dih y droporphyrin lacking the 6,y-cycloethano moiety (see [XXIII, XXVII, XXIXXXXI]}. In text, the term 'chlorin(s)' (note semi-quotes) will still refer to unspecified structures accord ing to the spectra l -type defin ition. 'Purpur in(s)': Chlor i n(s) with electron withdrawing groups (g.g. glyo xylic acid [XXIV], dicarbo xylic acid anhydrides [XXVI], formyl, etc.) in conjugat ion w ith t hey-meso position (see F i g. 2a). Band I absorption is maximal at >670 nm. 'Rhodin(s)': (1) Chlor i n(s) derived from chlorophyll-b Indi vidual forms identified by lower case letter suffix w ith arabic numeral subscript indicating the number of o xygen atoms (g.g. rhodin-g7 [XXVIII]). (2) Porphyrins which yield electr onic spectra with visible band order and shapes, remenescent of b-series chlor ins (i.g. 'rhodin(s) (1)). Structurally, porphyrins with keto-substituted 6-membered 'isocyclic' rings formed via acid catalyzed cyclization of a propionic acid to they-c arbon (see [XLI-XLVII]). These rhodins are identified by a prefix indicating the parental porphyr i n acid (see Appendix A). 'DPEP. DPEP-series. D': Named after the archetypical compound Qeox oQhyllogrythroetioQorphyrin (Fi gure 4a) Ser i es designat ion indicates pseudohomologs w ith the nominal mass of DPEP (476 amu) 14n (n is an integer: see section on Mass Spectrometry and Appendix A, [XXXII-XL]). 'ETIO. ETIO-series. E': Named after similarities to the 4 etioporphyr i n isomers (tetramethy l-tetraethyl porphyrins: Fischer and Orth, 1937; see Figure 4b, [LI-Lli]). Series designation indicates pseudohomologs with the nominal mass of an eti oporphyrin (478 amu) 14n (where n is an integer : see section on Mass Spectrometry).


a) c) N H b) d) R R R R Figure 4. Structures of archttpical compounds mentioned in text.a)DPEP[XXXVIII],b)ETIOLLII],c)pyrrole,d)'benzoetioporphyrin'(R=H or alkyl.see[CXVI]). 16


17 'Benzoporphyrin(s)': Porphyrins with a 4 carbon moiety cyclized and aromatized to adjacent B,B-pyrrole positions (viz. a butadiene substituent possibly formed from the cyclization/aromatization of adjacent methyl/n-propyl substituents: see Figure 4c-d). 'Benzo-DPEP. Benzo-DPEP-series. BD': A benzoporphyrin with a DPEP nucleus. Mass spectral series with nominal mass of M-8, where M = ETIO (see Yen et gl., 1969: cf. gl., 1967). 'Benzo-ETIO. BenzoETIO-series. BE': A benzoporphyrin with an ETIO nucleus (Fig. 4). Mass spectral series with nominal mass of M-6 where M = ETIO (see Yen et gl., 1969: cf. Baker et gl., 1967). 'Tetrahydrobenzo DPEP. THBD': Mass spectral series occurring at a nominal mass of M-4, where M = ETIO. Originally proposed as being a "di-DPEP" compound. That is, a porphyrin with 2 isocyclic rings (Yen et gl., 1969). Recently shown to be the reduced (tetrahydro) form of Benzo-DPEP (Barwise and Roberts, 1984). 'QYIQ=': Modifier used to indicate the ('thermal') loss of the 10-carbomethoxy moiety from the 'pheophorbides', 'pheophytins', or 'chlorophylls' (Fig. 3). 'meso-': (1) Prefix indicating the reduction of the 2-vinyl moiety to an ethyl substituent in any of the 7,8-dihydroporphyrins derivable from natural 'chlorophylls'. (2) Modifier to indicate a methine bridge position or substituent on a porphyrin (i.g. a meso position: Fig. 3). 'Oxy-deoxo. O.D. ': Designates the conversion of a carbonyl moiety to a hydroxyl, usually via analytical reduction with sodium borohydride (cf. Holt, 1959). In the present text 3 specific derivatives, given below, and a generalized "oxy-deoxo' designation are mentioned. The generalized form being used to relate positive conversion of carbonyl to hydroxyl (see Results-!) with geological tetrapyrrole pigments for which only a preliminary classification can be made. '9-0xy-deoxo. 9-0D-': Term employed herein to designate reduction of the 9-keto moiety of phorbides (Figure 5) be they synthetic standards ([V, VII, IX, XI, XVI, etc.]) or geologi c iso lates with similar physicochemical behavior. '3-Methanol-desformyl. 3-MDF-': Designates reduction of the 3formyl moiety to a 3-methanol substituent (cf. Holt, 1959) for chlorophyll-b derivatives (see [XVI, XVIII, XXIX] Figure 5.) '2-a-Hydroxy-ethyl-desacetyl, 2-aHE-DA-': Refers to the reduction of the 2-acetyl moiety of bacteriochlorophyll-a derivatives ( [ XXII]: See Figure 5). More detailed treatises on tetrapyrrole nomenclature can be found in Bonnett (1978), Fischer and Orth (1937), Fischer and Stern (1940), IUPAC-IUB (1978), Lemberg and Barrett (1973), Marks (1969), Seely (1966), Smith (1975), Tait (1968) and Willstater and Stoll (1913).



19 Tetrapyrrole Biosynthesis The following brief introduction to general tetrapyrrole biosynthesis was compiled from reviews of chlorophyll (Bogorad, 1966, 1976; Jones, 1979; Marks, 1969), heme/cytochromes (Lemberg and Barrett, 1973; Marks, 1969; Okunuki et gl., 1968; Tait, 1968) and general biochemistry (Lehninger, 1975; White et gl., 1964). Biosynthesis of porphyrin macrocycles begins with products of the Kreb's (tricarboxylic acid) cycle and amino acid metabolism/bio synthesis. Succinyl-CoA and glycine, respectively, are condensed into a-amino-B-ketoadipic acid which, in turn, is decarboxylated to yield 6-aminolevulinic acid (6-ALA). Two moles of 6-ALA are then united, via dual dehydration, to yield 1 mole of porphobilinogen (PBG), the primary pyrrole monomer. The union of 4 moles of PBG then yields the actacarboxylic acid uroporphyrinogen-III. As given earlier, a porphyrinogen (Fig. 2g) is an octahydroporphyrin containing saturated methine bridges In this paper (cf. Lehninger, 1975; Tait, 1968), subsequent product species will be given as the fully aromatized porphyrins. Thus, uroporphyrin-III (Fig. 6a) is the first porphyrin of this series. Uroporphyr.ins, as free-base pigments, are found primarily in the Mollusca (Fox, 1953; Rimington and Kennedy, 1962) and as the Cu(II) chelate "turacin" in the feathers of Turacus sp. (Aves: Fox, 1953). Decarboxylation of the (C-1, -3, -5, -8) acetic acid moieties of uroporphyrin-III (Fig 6a) yields coproporphyrin-III, which is wide spread in the worm taxa (Platyhelminthes, Nematoda, Annelida: Fox, 1953; Prota, 1980; Rimington and Kennedy, 1962). Within this series, continued decarboxylation of coproporphyrin-III at the C-2 and C-4 propionic acid groups, followed by oxidation of the resultant ethyl


a) b) COOH COOH C H 2 COOH CH2 CH2 C H 2 CH2 0 CH2CH2 C 0 Hf 0 0 0 0 H H H CH2 C 0 C H 0 Hf CH3 0 CH2 CH2 0 CH2 C H 2 H H CH2 CH2 CH2 CH2 COOH COOH COOH COOH c) -..-. -. d) C H=CH 2 H3C-{ '\., )-CH=C H 2 Hf C H3 H3C CH2 CH2 CH2 C H 2 COOH COOH Figure 6 Compounds involved in the biosynthesis of porphyrin(e. g heme) and chlorophyll structures. a) Uroporphyrin-III, b) coproporphyrin-III, c) protoporphyrin-IX, d) Mg-2,4-divinyl pheoporphyrin-a5 ME. N 0


21 moieties, yields protoporphyrin-IX ([LIII] Fig. 6c). ProtoporphyrinIX, as the prosthetic group of a variety of heme-and cytochrome-type complexes (Rimington and Kennedy, 1962; Okunuki et gl., 1968), is perhaps the most ubiquitous true 'porphyrin' in the biosphere (cf. Fox, 1953; Lehninger, 1975; Marks, 1969; Prota, 1980; Rimington and Kennedy, 1962; Tait, 1968). Though protoporphyrin-IX [LIII] is extremely widespread throughout the biosphere, it is not the most quantitatively abundant tetrapyrrole. The chlorophyll-to-heme weight ratio has been estimated to be about 100,000:1 (Corwin, 1960). Protoporphyrin-IX [LIII] gains additional importance in the overall scheme of tetrapyrrole biosynthesis as a key intermediate in the formation of the chlorophylls. That is, cyclization and oxidation of the 6-propionic acid moiety of protoporphyrin-IX ([LIII], Fig. 6c), in addition to the chelation of Mg(II), yields 2,4-divinyl-pheopor phyrin-a5 monomethyl ester (Fig. 6d), the precursor of chlorophylls (Lehninger, 1975; Jones, 1964, 1979). The above abbreviated synopsis of porphyrin biosynthesis reveals not only the basic macrocyclic structure of tetrapyrrole pigments but also serves as an introduction to their distribution as end-product biochemicals which are then available as geochemical precursors. Potential Geoporphyrin Precursors In accord with phylogenetic order, the chlorophylls evolve from a complex melange in the Prokaryota (bacteria: 15+ structures), through a more restricted mixture in the 'lower' Eukaryota (algae: 4[5?] structures) and finally to the relatively simple coupling of


chlorophylls-a and -b in the Chlorophyta and 'higher' plants (2 structures). 22 The most familiar structure of a 'chlorophyll' is that of chlorophyll-a ([I]; Fig. 7a) Since chlorophyll-a [I] is the most familar and widespread form of 'chlorophyll', we shall compare the alternate chlorophylls to it for the purposes of this section. In this light, chlorophyll-b ([XIV]; Fig. 7b) i s 3-formyl-3-desmethyl-chloro phyll-a and chlorophyll-d (Fig. 7d) is 2-formyl-2-desvinyl-chlorophyll a. There is some question as to the true in vivo e xi stence of chloro phyll-d (Holt, 1966; Jackson, 1976). Chlorophyll-d is neither wide spread nor even abundant when present in the Rhodophyta (Holt, 1966; Jackson, 1976). The occurrence of chlorophyll-d may reflect the high lipoxygenase activity of the Rhodophyta (Jacobi, 1962; Peterson, 1940). That is, the reported ease of chlorophyll-a vinyl group oxidation (i.g. mild permanganate: Holt, 1961, 1966) may, by analogy, facilitate 2formyl-2-desvinyl-chlorophyll-a formation (Fig. 7d) during extraction. During the present study, one fresh-water and several marine species of red-algae were extracted and, via spectrophotometric and chromato graphic analyses, no trace of 'chlorophyll-d' (11 = 688 nm: Smith and Benitez, 1955) could be shown. This may be the result of frozen samples in the present study. Freezing and other pre-treatments are known to deactivate certain enzyme systems associated with chlorophyll biosynthesis/degradation (Holden, 1976; Jeffrey, 1969). Future study of the Rhodophyta, including comparison between extraction of fresh and 'pre-treated' samples, may clarify whether 'chlorophyll-d' is biotic or an artifact.


H 3 C H 3 C 2 H S C 2 H 5 "c" d 1 H:P CH3 C H 3 Figure 7. Structures of algal and higher plant chlorophylls a)chlorophyll-a, b)chlorophyll-b, c)chlorophyllsc(alt. 'chlorophyllidesc '), d)chlorophylld 23


24 "Chlorophyll-c" [XIX], discussed at some length in Appendix A, is placed within quotes to emphasize major structural differences between its two isomeric forms (Fig. 7c) and the other chlorophylls. First, 'chlorophylls-c' [XIX] basic nucleus is a true porphyrin rather than a phorbide (Fig 2e) The presence of an isocyclic ring (Ring E in Fig. 2e) further classifies this macrocyclic type as a pheoporphyrin {7,8didehydrophorbide) or phytoporphyrin {cf. Bonnett, 1978; IUPAC-IUB, 1978). Second, 'chlorophylls-c' [XIX] lack an esterfied isoprenoid (phytyl, farnesyl) moiety, as found in the other chlorophylls. Rather, the #7 carbon is substituted with a free acrylic acid group (Fig 7c). The two main forms of 'chlorophylls-c' [XIX] are -C1 and -C2 with the difference being substitution at the #4 carbon by ethyl or vinyl, respectively (Dougherty et gj., 1966, 1970; Jeffrey, 1969). There are five principal forms of 'bacteriochlorophylls or, more properly, bacterial-chlorophylls. The subtle difference in nomenclature just forwarded is meant to draw attention to the time-honored usage of bacteriochlorin {Fig. 2d) to designate a nucleus comprised of a 3,4,7,8-tetrahydroporphyrin nature {Fischer and Stern, 1940; cf. Aronoff, 1966; Inhoffen et gj., 1967; Scheer and Inhoffen, 1978; Smith and Benitez, 1955). That is, of the 5 bacterialchlorophylls, or chlorophylls of bacteria, only the first 2 (-a, -b) are true bacteriochlorophylls while the remainder, variants among the Chlorobacteri aceae, all contain a phorbide (Fig. 2e: 7,8-dihydroporphyrin plus ring E), rather than a bacteriophorbide (Fig. 2f), nucleus. This may at first appear to be a moot point, but the usage of bacteriQchlorophyllc, -d and -e (cf. Bonnett, 1978; Brockman, 1976; IUPAC-IUB, 1978; Jones, .1979) infers the 3,4,7,8-tetrahydro-porphyrin oxidation level.


This is not the case. Thus, while all bacteriQchlorophylls are bacterialchlorophylls, the reverse is not true. These structural points are detailed below. 25 Bacteriochlorophylls, per se, e xist as two primary forms (-a, -b: Fig. B). Bacteriochlorophyll-a (Fig. Sa: see [XXI]) can also be considered as 2-acetyl-2-desviny l -3,4-dihydro-chlorophyll-a (Fig Sa; Fig. ?a). The principal form of this structure is more precisely designated bacteriochlorophyll-aP, with the 'p' subscript designating phytol as the esterifying (viz. C-7 propionic acid ester) isoprenoid. An alternate form is known and is designated bacteriochlorophyll-a99, with the 'gg' subscript indicatiRg the hex adehydro-form (precursor) of phytol, namely geranylgeraniol, as the esterifying isoprenoid (Olson and Stranton, 1966; Pfenni g, 1965; Seely, 1966; Strain and Svec, 1966; Svec, 197S). Bacterioch l orophyll-b (Fig. Sb), present in Rhodopseud o monas v i r idis, differs from the 'bacteria-a' form i n the presence of the unusual ethylidene group at C-4 (Fig. Sb: Svec, 197S). Bacterio chlorophyll(s) a is present in the purple sulfur bacteria while bacteriochlorophyll-b, to date, appears restricted to 1 species (viz. R. viridis : Strain and Svec, 1966; Svec, 197S). Thus, the suggested biochemical taxonomic differentiation between the Thiorhodaceae and Athiorhodaceae, based on the respective absence or presence of bacteriochlorophyll-b (Pfennig, 1965; Stanier et . 1970), appears tentative. In terms of structural diversity, the most complex chlorophylls are those of the family Chlorobacteriaceae, the green sulfur bacteria. The 'bacteriochlorophylls' -c, d and -e are present in species of the Chlorobium genus and were first called Chlorobium-chlorophylls.


a ..... . : H C 0 .. 3 : M . . M b H 3C, ,.0 c;.o" M Figure 8. The structures of bacteriochlorophylls-a (a) and -b (b). . . . . M N (j\


27 The investigation of the Chlorobium-chlorophylls has spanned more than twenty years {Archibald et gl., 1966; Brockman, 1976; Holt, 1961; Holt and Hughes, 1961; Hughes and Holt, 1962; Jones 1964, 1979; Kenner et gl., 1976; Purdie and Holt, 1965; Smith and Calvin, 1966; Smith et gl., 1982, 1983; Stanier and Smith, 1960) and is detailed elsewhere {Baker and Louda, 1986a). The 'bacteriochlorophylls' -c and -d correspond to Chlorobium chlorophylls -660 and -650, respectively gl., 1980, 1982, 1983; Stanier and Smith, 1960: cf. Bonnett, 1978; IUPAC-IUB, 1978). Designation of the Chlorobium-chlorophylls as either -"660" or -"650" reflects the (nm) position of band I (red) absorption for spectra taken in ethyl ether {Stainer and Smith, 1960). The structures of 'bacteriochlorophylls' -c and -d series, also given in the Chlorobium spectral type nomenclature, is presented as Figure 9. Unique features of these chlorophylls include the 2-a-hydroxy-ethyl group, lack of the C-10 carbomethoxy moiety (viz. pyropheophorbide nucleus), existence as various alkyl homologs at C-4 and C-5, esterification of the C-7 propionic acid with farnesyl {C15 ) rather than phytyl (C20 ) and, in the '660' (BChl-c) series, meso-methylation at the 6-bridge {Holt and Hughes, 1961; Holt et gl., 1966; Hughes and Holt, 1962; Purdie and Holt, 1965; Smith et gl., 1980, 1982, 1983). The structural assignments given (Fig 9) are the most recent 1980, 1982, 1983). The 'bacteriochlorophylls' -c and-dare present in various strains of Chlorobium thio-sulfatophilium (Stainer and Smith, 1960). 'Bacteriochlorophylls' -d, isolated from Rhodopseudomonas spheroides, appears to differ from the primary Chlorobium type pigments in the presence of the C-10 carbomethoxy moiety (Jones, 1964). Chlorobium


28 Calculated SUBSTITUENTS11 DPEP-derivative Chlorophyll 11 RJ carb"on.j molec. Designation R1 R2 number weight u ....... Fr-a Et Me Me 33 490 '0 Fr-b Et E t Me 34 504 l/)<0 _)<0 Fr-c nPr Me Me 34 504 I, UE Fr-d nPr Et Me 35 518 Q::l O::ii Fr-e i8u Me Me 35 518 wo Me 36 532 1-L Fr-f i Bu Et uo Me 36 532 <.( Fr-g neoPent Me cnu Fr-h neoPent Et Me 37 546 ._., -o-' 0 650-1 i Bu Et H 35 518 I

29 phaeovibroides yields its main chlorophyll as the 3 formyl-3-desmethyl analog of 'bacteriochlorophyl l-c' fraction-6. This compound has been designated 'bacteriochlorophyll-e' (Brockman, 1976). The heme-type compounds are the most ubiqu i tous tetrapyrrole pigments in the biosphere through their intimate involvement as respiratory pigments, electron transport nuclei and as t h e prosthetic groups of numerous enzymes (Fox, 1953; Lehninger, 1975; Lemberg and Barrett, 1973; Okunuki et gJ., 1968; Sane, 1979; Stanier et gl. 1970; Wilson and Erecinska, 1979). Classification, nomenclature and structures of cytochrome prosthetic groups (viz. hemes) follow that given by Lemberg and Barrett (1973). Additional information derives from the works of Bonnett (1978), Rimington and Kennedy (1962), Marks (1969) and Tait (1968). The metallo-chelates, mainly Fe, of protoporphyrin-IX (Fig. 6c) form the prostetic groups of heme (Fe(III)Cl) in hemoglobin, protoheme or cytoheme-b (Fe(II)) in the b-series cytochromes, and various enzymes (Okunuki et gj., 1968; Riminton and Kennedy, 1962). Variations of the structure of protoporphyrin-IX (Figs. 6c, lOb) i nclude those shown in Figure 10. Heme-a, or cytoheme-a (Fig. lOa), differs protopor phyrin-IX in being the C-8 formyl-desmethyl analog and containing a C17H290 moiety in place of the vinyl at C-2. This long isoprenoid chain (ethyl plus farnesyl) is also unique among natural tetrapyrroles. The heme-c pigments (Fig. lOc) are basically ferroprotoporphyrin-IX which is reduced to ferromesoporphyrin-IX and linked to peptides via thioether bonds to the terminal carbon (B) of the (C-2, C-4) ethy l moieties. Cytoheme-d (Fig. lOd) differs from protopo r phyrin-IX (cytoheme-b: Fig. lOb) in being a chlorin (7,8-dihydroporphyrin:


30 a) H2 H c c-c C =C/ '-c/ '-cH3 H \CH H2 3 b) H3C CH=CH2 C=CH2 H H,c O'' C H3 CH2 CH2 CH2 CH2 CH2 CH2 COOH COOH CH2 CH2 COOH COOH c) d) R I H-C-OH HC C=CH2 H H3C H3C CH3 CH2 CH2 HH C H2 CH2 CH2 CH2 COOH COOH CH2 CH2 COOH COOH Figure 10. Heme(alt. haem) type a ) Heme -a(cytoheme-a), b) protoheme (cytoheme-b), c) heme-c (cytohemec), d ) c ytoheme-d.


31 Fig. 2c) and having an alkyl-substituted methanol group in place of the C-2 vinyl. Derivatives of biotic tetrapyrroles are of keen interest in geochemistry as standards and analogs for comparison to geoporphyrins. Certain in vitro derivatives of protoporphyrin-IX [LIII] are given in Figure 11. Beginning with protoporphyrin-IX ([LIII] Fig. 11a), reduction of both vinyls (C-2, C-4) yields mesoporphyrin-IX [LV] and, following decarboxylation (C-7, C-8), etioporphyrin-III [LI]. Alternately, devinylation and decarboxylation provides deuteropor phyrin-IX [LVI] and deuteroetioporphyrin [LVII], respectively. Aside from the widespread chlorophyll and heme-type compounds, several unusual tetrapyrroles are known and, as potential geoporphyrin precursors, require mention. 'Spirographis porphyrin', also called chlorocuroroporphyrin (2formyl-6,7-bis [2-carbonylethyl]-1,3,5,8-tetramethyl-4-vinyl porphyrin: Fig. 12b), is found in the greenish 'blood' (hemolymph) of certain polychaetes (Jackson et 1974; Prota, 1980; Rimington and Kennedy, 1962). In essence, 'Spirographis porphyrin' is the 2-formyl-2-desvinyl-derivative (Fig. 12b) of protoporphyrin-IX ([LIII] Fig. 11a). The green dermal integuments of the echiurian Bonellia viridis yields the pigment bonellin (Fig. 12c). This compound is unusual in nature since it possesses gem-dimethyl substitution at C-8 (Pelter et 1976, 1978). Structurally, bonellin can be considered as a 7H-8-methyl derivative (Fig. 12c) of deuteroporphyrin-IX ([LVI] Fig. 1lc). Neobonellin, present in the proboscis viridis, is found to be the 7-d-isoleucine conjugate of bonellin . 1979). Alternately, bonellin (viz. neobonellin) is found conjugated to valine,


a) b) H C=CH2 H t ")-H ( DEVINYLATION) C H 2 C H 2 C H 2 CH2 CH2 CH2 CH2 CH2 COOH COOH R R b I) R=COOH b 2 ) R= H (R EDUCTION) c) t d) C H 2 C H 3 C H 3 1 { J C H 2 C H 3 N H N_ (DECARBOXYLATION) C H 2 C H 2 C H 2 C H 2 C H 2 C H 2 C H 3 CH3 COOH COOH Figure 11. S tructural relationships in the protoporphyrin-IX series. a)Protoporphyrin -IX, b) b1 ; deuteroporphyrin and b2 ; deuteroetioporphyrin, c) mesoporphyrin-IX, d) etioporphyrin -III. w N


o) 0 jJ b) CH3 H C 3 'LN; . N_/ H 0 H f C H < 0 CH2 CH2 CH3 CH2 CH2 COOH COOH c) d) H C:H H H H3c-/ 1 H 2o C H 3 H,C-,<. 1 \ I Figure 12 Structures of certain unusual biologic tetrapyrroles. aO F430M, the nickel tetrapyrrole nucleus of F430 in methanogenic bacteria(Pfaltz al.,1982), b) Spirographis porphyrin, c) bonnellin, d) anhydrobonnellin. w w


34 and alloisoleucine (Pelter et 1978). Anhydrobonellin, given as Figure 10d, is the 73-keto-7,y-cyclopropano derivative of bonellin formed by a dehydration-cyclization reaction (Agius et 1979; Pelter et 1978). Such cyclizations of propionic acid groups to adjacent unsubstituted methine bridges are well known in vitro reactions, as typified by the cyclization of mesoporphyrin-IX [LV] to yield meso-rhodin-IX [XLI] (Fischer and Orth, 1937; Fuhrhop, 1978). The molecular 'fingerprints' of the methanogenic Archebacteria are becoming increasingly identified within the organic matter of sediments and bituminous products (i.g. oil: Brassell et 1981; Chappe et 1982; Michaelis and Albrecht, 1979). Recently, the nickel (II) tetrapyrrole prosthetic group of F-430, required for C02 reduction (Diekert et 1979, 1980a-b; Fassler et 1982; Whitman and Wolfe, 1980) by the methanogens (g.g. Methanobacterium thermoauto trophicum), has been structurally defined by A. Pfaltz and co-workers (1982). The structure of F430M, a methanolysis product of F430, is given as Figure 12a. This unique compound might be a geochemical precursor in methanogenic sediments. However, conversion to known geoprophyrin structures would appear tenuous, given the highly reduced nature of macrocycle and the uniqueness of the periphery. "Haemovanadin," present in the blood of sea-squirts (Ascideacea: Califano and Boeri, 1950; Fox, 1953), had been considered as a potential source for the vanadium tetrapyrroles in shales and petroleum (Glebovskaya and Vol'kenshtein, 1948; Moore and Dunning, 1955). However, recent studies reveal that the V(III) compounds are not tetrapyrroles (Prota, 1980) and substantiates an earlier report (Webb, 1939) with the same conclusion. Thus, "haemovanaqin" is discarded as a


35 precursor to vanadyl petroporphyrins and even as a vanadium-enriching route, given recent studies on metals during sediment forming processes (lead references in Lewan and Maynard, 1982). The above biologic tetrapyrroles are considered as the potential precursor pool from which geoporphyrin products ultimately evolve. Geologic Metalloporphyrins Biologic tetrapyrrole pigments which escape total remineral i zation eventually evolve into geologic metal loporphyrins, also known as "petroporphyrins (Corwin, 1960). During diagenesis biologic metallochelates, primar i ly Mg(II) and Fe(III), eventual l y loose their enzymatically 'chosen' metal, form free-base pigments and subsequently (Baker and Louda, 1983, 1986a) complex with an alternate metal. Six metals have been adequately identified as being chelated by geoporphyr i ns, that is, discounting identifications based solely upon e l emental analyses (g g atomic absorption) of crude fractions which conta ined tetrapyrroles (lead references in Dunning, 1963). That is, identification of a metal(s) and tetrapyrrole pigments (via demetalla tion techniques) from a crude fraction or extract does not necessarily imply that all the metals found e x isted in situ as metalloporphyrins These 6 metals (Table 1) are; V(IV), Ni(II), Fe(III), Cu(II), Ga(III) and Mn(II). The first two metals indicated in geologic 'porphyrin complex salts' were V and Fe by Treibs (1935a). Vanadium was essential ly confirmed by the syntheses of vanadium chelates of mesoporphyrin-IX [LV], protoporphyrin-IX [LIII] and "etioporphyrin" ([LII]?) for spectral comparisons (Treibs, 1935a). The second complex was


Table 1. Metals reported in geologic metalloporphyrins. METAL11 V V( IV), as VOZ+ 'Fe' 'Fe' Fe(III) Ni (II) Cu(II) Ga(III) Mn(III) REFERENCE Treibs, 1934 a-b al., 1956 b Treibs, 1934 a-b Moore and Dunning, 1955 Bonnett and Czechowski, 1981 Glebovskaia and Vol'kenshtein, 1948 Palmer and Baker, 1979 Bonnett and Czechowski, 1980 Bonnett and Czechowski, 1981 11 Semi-quotes refer to reports which lack, by today's standards, sufficient proof and/or do not show the valence of the metal. 36


as the "Hamochromogentyp" (Treibs, 1934a-b}, or i ron porphyrins. 37 At the time of Treibs' identification of vanadium, he ascribed two possible valences (IV and V} and four potential, o xygen contain ing, stru ctures. The two favored structures (PH = porphyrin -2H) were PH=V(OH)2 and PH=V-0-0-0-V=PH for the tetra-and penta-valent vanadium cat ions, respectively ( Treibs, 1934a). These forms were, for the most part, accepted and quoted by F i scher and Orth (1937). The p r esence of V-geoporphyrins was later conf irmed (Glebovskaia and Vol'kenshtein, 1948), reconfirmed (Skinner, 1952) and became increasingly well studied (Baker and Louda, 1986a; Baker and Palmer, 1978; Dunning, 1963; Hodgson et gl., 1967). Erdman and co-workers (1956b} determined that the form of vanadium is tetravalent and exists as chealted vanadyl [(V0)2+] radica l (Table 1). Even more e x acting confirmation of V(IV) porphyrins being vanadyl chel ates arose with the synthesis (Baker et gl., 1968) and c rystallographic descr i p tion o f VO-DPEP (Pettersen and A l e x ander, 1968). The se cond metalloporphyrin type isolated by Treibs (1935a-b) was ascribed to Fe-chelates and reported a-band absorptions ranged from 547.6 to 553.0 nm for "Hamochromogentyp" compounds from oilshales petroleums and a wide variety of coals. Later, Glebovskaia and Vol'kenshtein (1948) e xamined sedimentary bitumens and petroleums and concluded: "The e x istence of t w o types of metallic complex es of p orphyrin-vanadium and, apparently nickel has been established."* *From translation of Glebovskaia and Vol'kenstein (1948) by G. Belkov (1951, Ottawa: National Research Council of Canada, Technical Translation #TT-281).


38 Numerous studies during the 1950's (references in Baker, 1969; Dunning, 1963; Hodgson et 1967) have confirmed their identification. While 'the Fe-porphyrins identified by Treibs (1935a-b, 1936) were later reported by Glebovskaia and Vol'kenshtein (1948) to be the nickel species' (Baker, 1969; Baker and Palmer, 1978; Dunning, 1963; Dunning and Moore, 1957; Hodgson, 1973; Hodgson et 1967; Maxwell et 1980; etc.), it is highly probable that Treibs (1935a-b) did, in fact, isolate both Fe and Ni porphyrins (cf. Baker and Louda, 1986a; Bonnett et 1984). In retrospect, the presently known Fe and Ni species probably were the "Hamochromogentyp" pigments Treibs (1935a-b) found in coal and bitumens, respectively. Moore and Dunning (1955) again postulated Fe porphyrins as geochemical entites. However, these identifications rested upon the relative amounts of porphyrins (demetallation technique) and metals (elemental analyses) available (Dunning, 1963, Dunning and Moore, 1957; Moore and Dunning, 1955). The first definitive identification of iron porphyrins (viz. Fe(III)) is ascribed to Bonnett and Czechowski (1981). All of the Fe(III) porphyrins positively identified have been isolated from humic coals (Bonnett and Burke, 1985; Bonnett and Czechowski, 1981; Bonnett et 1984: cf. Treibs, 1935a-b). Recently, the occurrences of Ga(III) and Mn(III) porphyrins, also from humic coals, have been reported (Bonnett and Czechowski, 1980, 1981; 1984; Bonnett et 1983, 1984). Copper porphyrins were first definitively identified as trace constituents of deep-sea sediments by Palmer and Baker (1979). In that same report, it was suggested that Cu(II) porphyrins accompanied redeposited-oxidized terrestrial organic matter Louda and Baker


39 (1981) subsequently showed that these Cu(II) complexes of "highly gealkylated gtioporphyrins" (HOEs): gave rise (via de-and re-metallation) to coincident arrays of Ni(II) HOEs; correlated with erosional/ redepositional events accompanying oceanic regressive-low stand events; and probably evolved from porphyrins found in terrestrial organic accumulations (peats, coals: Louda and Baker, 1981). Further studies on the occurrence and probable history of the Cu(II) porphyrins, including data from the present study, have been completed and reported (Baker and Louda, 1984, 1986a-b). Geoporphyrin Heterogeneity Between the initial studies on geologic tetrapyrrole pigments (Treibs, 1934a-b, 1935a-b, 1936) and the application of modern mass spectrometric technique (Baker, 1966; Baker et gl., 1967) the heterogenous nature of geoporphyrins was all but unknown. During this span, however, Corwin (1960) coined the term 'petroporphyrins', which served to segregate consideration of porphyrins of geologic samples from those of biotic sources Aside from a difference between 'plant-type' (DPEP 'phyllos') and 'animal-type' (ETIO) porphyrins, the only diversity thought to e xist among geologic porphyrins in the 1950-1960s was the random presence of their carboxylated precu r sors (Dunning, 1963). The presence of carboxylated porphyrins in bitumens has been alternately shown via HCl-number (Treibs, 1934a, 1935b), water-spray extraction (Dunning, 1963; Dunning et gJ., 1953), paper chromatography (Blumer, 1956; Dunning and Carlton, 1956) and recently by MS and/or NMR analyses (Bonnett et gl., 1983; Habermehl and Springer, 1982; Louda-this


study; Louda and Baker, 1981; Ocampo et 1985a-b; Smith et . 1980). Preliminary ind i cations that the metalloporphyr ins in petroleum crudes might e xist over a large molecular weight range evolved from 40 industrial observations that the catalyst poisons V and Ni were spread throughout various d istillate fractions and that they were present as porphyrin complex es (Erdman et 1956a) Such observations were to lead to var ious methods (Erdman et 1959) to dest roy the metallo porphyrins prior to the catalytic cracking of petroleum Beach and Shewmaker (1957) performed detailed distillat ion-cu t analyses and defined two main types of petroleum metallo -complexes. Class I complex es i ncluded mainly monomeric VO-porphyrins (b.p. 585-650C, m.w. <500-800 a m u.) Class II complex es included high molecular weight VO-porphyrins (b.p. >650C, m .w. 800+ a.m. u ) and unspecified nonporphyrin V-compounds (Beach and Shewmaker, 1957). Gel permeation chromatography (GPC*) performed on oil-shale bitumens revealed that, in addition to VO-porphyrin monomers ('regular': ca. 4 50-650 daltons), dimers (mw 1,000 daltons) and complex es (2,000-20,000 daltons) e xisted (Blumer and Rudrum, 1970; Blumer and Snyder, 1967). These "complex es" of VO-porphyrins were also reported to max imize at ca 2,000 or ca. 20,000 daltons depending upon analysis of 'maltenes' or 'asphaltenes,** respectively (Blumer and Snyder, 1967). Alternately referred to as 'size e x clusion chromatography' (SEC). **Defined by asphaltene precip itation with n-pentane (Blumer and Snyder, 1967).


Mass spectrometry provided the breakthrough in unraveling the heterogeneity of geoporphyrins. 41 The first use of MS technique for the study of (geo-) porphyrins was the determination of the standard pigments Ni-etioporphyrin-III (Ni[LII]: Hood et gl., 1960) and VO-etioporphyrin-I ([CVII]: Mead and Wilde, 1961). Dean and Whitehead (1963) were apparently the first to use MS for geoporphyrin analysis. They reported MS evidence for metallo-porphyrin 'homologs' extending from at least C28 to C33. However, they qualified their conclusion by stating that they" ... accepted with reserve ... "this conclusion on the basis of the high temperatures required to distill the pigments within the mass spectrometer (Dean and Whitehead, 1963). Their results did, however, lay the first firm foundation for the consideration of geoporphyrins (alt. petropor phyrins": Corwin, 1960) as anything but the single carbon numbered entities (i.g. C32) predicted by the Treibs' scheme (Treibs, 1936). Independent reports on the mass spectral distributions of porphyrins from oil-shales came from Morandi and Jensen (1964, 1966) and Thomas and Blumer (1964). Both of these reports employed high voltage (70 eV) technique, yet made mention that "low-voltage" (? eV) spectra contained primarily molecular i ons. Systematic studies by Baker (1966) on the mass spectral characterization of 'petroporphyrins' provided the description of four main geoporphyrin series (DPEP, M-2; ETIO, M; rhodo-DPEP, M-8; and rhodoETIO, M-6) which were defined by hydrogen deficiencies when related to the ETIO-series (478 m/z 14n, where n is an integer: gl., 1967). In addition to the description of geoporphyrin series, the need


42 for low-voltage (12-14 eV) technique and care during sample volatilization, in order to achieve reproducible data, was shown (Baker, 1966; Baker et gl. 1967). These studies defined the first two levels of geoporphyrin heterogeneity: These being; (1) the e xistence of several 'series' or types, and (2) the presence within these 'series' of carbon numbers ('homologs') ranging from at least C -26 to C-38. Even though mass spectrometry has proven to be a powerful technique for geoporphyrin description, the more routine methodology of ElMS* tells little of the third and most complex level of geoporphyrin heterogeneity. That is, within each 'series', as defined by electronic and mass spectral data, a wide variety of isomeric forms (exacting structures) has been shown in the last several years. Prel i minary evidence for of more than one isomer for each carbon number, most notably amongst the DPEP-series, derived from a combination of thin-layer chromatographi c (TLC) and MS techniques (Alturki et gl., 1972; Blumer, 1974; Didyk et gl., 1975a; Thomas and Blumer, 1964). The analytical oxidation of porphyrins with chromi c acid to yield maleimides, a val uable technique synthetic and biotic tetrapyrroles (Bonnett and McDonagh, 1969; Ellsworth, 1970; Ellsworth and Aronoff, 1968; Morley et gl., 1959), was nex t applied to geoporphyrin analyses (Baker and Palmer, 1978; Barwise and Whitehead, 1980; Hodgson gl., 1972; Quirke et gl., 1980a). Notable findings obtained through maleimide analyses include; proving the proposed benzo-porphyrin nature of geologic 'rhoda-porphyr ins' gl., 1967), through the *ElMS = electron impact mass spectrometry.


43 identification of phthalimides (Barwise and Whitehead, 1980), and revealing that the higher alkyl 'homologs' contain primarily n-alkyl Bpyrrole substituents, up to at least n-C11 (undecane) and likely beyond (Baker and Palmer, 1978; Barwise and Whitehead, 1980; Quirke et gj., 1980a). During the past decade, there have been efforts to unequivocally identify single geoporphyrin isomers. These studies employed gas chromatography (GC: Boylan and Calvin, 1967), GC-MS (Boylan et gJ., 1969; G allegos et gJ., 1983), computerized GC-MS (Eglinton et gj., 1984; Hein et gJ., 1985), derivatization of 'open' B-py rrole (i.g. B-H) positions (Antipenko et gJ., 1979; Quirke, 1983), high-performance liquid-chromatography (HPLC: Barwise and Park, 1983; Barwise and Roberts, 1984; Hajibrahim et gJ., 1978, 1981; Quirke et gJ., 1979, 1982; Sundararaman, 1985), nuclear magnetic resonance (NMR: Chicarelli and Maxwell, 1984, 1986; Chicarelli et g}., 1984; Fookes, 1983a-b; Krane et gj., 1 983, 1984; Ocampo et gJ. 1984; Quirke and Maxwell, 1980; Quirke et gj., 1979, 1980a-b; Storm et gJ., 1984; Wolff et gJ., 1983), chemical ionization (CH4H2 ) MS (CI-MS: Eglinton et g}., 1979; Shaw et g}., 1978, 1981), H2-CI -MS-MS (Sundararaman et gJ., 1984) and X ray diffraction (XRD:Ekstrom et g]., 1983a). A summary of the reports of geoporphyrin structures is given as Figure 13 and Table 2. Five of the structures shown in Figure 13 are not definitive and include two possible artifacts (Fig. 13c6.7) and three structures (Fig. 13d,j,o) narrowed to two possible isomeric forms. Inc luded among the identified geoporphyrins are 20 alkylporphyrins (Fig. 13a-j) and 10 porphyrin carboxylic acids (Fig. 13k-o). The isolation of most of these pigments occurred primarily (30 out of


al b) C) d) el f) g) h) ll Jl R k) I) ml nl ol R COOH COOH Figure 13. Structures of geoporphyrins for which uniquivocal identification is reported in the literature.References are coded by letter to those Riven in Table 2. Abbreviations;Me= methyl,Et= ethyl, nPr= n-propyl,iBu= iso-butyl, ?"= 2 possible isomers.STRUCTURES:(a) -(ref.A)J(b)-(Refs.B,D,E); (c) #c1/R1=H,R2=Me #c2/ R1=Me,R2=Me,R3=Et(Refs.F,G,P)J R3=nPr(Ref.M),#c5/R1=Et,R2=H,R3=Et(Ref.I);(d)R1=Me(or Et),R2 =Et(or Me)-(Ref.P);(e) #e1/ R=Me(Ref.L);#e2/R=Et(Ref.l);(f) #f1/R=H(ref.n);#f2/R=Me(Refs.L,N);#f3/ R=Et(Refs. L,N,O); (g) #g1/R=Me(Ref.R); #g2/R=Et(Ref.R); (h)-(Ref.Q); (i)-(Ref.K)(j)-(Ref.U); (k) #11/R=H (ref.S),#l2/R=Et(refs.S,T);(m) #m1/R=Et(Ref.T);#m2/R=nPr(Ref.T);#m3/R=iBu(Ref.T);(n)(Ref.S); (o}-Ref.U). +=" +="-


Tabl e 2. G eoporphyrins for which u nambigu o u s assignment of structure has 1-( :) g SEniES(4 ) >lo S o (S) NATIVE (b) \o r171 t- Cl sn1F.S HETH SOURn TOTAL "' F.::!9 (>I) Ml C. I L 1) Ml J CO S 9 cl lIIDE-DPEP(XL) Oll (M 2) HI JCOS 12 1.: 00 00 HI ABEL 100 c1 00 .. v sos -f I 1, l S &-t et raac th)" J .. _. -cthyt-1, y -eye: 1 o-porph I n OlO (H l) Ml JC05 I o 5 f2 ,t,, 2, 3, S, 1-pent thyl-.t-e:thy 1, y C yc I l n OJI ( II Z) \'(! ) sos < rz .. 00 Ml z 0 s fl ,t, ,l, S, I t c t raact hyl -2 ,.t-di ethyl -7 ,ycrc in 032 ()1 2) V(!) sos fl .. .. Nl JCOS 0 0 fl .. .. .. v sos II S S -t ct ro111u hy 1-7 c thy l-6, y eye I oct hano .. J, -1 .. eye Jobut,.d i cnOJiorrld n 8Dl2 (II S) v BOSC )I I 2 1, S, 1-t r l cthy 1 2, 7 J I et hy 1-6, y-cyc I oct, 4 i cno-porph I n BOll ()I I) v BOSC h l,l,S,!, 9 -pcntar.:acth)'l-2,4-dtcthyl-7,10-c)'Cioprop(7 )cnc-7,5-dtdchyJro-phorbidc Btl.a (11-6) v sos I I l,l, S ,.S-tet r a111uhy 1-2, .t-1.1 I cth)'l-7,y-cyc I oct .. ,.cthyl) -porrhin OJI (II 2) Nl I I OS I J l ,l. S-t rlc t hyl -2,-' -J I et hy 1 -6, y C)'t I oct hnno-7, &-ere 1 abut ano-rorrh t n TUllO j l ()I 4) y ELS ? Er\ ll Nl HOS I kl ploy lloporph)' rl n ( L \ olll.i) EAll Nl II OS ? l''ll l i DS I ::::; 11 PAll HI HOS ? < 12 orE(XXX1IIl I> .Ill Nl I I OS >SO "'II .. 00 HI )lOS ; I S-EOH-ort II All N l HOS ? .... z S-( D > l .J n r 0 C-0 r t: 0.\l..& HI I l 5-EOll<. I roE-OPE 11.\lS Nl HOS I g n I l S, S-t e t r ::ar.1 c thy 1 -! -1 d l c l h y I 7 y-c rc 1 oct h :n o ( 7 I -:a c: e t I c ::a c l d) -J1 or ph i n IJ,\J 2 Nl II OS '-0 csoro q hy r l n-I l (I.\' ) t:Ji.\34 Fe ALis >70 l)As o( June 199C';2)Scc .1lso Fi,urc ll; l)Civc n in the FischerH)'lc no u :ncJal\lre(s c c Introduction) .Abbrcvletlons: UrEPdesoaoph>"ll oe fyt h roet 1 opoq1h)'r In ,HO[ e t h)'l-dcs e t hy I, I'DEpr o p y I -Jc: s e t h)'l, OPEdc so1.ophy 11 oerythr 1 n, EDtc t hyl -dc sc t h y I. :!_) t ct io (.a 78! 14/:), OOrEr ( 4 76J 14a/::: 11CAr"cyc I 0.1 rphrr1 ns; ...!.Chi ca r c IIi and Mu: wc 11,1916) ,00 oxr-Dr[r (ODO &0 :o-0r1 : r ( 470t 1 t), DF.llcn:o-r:T TO ( 72! U11/ z), TIIBU tet r.lh)'ll rohonzo-DrEP ( 74 .t 14 /,), Aca rboa y 1 1 c .lCid. in of hydroecn Jefcclrncles as to the ct a1.,1967:Yen et :at -;-196!:1).6) oil Hill coai,.JCOS.Iulll Creel.. oil shalc,ABELAhft'i'Onl,c, rcls;-J'c:rpiano Oil sh.l)c bOSCBoscJn crude oi I,ELSEI J.ujan $h.l lt',All,Ausrrallan llgnltc, 7)J\s the I U thor w :as htst a b l e to ascertain fro the l l tcr:'lturc ciceJ. S)M.cferrnces tlvC"n bdow by lcttrr co,lc. -:\(Quir\c ..:t a l.,l97!)),ft(t}ulrl.c and et ct :ai .,I9Sl),F.(8onnett and l:cc how sti.198ol),fTf.O"nkcs,t98.ii),r.(Stor :t1.,1!18.,),11(E[HrO ct-;l:-7"198l),I(C:iilr:trcl11-.j;'J taawrll:-t9SJ),J(t::r:anc ct :a1.,1984/1JIOJ$Ihlc-;arrif:.ct,),lr:(OC';aI'O ct-;-I-:-:-J!)II4),1.(Chicarclli'"Ct""""il l94.&):-H(\'crnt'-ti!>Cr cl oai .. c:s T9Slb),O(to:olff ct c t :ai.,Tisl),Q(Chic.:arclli 1nd JlouW"CJT;l9S6).R(to1u r ct ;ai.,J9Bb'T,S7oc3f!o ci" !COcapO !_! !1_.,191'1). ------been E/! F F c p C/1 0 F F II If I J/1 J/1 P/1 L L N L H L H 0 R R Q K U/1 s s s s s T T T T s V/1 Vl


46 40 identifications) during the in-depth study of a few immature oilshales (Table 2). That is, there are 13 identified components from the Messel shale (10 porphyrin acids and 3 alkyl porphyrins), 9 structures (all alkyl porphyrins) from the Julia Creek formation and 8 geopor phyrins known from the Serpiano oil-shale. The majority (16/28) of the reported structures (Figure 13) agree with modified Treibs' scheme (cf. Baker and Louda, 1986a; Baker and Palmer, 1978; Corwin, 1960; Treibs, 1936) diagenesis. That is, by invoking only the in vitro degradative chemistry of chlorophyll (vinyl scission, devinylation, oxidative isocyclic-ring opening, etc. Fischer and Orth, 1937; Fischer and Stern, 1940) and the structures of the bacteriochlorophylls, the majority of the known geoporphyrin structures are potentially explainable by the 'Treibs' scheme' (Baker and Louda, 1986a). Enlightening information as to the participation of chlorophylls other than the 'a-form' [I] has also evolved. For example, chloro phyll-b is the suggested precursor to the 3-desmethyl-DPEP isomer (Fig. 13c5: Chicarelli and Maxwell, 1984) and the bacterial-chlorophylls (alt. Chlorobium chlorophylls-660) are seemingly valid precursors to the OPE [XXXVIIa] analogs with variant alkylation at C-4 and C-5 (Fig. 13m1,m2,m3: Ocampo et gl., 1985b). Much harder to explain are the evolution of several 'pseudo-DPEP' compounds (Fig. 13d,e1-e2,f1-f3,hi,i) and the 2 benzo-DPEP structures (Fig. 13g1-g2) presently known. Each of these latter compounds has received preliminary speculation that unusual side-chain cyclizations C-7 acrylic to C-10 in chlorophylls-c, C-7 propionic acid to C-10 in chlorophyll a: references to Fig. 13) may form a significant portion of overall


tetrapyrrole geochemistry. Thus, this third and only recently proven level of geoporphyrin heterogeneity (isomer existence) vividly demonstrates just how complicated tetrapyrrole geochemistry truely is. Considering only 3 mass spectral 'entities' (viz. 030-032) one finds (Fig 13, Table 2) that these can represent, to-date, eleven separate compounds 3 ea./030; 5 ea./031; 3 ea./032: Table 2). 47 Every effort has made to glean some indication as to the quantita-tive importance of each structure to the overall geoporphyrin array from which it was isolated and reported. Examination of Table 2 reveals that only 7 specific quantitative estimates, out of 42 possible (16.6%), have been made. Thirty of the 42 make no inference as to quantitation and the rest are extremely vague (major vs. minor, etc.). Further, none of these studies (Fig. 13 references) report an overall 'fingerprint' (M.S. and/or HPLC) for the melange from which each structure was isolated and for 6 of the 42 reports the reader must even assume the chelated metal. These shortcomings in geoporphyrin analytical reports points to a lack of standardization in the field that is addressed elsewhere (Baker and Louda, 1990;Louda and Baker, 1987; 1990). The Controversfal OPEP-to-ETIO Crossover Presently, the most widely determined and applied geoporphyrin parameter or index is the OPEP-to-ETIO ratio (see Baker et gJ., 1967) or the more recent expression of identical data sets, the percent-OPEP (Barwise and Park, 1983: cf. Baker and Louda, 1986a). While the phenomenon of decreasing% OPEP (alt. 0/E) occurring in concert with increasing integrative temperature-time exposure ('thermal


48 stress') is well known in geochemistry (Baker and Louda, 1983, 1986a; Baker and Palmer, 1978; Didyk et gl., 1975a-b; Mackenzie et gl., 1980), the mechanisms and driving forces are currently an area of active debate (Baker and Louda, 1986a; Barwise and Roberts, 1984; Burkova et gl., 1980a; Didyk et gl. 1975a-b). As initially proposed by Treibs (1936), chlorophylls would give rise ('geochemically') to the DPEP-porphyrins and the heme-pigments would evolve into the E T IO-type (Fig. 1). Thus, on this basis alone, one might expect the DPEP-to-ETIO ratio to reflect the relative amounts of precursor biotic pigments. As given above, it is known that, for maturing to mature organic matter, the relative abundance of the DPEPseries decreases This does not, of course, preclude a pre-catagenetic imprint of the original precursor biological pigment types. However, excepting the special cases of humic coals (cf. Bonnett and Burke, 1985; Bonnett et gl., 1983, 1984), little is known beyond the fact that anoxic aquatic sediment bitumens are vastly dominated by phorbide-type pigments characteristic of reductive chlorophyll degradation (Louda and Baker, 1986). On the bases of relative biotic precursor abundance (i.g. chloro phyll vs. heme "tonnage" : Corwin, 1960), apparent therma 1 conversion of DPEP-to-ETIO type structure during the retort of an oil-shale (Morandi and Jensen, 1966) and both observed natural trends and the results of in vitro heating experiments (Didyk et gl., 1975a-b), the concept of a thermal or thermocatalyt i c DPEP to ETIO "crossover" evolved (Baker and Palmer, 1978). Recently, the identification of mesoporphyrin-IX [LV], the quintessential precursor to etioporphyrin-III [LI], in immature humic


49 coals (Bonnett and Burke, 1985), and proposals that the isocyclic ring of chlorophyll(s) derivatives can be oxidatively opened during early diagenesis (Baker and Louda, 1980a, 1982, 1983, 1986a; Barwise and Roberts, 1984; Corwin, 1960; Louda and Baker, 1981, 1986) has fueled debate over both the ultimate source of ETIO-porphyrins and the mechanisms underlying observed maturational decreases in the OPEP-toETIO ratio (0/E: alt. % OPEP). In the first case, valid heme-derived precursors (viz. mesoporphyrin-IX [LV]) to etioporphyrins are now known to exist in geologic settings. Secondly, the probable conversion of chlorophyll(s) derivatives into likely ETIO-series precursors (g.g. phylloporphyrin [LVIIIa), pyrroporphyrin [LX) cf. Ocampo et gj., 1985a) may well lead to an etioporphyrin pool sufficient to explain the quantities found in more mature organic matter Thus, thermal or thermocatalytic OPEP-into-ETIO conversion does not need to occur in order to explain observed decreases in % OPEP (alt. 0/E-value). That is, it has been suggested that only a faster rate of OPEP-series destruction (k1 in Fig. 14), relative to that for the ETIO-series (k2 in Fig. 14), is requisite to produce the observed maturational (-%0) trends (Barwise and Roberts, 1984; Burkova et gj., 1980a,b). Thus, as is summarized in Figure 14, the source of ETIO-series may be from either heme-pigments and/or chlorophyll(s) during early diagenesis and/or by later diagentic/catagenetic conversion of OPEPtype into ETIO-type skeletons. Likewise, observed maturational decreases in the %0PEP (0/E-value) may be due to a faster rate of OPEPseries destruction, relative to that for the ETIO-series, and/or a true thermal/thermocatalytic conversion of OPEP-into ETIO-type pigments.


C H LOROPHYLL VO DP EP thermo-I catalyti c ;; "' ""' oxidative gases I TBP \ 'coke' t :o o=c HO OH 'HEME-TYPE' VO ETIO-lll

The above dilemma is the greatest single unsolved mystery of tetrapyrrole geochemistry and potentially spans the entire evolution ary-maturational continuum. Purposes and Goals of These Studies 51 The ultimate goal of these studies is to attain an overall understanding of tetrapyrrole geochemistry and the various ways that natural processes (g.g. organic source, paleoenvironment, heat flow, oxygen tension, tectonics, etc.) control and modify the underlying reaction mechanisms and rates. This grandiose plan, given the complexities revealed earlier in text, is certainly not attainable in a few years. More realistically, two major tasks aimed specifically at fulfillment of the ultimate goals were undertaken. Essentially, these tasks are: (1) The development or refinement of analytical techniques employed in the analyses of geopigments during all stages of their evolution, and; (2) The application of these methods to the study of tetrapyrrole geochemistry in a sufficiently wide array of samples and sample suites to attain general yet reasonable conclusions. Except for "Materials and Methods" and "Conclusions", the text is divided into two sections; (1) "Analytical Experimentation and Results," and (2) "Tetrapyrrole Geochemistry." Each of these 'Results' sections is appropriately subdivided into individual specific topics. By melding 'methodology' and 'application' it is hoped to develop a baseline study from which standardization of technique and an overall understanding of the multifaceted phenomena involved in tetrapyrrole geochemistry, respectively, will ultimately emerge.


CHAPTER 2 MATERIALS AND METHODS This deals only with analytical techniques and materials which the author considers truely 'routine' and/or 'accepted.' General Laboratory Procedures 52 Pigments, by definition, abso rb light and must be protected from harmful irradiation. However, it is often not just the presence of light which can lead to photo-induced alteration of pigments in vitro but, rather, the concerted effects of light, heat and oxygen. Thus, every effort was made to minimize the exposure of tetrapyrrole pig ments, whether synthetic standards (see Appendix A) or geochemical extracts/ isolates, to any two of these agents at the same time Samples, extracts and isolates were maintained in the dark, frozen and under N2 except during actual procedural manipulation. During analytical handling, most notably chromatography, exposure to light was minimized in accord with the dictates of the Roscoe-Bunsen reciprocity law. Metalloporphyrins, the most stable pigments handled herein, were the only compounds analyzed under more than brief exposure to 'white' light. Free-base porphyrins and all extracts or containing dihydroporphyrins (chlorophylls, phorbides, chlorins, etc.) were handled under dim yellow (l-550-585 nm) light. Because 'pure' yellow lamps were not available, market variety 'bug-lights' were substituted. Testing by prismatic diffraction revealed that 'bug-lights' emit almost


53 no 'reds' (l > ca. 650 nm) or 'blues' (l < ca. 500 nm), the wavelengths of concern. Standards The present study relies heavily upon physicochemical comparisons of geologic pigments with authentic tetrapyrroles. One hundred and nineteen compounds were either prepared {82 compounds) or obtained from alternate sources (gifts, purchases) and purified. Descript io n of these procedures is included i n Appendix A. Solvents All solvents, including 'house-distilled' water, were fresh l y redistilled in all-glass apparatus. Ethers (ethyl ether! tetrahydrofuran) were depero xided by stand ing over grade 'super-I' (0% H20) basic alumina (Merck #1076) and filtered through a pad of the same just prior to use Testing fo r peroxides using the ferrous thiocyanate method (sensitivity -0 .001 % as peroxide) revealed that the basic alumina technique for 'de -p ero xi da t ion' was valid. Towards the end of this project it was decided that, if ethers were purchased in glass containers, only treatment with basic alumina was required. Thus, distillation of ethers was dropped, as no artifact formation could be shown if only freshly treated ethers were used. This method does, however, retain the peroxide inhibitor BHT (butylated hydro x y toluene = 2,6-di-tert-butyl-p-cresol) with i n the product ether. The presence of BHT (bp = 265C, mw = 205 daltons) in the concentrations ( 0.025 % wt.) used by industry posed no problems and might be considered advantageous because it slows pero xide formation.


54 Extractions Since a variety of sample types and sizes had to be dealt with, extraction methodologies had to fulfill one of two criteria. First, in most cases ('routine' analyses), extraction of the vast majority (g.g. 95-98%) of the bitumen in a non-injurious manner was desired. Second, in those special cases where post-extraction treatment of sediment was aimed at releasing bound materials ('new' bitumen: hydrous pyrolysis), exhaustive (ca. 100%) extraction was required. To fill these needs, (1) N2-flushed ball-milling ('large' samples, >10 g) or grinding in an all-glass chilled 'hand-homogenizer' ('small' samples, <10 g) and (2) conventional soxhlet extraction of pre-extracted material were employed, respectively. Ball-Mill Extractions Ball-mill extractions were performed by placing sub-divided samples (frozen sediment, peat, tar-sand etc.) into an appropriately sized ground-glass closured reagent bottle (g.g. sample/bottle val.: 10-50 g/500 ml, 50-125 g/1 125-300 g/2 ), adding several (5-9, by size of vessel) cylindrical corundum grinding pieces and a volume of extractant equal to 125-150% of the sample weight (v/w). Once charged, the ball-mill bottle was flushed with N2 stoppered, taped shut and rotated on a commercial tumbler in the dark at room temperature for periods of 15 minutes to 2 hours per extraction. The longer periods were used during the first several extractions in order to allow better dissemination of the sample. At the end of an extraction period the solvent/bitumen was retrieved by filtration and the sediment with fresh solvent added back to the bottle for subsequent extraction as above.


55 Extracts were evaporated in vacuo ('rotary-evaporator') at temperatures below 40-45C and sequentially pooled in like manner. Extraction was continued until further extracts were colorless and void of fluoresence when viewed under long-wave (366 nm) ultra-violet light. Solvents employed during ball-mill extractions were, in order of usage values indicate number of e xtracts; N = until completed) : Ace/MeOH, 1 : 1 (3); Ace/MeOH, 9:1 (3); and Ace/Benz, 1:1 (N). The first solvent mixture was included in order to assure complete dehydration Acetone-benzene mixture was used as the final solvent for complete extraction stems since inclusion of benzene was found to release additional tetrapyrrole pigment from sediments (Louda and Baker, 1981**), relative to acetone-methanol alone (cf. Baker and Louda, 1980a; Louda et gj., 1980). In retrospect, the additional e xtraction 'power' of benzene may indi cate an intercalation of layered structures, such as asphaltene sheets or silica m i nerals Hand-Homogenizer E xtractions Small samples ( < 1-10 g) were e xtracted in an all-glass hand homogenizer. The solvent regime given above was used e x cept where chlorophylls might be encountered Such samples included v i able algal cultures, sediment trap samples (Per u upwelling system) and sediments beneath 'short' water columns (Mangrove Lake, Pond Apple peat and Big Soda Lake). In these cases, MgC03-saturated acetone was used in place of pure acetone (cf. Jeffrey, 1974, 1980; Yentsch, 1965). However, the *Ace= acetone, MeOH =methanol, Benz = benzene, all proportions in v/v measure. **Abscissa of figure 2 in Louda and Baker, 1981 should have the units of not mg.


56 usage of basic substances ( MgC03 ) in order to counteract the 'pheophytinization' (-Mg) of chlorophylls by cellular-acids (Holden, 1976; Jeffrey, 1974, 1980) has come under question (Holden, 1976; Svec, 1978). Soxhlet Extraction Soxhlet extraction with a benzene:methanol azeotrope (60.5:39.5, v/v, b.p. = 58.3C) is often cited for 'exhaustive' e xtraction of bitumen (lead references in et gl., 1983; Johns, 1986; Leythaeuser and Rullkotter, 1986; Schenck et gl., 1984). The use of Soxhlet extraction for the 'routine' analysis of tetrapyrrole pigments was avoided since the process involves heat and air. However, Soxhlet technique was used as a 're-extraction' method pr i o r to hydrous pyro lysis experiments (cf. Lewan et gl., 1979; Schiefe lbein 1983; Winters et gl., 1983). Separation Techniques Two main methods were used to separate pigments or classes of pigments: (1) chromatography over/through various media and (2) liquid:liquid partition between two immiscible solvents. Refinements are presented, but the basic techniques are standard. Chromatographic Techniques hromatography (CC) is the 'classic' method used on plant pigments by Tswett (1906a-c) and Strain (1958). Herein, 'CC' refers to the separation of mixtures over an adsorbent bed (silica gel, alumina, cellulose) during which the solvent flows by gravity assist alone.


57 Adsorbants employed during these studies include; sugar (4X and lOX confectioners, Dixie Crystals), microcrystalline cellulose (Merck #2331), silica-gel 60* (Merck 40-63 pro, 230-400 mesh), and neutral alumina** (alt. aluminum oxide: Merck #1077; 63-200 pro, 70-230 mesh). In the case of alumina, the activity employed was grade 'Super-!' (0% H20) adjusted to Brockman grade II-III with the addition of 3.5% water, by weight. Other adsorbants were used as received. Columns were packed either with an adsorbant slurry (cellulose, silica. sugar) or by gravity settling (alumina) through a column of solvent. Bed dimensions used herein were typically of the following dimensions; 4.0x35-40 em (large). 2.5x35 em ('medium'), l.Ox17.5 em ('smal1') and 4.5x50 mm ('micro':Pasteur pipet). In all cases a ratio of 10-20:1 {h/w) was desired and utilized. Given the extreme heterogeneity of the majority of the geochemical extracts analyzed. it was deemed important to assure the total transfer of all material to the chromatographic bed being used. TransferH* of extracts or fractions to a chromatographic bed was performed by dissolution of the material in a minimal amount of a strong solvent (THF, benzene. CH2Cl2 ) and tr&nsfer into a solvent 'head' of such low polarity (n-pentane, P.E.) that precipitation of the more polar components occurred. Subsequent elution involved the low polarity flushing (3-5 bed volumes) of the column followed by the stepwise *Purchased and used only from polyethylene lined containers to avoid 'plastics' or 'mold-release' contaminations. **Purchased and used only from all-glass containers. H*Excepting those cases of 'analytical' chromatographies in which the 'injected or 'loaded was determined by difference (original minus remainder equals amount analyzed).


gradient (re solvent polarity) development to affect fur th er separations. 58 Typical solvent-systems employed during column chromatographies included: increasing percentages of acetone {ACE) i n petroleum ether (PE, 30-60C), for various dihydroporphyrin classes with cellulose and metalloporphyrins with alumina; tetrahydrofuran {THF) for GPC over/ through Sephadex LH-20; n-pentane (nC5)/PE (1:1, v/v) fo r Cu/Ni-highly dealkylated etioporphyr ins {HOEs: Baker and Louda, 1982, 1984, 1986b; Louda and Baker, 1981, 1986) during LPHPLC over methanol-deactivated silica (Baker and Louda, 1982, 1984; cf. Purcell, 1958); and others given 6more specifically within the text. Thin-layer chromatography (TLC) was employed primarily to test purity of both analytical isolates ('geopigments') and in vitro synthetic standards {Appendix A). TLC media in cluded; 100 pm silica gel on plastic (Eastman #13179), 100 pm cellulose on plastic (Eastman #13255), 200 pm neutral alumina on plastic (Eastman #55B1) and 250 pm silica-gel on glass (E. Merck #5763). Typic al solvent regimes ('developers') are given by pigment classes and TLC media below. Extremely non-polar pigments, such as carotenes (tetraterpenoid hydrocarbons), pheophytins and the methyl esters of dihydroporphyrins (pheophorbides, chlorins, purpur ins etc.) were checked for purit y u sing cellulose TLC developed with 0.5-5.0 % ACE in PE, depending upon intrinsic pigment polarity, so as to yield an Rf of ca. 0.5-0 .7. Dihydroporphyrin acids (g.g. pheophorbide-a [VIa], purpurin-18 [XXVIa]) or tri-acids (g.g. chlorin-e6 [XXIIIb], chlorin-p6 [XXVIIa]), and s imilar geopigments, were also checked with cellulose TLC except development was w i th 10-15% or 25-35% ACE in PE, respectively. Certain dihydroporphyrin acid


59 methyl esters (pyro-pheophorbide-a ME [VIII], DOMPP-a ME [XII], pheophorbide-b ME [XVII] etc.), free-base porphyrin aci d methyl ester s (phylloerythrin-ME [XXXII], deoxophylloerythrin-ME [XXXVIIb] etc.) and free-base 'alkyl' porphyrins, primarily as synthetic standards (DPEP [XXXVIII], 7-PDE-DPEP [XXXIX], OEP [L], deuteroetioporphyrin [LVII] etc.), were checked using TLC over silica using developing solvent systems such as 5-10 % ACE in PE, CH2Cl2 or benzene/cyclohe xane (1:1, vjv) Alumina TLC was utilized mainly during the development of appropriate solvent-systems for transfer to CC methods to puri fy Ni(II)-and V(IV)O-alkyl porphyrins. In these cases, 3.5 -5.0% and 12.5-15 .0% .ACE in PE, respectively, were effective Another e x ception was found with the 'standard' practices of analytical chemistry when applied to pigments. TLC loading and development is routinely performed in air While it is possible to flush a TLC chamber with an inert gas (N2 Ar), the actual loading of the plate is usually still in air. Thus, it was necessary to protect pigments from the adverse effects of oxygen. Towards this end, an inert-gas (N2 ) TLC-loading chamber was designed and fabricated (Figure 15). Gel-permeation chromatography (GPC) was utilized herein essentially as described previously by Baker (1970), with a few exceptions. GPC conditions involved the dissolution of the sample in a minimal volume of THF and developed with the same solvent over Sephadex LH-20 (Pharmacia). GPC was not performed on crude e xtracts but on fractions determined to contain only metalloporphyrins or unidentifiable species (g.g. 'chlorin-660 complex': cf. Baker and Louda, 1982). Thus, the more labile free-base dihydroporphyrins and porphyrins were never


6mm plastic tubing TOP BASE \ \ 0 0\._ -2mm gas vent f\ holes spotting access holes \ J \ 0 o \ o \ -------2 2 3 c m __ ____:..,.... \ retaining cot ferdam 6mmx6mm (acrylic) TLC plate supports Figure 15. Diagrammatic sketch of an inert gas chamber for loading of oxygen sensitive pigments oqto thin-layer chromatographic (TLC) plates. 60


exposed to even a trace of pero xide s (THF). Further, yields of monomeric metallo (Ni, VO) porphyrins from oil-shales and petroleum crudes were found to increase if GPC was employed after a complete separation of Ni-from VO-porphyrins. Liguid:Liguid Separations 61 The electronic absorption spectra of free-base porphyrins typically exhibit band I (i.g. lowest energy) absorption between 615-630 nm (see Appendix A and Results I: Analytical Experimentat ion ; cf. Fischer and Orth, 1937; Fischer and Stern, 1940; Fuhrhop and Smith, 1975; Treibs, 1973). Fortuitously, this absorption falls between the band I (650-680 nm) and band II (600-610 nm) absorption of dihydroporphyrins, as well as being bathochromic to the a-band maximum of metallo-geopor phyrins (Ibid.). Thus, the electronic spectra of crude (total) extracts offer reliable clues (absorption maxima or inflections at ca. 622 8 nm) as to the presence of even minor 5 % ) amounts of freebase porphyrins. Figures 16a-b provide e xamples of the spectra of crude e xtracts from which free-base porphyrins were iso l ated via HClextraction. In these cases, free-base porphyr in s were found to b e present with dihydroporphyrins or metalloporphyrins, respectively. Subsequent isolation of the free-base porphyrins, with further purification (cf. Baker and Louda, 1982, 1986a-b; Louda and Baker, 1981), yield free-base geoporphyrin isolates. Willstater and Stoll (1913) developed aqueous HCl e xtrac tion of free-base tetrapyrroles out of ethyl ether solution. They defined the "HCl-number" as "the weight percent of hydrochloric acid" (aqueous) "which will e xtract two-thirds of a chlorophyll derivative from an equal volume of ether" (ethyl: cf.


w u z CD 0: 0 1.0 a) 0.8 0 6 0.4 0.2 CHL-I (:660-670) I b) 350 450 550 650 750 350 Fi? /Ni-5(=395 -400) 450 Ni-

63 Seely, 1966; Smith, 1975). Going on the basis that concentrated hydrochloric acid (aq.) is 37.5% wt. HCl, or a nominal 12M (Sienko and Plane, 1966), the weight percent of more dilute HCl solutions can easily be calculated. Table 3 contains selected weight percents of aqueous HCl and the amounts of concentrated HCl which, when made up to 100 ml (0.1 with distilled water, will yield the desired concentration. Typically, extracts of very recently deposited sediments, especially from beneath productive waters, contain not only the tetrapyrrole pigments (chlorophyll derivatives) but also a wide variety of carotenoids (cf. Baker and Louda, 1982; Fox, 1944; Fox et 1944; Louda and Baker, 1986; Repetta and Gagosian, 1982; Tibbets et . 1978; Watts et gj., 1977). During the separation and purification of tetrapyrrole pigments, the hydrocarbon carotenoid pigments (carotenes) are relatively easy to remove, as they are less polar than most of the dihydroporphyrins with which they co-occur (Baker and Louda, 1982; Louda and Baker, 1986: cf. Holden, 1976; Liaaen-Jensen, 1971). However, the majority of the oxygen-containing carotenoids ('xanthophylls'), especially the carotenols, can interfere greatly with the purification of functionalized dihydroporphyrins, such as early diagenetic chlorophyll derivatives (Baker and Louda, 1982; Louda and Baker, 1986). Thus, following a preliminary mild chromatographic fractionation (microcrystalline cellulose: see Chapter 3) the 'non polar pigments' (non-carboxylic acids: g.g. pheophytins, decarbox ylated tetrapyrroles, carotenoids) were dissolved in an organic solvent (n-hexane, ethyl ether or petroleum ether) and the carotenols removed by successive liquid:liquid extraction with 90% (aq ) methanol.


Table 3. Preparation of various weight percentages of aqueous hydrochloric acid for usage in the determination of the "HCl-number"(1l) of tetrapyrrole pigments(). mL HCl(con) mL HCl(con) % wt. HCl lOOmL Volt % wt. HCl lOOmL Volt 1.0 2.7 12.5 33.5 2.0 5.3 15.0 38.8 3.0 8.0 17.5 46.6 3.5 9.3 20.0 53.2 5.0 13. 3 22.5 59.8 7.5 20.0 25. 0 66.5 10.0 26. 6 30.0 79.8 FoarNOTES: 1l The "HCl" or ''Willstater" number is the weight percent aqueous hydrochloric acid which will extract two-thirds of a porphyrin from an equal volume of ethyl ether solution. (See Smith,1975;Willstater and Stoll,1913). Calculated using 37.5%wt. as the concentration of 'concentrated'HCl[HCl(con)].Volt= total volume,made up with deionized water. 64


65 The above extraction technique to remove carotenols and, to a lesser extent, carotenones from less polar pigments (carotenes, pheophytins) is derived from a common carotenoid technique referred to as 'hexane:aqueous methanol partition' (Davies, 1976; Krinsky, 1963; Foppen, 1971; Louda, 1978; Petracek and Zechmeister, 1956). Non-polar pigments remain in the organic epiphase while the more polar oxygenated carotenoids, most notably the carotenols, are e x tracted (partition) into the aqueous methanolic hypophase. The separation of carotenoids from chlorophylls of common spinach (Spinacea oleracea) provides an example of the separative 'power' of organic :aqueous methanol partition, as applied to preliminary tetrapyrrole* purification. This example also directly applies to techniques used to isolate certain standards (chlorophyll a [I] chlorophyll -b [XIV]; bacteriopheophytin-a [XXI]: Appendix A). The total extract of spinach yields an electronic absorpt ion spectrum (Figure 17a) in which an abundance of carotenoids can be seen in the characteristic absorp-tion in the blue to blue-green region (g.g. 425-525 nm). Successive extractions of the total mixture in petroleum ether with 90% aqueous methanol effectively removed much of this carotenoid (viz. carotenol: Fig. 17b) interference and left a tetrapyrrole concentrate in the organic epiphase (Fig. 17c). The carotenoids primarily removed were neoxanthin, lutein and viola x anthin (cf. Goodwin, 1965; Strain, 1958), as these are the most abundant o x ygen-containing carotenoids in *The reverse also holds. That is, this partition technique is of great importance in the removal of tetrapyrroles during carotenoid analyses in which alkali-labile pigments (tetraterpenoid) are present and saponification tech nique must be avoided (cf. Davies, 1976; Liaaen Jensen, 1971).


Figure 17. oleracea), (aqueous), 1.0 Ia} I I I I 1.0 I b) I I I I 1.0 lc) 429 A I 0 .8-l 0 8 I 430 I I I A 660 0.6 0 6 w w I r1 660 u 438 u z z

67 s. oleracea. Similar results were obtained during the purification of bacterial tetrapyrrole pigments {bacteriopheophytin-a [XXI]: Appendix A, Fig. A9) and immature sediments. The minor absorption at ca. 660-665 nm found in the methanolic extracts {Fig. 17b) probably does not represent 'true' partition of the chlorophylls (-a, -b) but, reflects a correspondingly minor entrapment (emulsification, 'lake,' or inadequate phase separation) of the epiphase in the hypophase. Analytical Derivatizations During the course of these studies tetrapyrrole pigments were converted to certain selected in vitro derivatives, including the methyl and dihydrophytyl esters of carboxylic acids. Methyl Esters The most common derivatization required during the analyses of geologic and water column detritus-borne tetrapyrrole pigments is that of forming the methyl esters of carboxylic acid pigments. Methyl esterification is needed to alter the polarity (i.g. 'protect' or 'weaken') of the free-acid and to allow mass spectral analyses. Essentially, three methods for the preparation of tetrapyrrole acid methyl esters {ME} were tested or utilized during the present studies. These include mineral acid/alcohol, diazomethane and Lewis acid (BF3)/alcohol. A quite common method for the preparation of carboxylic acid methyl esters is the treatment of the free acid with an a l coholic (methanol, CH30H} solution of a mineral acid ( H2S04 HCl} in which the acid is present at about 3-7% by weight Although this method ts


68 simple, it is essentially a reversible (equilibrated) reaction, though mass action related excesses of the acidic alcohol will favor the ester (product) side (cf. Morrison and Boyd, 1966). Thus, in these studies (Appendix A: g.g. pheophorbide-a ME[Vlb] etc.), the mineral acidalcohol technique was applied only to semi-synthetic standards, as has become relatively routine in tetrapyrrole chemistry (Fischer and Orth, 1937; Fischer and Stern, 1940; Fuhrhop and Smith, 1975). The application of the 'mineral-acid/alcohol' method of esterifying tetrapyrrole acids went as follows: To dried (evaporated from solution) tetrapyrrole carboxylic acid, a 100-1,000 fold excess (10-100 ml/100 mg: vjw) of 5% H2S04 in methanol (w/v) was added under a nitrogen blanket. The solution was stoppered, still under N2 and kept at ca. 4C in the dark 'overnight' (10-18 h.). The reaction was stopped and the product (ME) isolated by dilution with copious amounts (-100X, v/v) of water, followed by extraction of the resultant aqueous solution with ethyl ether or dichloromethane. The resultant organic solution of tetrapyrrole ME was washed once with dilute* aqueous sodium bicarbonate, in order to neutralize any remaining acid, and 3-5 times with water, to remove any generated salts (Na2S04 etc.). Evaporation of the organic solution and subsequent chromatography over microcrystalline cellulose (see Chapter 3) was usually all that was required to obtain a pure, as shown by TLC and/or LPHPLC assay, product of the desired tetrapyrrole acid ME standard (see Appendix A). Diazomethane (CH2N2), an effective source of the C1 carbene 'methylene', is a very efficient and commonly used reagent for the *"Dilute," in this case, refers to a solution made of 1 volume saturated NaHC03 (aq.) plus 9 volumes of water.


methyl esterification of carboxylic acids. In general, CH2N2 is generated by the alkali hydrolysis of suitable precursors such as; nitrosomethylurea, Q-toluenesulfonylmethyl-nitrosamide ("Diazald"TI1, Aldrich Chern. Co.) or N-methyl-N'nitro-N-nitroso-guanadine. 69 During earlier studies on DSDP core-samples, (Baker et (1976, 1977, 1978a) employed CH2N2 for the routine methyl esterification of tetrapyrrole acids. The laboratory method for this technique (S. E. Palmer, pers. commun. 1978) involved overlayering aqueous alkali (2.0 ml of 50% KOH) with ethereal pigment (-5.0 ml), in an ice-bath, and stirring-in the reagent (N-methyl-N'-nitro-N-nitroso-guanadine, ca. 0.1 g). The reaction was stopped by titration to neutrality of the aqueous layer with 5% (w/v) aqueous HCl. As Louda et (1980) reported, this technique was found to generate artifacts, most notably purpurin-18 ME [XXVIb]. Subsequent testing revealed these artifacts could be avoided if the CH2N2 is generated, trapped in pure ethe r washed with water (3X) and then added to the pigment (cf. Fuhrhop and Smith, 1975). The electronic spectra of pheophorbide-a free acid [VIa] and the products obtained by the overlayering technique described above are presented in Figure 18. TLC tests of the mix tu re obtained (Louda, this study : cf. Louda et 1980) revea l that a large proportion of the original pheophorbide-a [VIa] had been converted to purpurin-18 ME [XXVIb] and other chlorins (g.g. [XXIV], [XXVII]: see Appendix A). The generation of compounds such as purpurin -18 ME [XXVIb] may explain, partially, the presence of absorption maxima at ca. 690-695 nm in some of the earlier reports on DSDP samples (cf. 1978a). However, following the report of Louda et 1980, the presence of


1.0 -.----,-----.-------r-----. w u z <( rn 0:: 0 (./) CD 0.4 <( 0 2 405 -409. 5 (\ I I / I ; I ( I \ \ 666.5 350 450 550 650 "750 WAVELENGT H(n .m) 70 Figure 18. Electronic absorption spectra of the reaction products (solid) formed by the treatment of authentic pheophorbide-a free acid (dashed) with diazomethane in the presence of alkali. Absorption at 696nm is indicative of purpurin-18[XXVI].


71 purpurin-18 [XXVIa] as a diagenetic product has been re-affirmed (Baker and Louda, 1982; Louda and Baker, 1981, 1986). Besides the potential of artifact formation, through the presence of even trace alkali with the CH2N2 method, the use of diazomethane with tetrapyrrole pigments was avoided for two other reasons. First, even with highly purified* ethereal diazomethane, molar yields of methyl ester products were rarely found to e xceed 85%. This finding is surprising and, as of yet, unexplained since the pigment never left the vessel in which it was reacted That is, transfer errors are ruled out. Second, CH2N2 is known to react with certain tetrapyrroles to yield nitrogenous adducts. One such example is the addition of CH2N2 to exocyclic double bonds, such as shown in Figure 19, to yield products by an Azzarello-type pyrazoline ring forming reaction (Fuhrhop, 1978). This latter point takes on added significance with the recent identification of a geoporphyrin containing an e x ocyclic double bond (Chicarelli and Maxwell, 1986: Table 2; Fig. 13h). The Lewis acid, boron trifluoride (BF3)-methanol (cf. Hickinbottom, 1957; March, 1968) was found to be the most efficient and artifact-free method of converting geotetrapyrrole acids to their corresponding methyl esters. The technique is e x ceedingly simple and, thus, elegant. The technique involved only the addition of 100-1,000 fold excess of BF3-MeOH (12% BF3 in MeOH: Kodak #3706) to the dried pigment or fraction in a small Erlenmeyer flask (10-25 ml) or test tube with a blanket of dry N2(g), closure under N2 and allowing the reaction to proceed overnight (10-16 hrs) in the dark at refrigerator *That is, washed and tested free of residual alkali.

PAGE 100

CH2N2 ..... -N-N COOCH3 Figure 19. The addition o( diazomethane (CH2 N 2 ) to porphyrin exocyclic double bonds. Example given is mesoverdin-IX ME[XLIII](after Fuhrhop,1978). -......! N

PAGE 101

temperatures (ca. 4-6C). The reaction was stopped b y evaporation of the solution with a stream of N2 in a water bath. Yields were ess e n tially quantitative (%). Boron trifluoride was also employed as the etherate (53% BF3 in ethyl ether, w/v: Kodak #4272) for the formation of dihydrophytyl esters (cf. Marshall et gl., 1970) as detailed in Appendix A [lib]. Metals and Metalloporphyrin Formation 73 The nickel (II) and copper (II) chelates given in Appendix A were formed during the present study, with two exceptions. That is, Ni DPEP ([XC]; Baker et gl., 1968) and Abelsonite ([XCII]; Storm et gl., 1984) were available as the Ni(II) chelates. Three isotopic forms of Ni and two isotopic forms of Cu were used, as detailed below. Naturally occurring nickel consists of five isotopes (5 8N i [ 67.8%], [ 26.2%], MNi [1. 2 %], [3.5%], [1.2 % ]) of which two (58Ni, are overwhelmingly dominant (Heath, 1966). As will b e detailed during a subsequent section on the mass spectrometry of tetrapyrrole pigments, the relationship of 58Ni and = 0.3864 x 58Ni) is of vital importance in the valid estimation of nic kel porphyrin and dihydroporphyrin series. In order to synthesize the authentic nickel chelates required for use as standards, natural isotopic abundance 'NinatL, and 2 monoisotopic (58Ni, forms were obtained. 'Natural' n ickel was 'offthe-shelf' reagent grade nickel (II) sulfate (NiS04 ) and nic k el (II) acetate [Ni(OOCH3 ) 2 ] Mono-isotopic nickel species were purchased from the Union Carbide Corporation Oak Ridge, Tennessee, U.S.A. and were received as the ground state metals. 58Ni was certified 99.927 % pure

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and contained only 0.065% as was certified 99.62% pure and was 0.34% as 58Ni. To form the required divalent nickel cation {Ni*, nickelous) it was necessary to oxidize the ground state isotopes (58Ni0 and form an easily dissociated salt. A hot nitric/ sulfuric acid mixture was used, with nickelous sulfate as the desired end product. 74 The reaction of 58Ni(s) and to the nickelous cation, as the sulfate, was performed as follows: To 50 mg nickel metal (58Ni0 or in a 10 ml round-bottom boiling flash was added 1 ml concentrated (93% wt) H2S04 and 3 ml concentrated HN03 A short reflux column was afixed anp the mixture heated 80-90C, with stirring, in an oil bath for 2 hours, at which time the nitric acid was driven off as the negative azeotrope HN03/H20 {br -120-125C) over a Bunsen burner. After cooling, 1.5 g NaOH (37.52 m equiv.)* was added to neutralize the residual sulfuric acid. Final pH adjustment to 7.0 was achieved by back titration with 0.1 M H2S04 (aq.) and the solution taken to dryness over a gas flame. The final, albiet crude, preparation was a pale green crystalline solid and, as such, most likely consisted not only of NiS04 but Ni{OH)2 and NaS04 as well. The probable impurity of the final preparations was of little consequence as the desired oxidation of both mono-isotopic species (58Ni, and the formation of a readily dissociated nickelous salt (NiS04 ) was affected. Mass spectrometric analyses of the corresponding nickel (II) using only the first dissociation 9f H4S04 H+ + HS04-Ksp = 1.0x103 ) and ignoring the second {HS04 H' + S04 'Ksp = 1.3x1u-2 [Sienko and Plane, 1966]).

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75 chelates of several tetrapyrrole pigments reinforces the success of the above reactions (g.g. 58Ni OEP, M = 590 m/z: M = 592 m/z). Natural copper exists as 2 isotopic species, (69.09%) and 65Cu (30.91%: 65Cu = 0.447x63Cu). Two forms of copper were used during the present studies, these being of natural isotopic abundance ('Cunatl,) and a mono-isotopic species Natural isotopic abundance was 'off-the-shelf' reagent grade copper (II) sulfate (CuS04). Monoisotopic copper-63 (63Cu) was purchased from the Union Carbide Corporation, Oak Ridge, Tennessee, U.S.A. and was supplied as cupric oxide (63CuO:tenorite). Even though the oxidation state of was correct (63cu2 cupric) as received, the insolubility of its form (CuO) forced alteration, using a similar reaction to that given for the nickel metals. To 455.2 mg 63Cu0 in a 25 ml round-bottom flask was added 2 ml concentrated H2S04 and 6 ml concentrated HN03 and the reaction ran as given above for nickel. Following removal of HN03 and titration of the solution to pH 7.0, water was added to yield a mediumdeep blue aqueous solution of present as hydrated cupric ion (Cu++ + H20 CuoH + H), of about 72 mM concentration (100 ml). Because tetrapyrrole pigments readily chelate cupric ions (Erdman et gj., 1957; Louda, unpublished; Zelmer and Man, 1983) it was not necessary to continue this preparation to a stage yielding crystalline salts. The formation of the Ni(II) and Cu(II) tetrapyrroles required as standards (Appendix A) essentially followed the techniques given in the literature (Buchler, 1978; Fischer and Orth, 1937; Fischer and Stern, 1940; Fuhrhop and Smith, 1975), with minor exceptions. That is, a

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76 constant blanket of N2 was used and the chelation of Cu(II) was at room temperature. The ana lytical metallation of geopigments with 63Cu was performed by the addition of a few drops of the aqueous 63Cu(II) solution, described above, to the pigment i n ethyl ether. The conditions of this reaction were such that some salts did precipitate. However, the chelation of Cu(II) by tetrapyrrole pigments was found to be so facile that 100 5 % yields were obtained

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CHAPTER 3 RESULTS AND DISCUSSION I: ANALYTICAL EXPERIMENTATION 77 Pri or to investigating the geochemistry of tetrapyrrole pigments, valid and reproducible methods for their analysis must be attained. Given the heterogeneity of geolog i c tetrapyrrole arrays, especially during 'early diagenesis' (Louda and Baker, 1986), this has proven to be a formidable task. During the development of analytical schemes w i th which to better iso late, t ype and identify geologi c tetr apyrro l e p igments seve r a l goals emerged. These included: (1) the determination of the validity of tetrapyrrole quant itation methods; (2) the development of separation schemes which would allow the quantitative splitting of tetrapyrrole arrays into s i milair functiona l c lasses (alky l mono-carboxylic acid di-and tricarbo x y lic a cid); (3) the establishment of an electronic absorption spectral (UV/VIS) data base for tetrapyrroles with known or suspected c ounterparts in the geo sphere ; (4) the appli c ation of 'soft' derivatization techniques (BH4 63Cu chelation) to geologi c isolates for chromophore identification; (5) the e x tension and refinement of mass spectral analyses in order to yield reproducible data; and (6) .to provide a basis for a standard method ([. Louda and Baker, 1987) in the reporting of geoporphyr i n results.

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78 Since several of the above goals overlap, the present chaper is divided into the following sections: Initial Assay of Bitumen; Separa tion and Isolation Studies; Electronic Absorption Spectroscopy and Chromophore Identification; and Mass Spectral Analyses. Initial Assay of Bitumen In order to select the most appropriate analytical route to follow, one must first ascertain both the quality and approximate quantity of tetrapyrrole pigments in an extract (EOM, bitumen) or liquid (tar, petroleum) sample. The examples of tetrapyrrole mixtures and conclusions given here are generalized from the results of the following chapter 4. The first clues as to the presence or absence of tetrapyrrole pigments in an extract or liquid sample evolves from the qualitative analysis of the electronic absorption spectrum (UV/VIS) of these materials. Further, if performed in a quantitative manner (i.g. known volume and/or dilution), the initial UV/VIS spectrum can also yield an, albiet crude, estimate of pigment abundance. Thus, this initial spectral assay is useful both in the choice of separation methodology (pigment types) and the scale (pigment amount) of the operations to follow. Figure 20 contains electronic absorption spectra chosen to represent tetrapyrrole pigment arrays characteristic of essentially all stages of geochemical evolution, as presently defined (cf. Baker and. Louda, 1983, 1986a). Only brief discussions of subsequent analytical methods are given, as these are detailed in the next section.

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1.0--.-0 8 0 6 0.4 w 02 u z <( &? 0 0 ,1-.-1 --,1,--.-1 -rl ::::1::::._1 0 1 .01,-----<(
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Water Column Detritus and Very Recent Sediments 80 Extracts of algae, water column detritus ('sediment trap' samples) and recently deposited ('surface') sediments typically yield UV/VIS spectra as given in Figure 20a. Characteristic absorption maxima and 'hidden' maxima, namely inflections, present in these t ypes of spectra include: peaks at ca. 411, 535, 610 and 667 nm; and 450, 475 and 500 nm. These represent 'early' chlorophyll derivatives pheophytin-a [IIa], pheophorbide a [VIa]) and carotenoids, respectively. In cases where chlor ophyll-a [I] is dominate, the major tetrapyrrole absorption is found at 429 and 663 nm. It is known that algae (Fenical, 1982; Goodwin, 1976; Liaeen Jensen, 1978; Moore, 1981), primary consumers such as crustaceans (Fox, 1976; Jeffrey, 1980; Louda, 1978; Thommen, 1971), water column detritus (Louda and Baker, 1986; Repetta and Gagosian, 1982) and recent sediments (Baker and Louda, 1982; Fox 1944, 1953; Fox et gl., 1944; Louda and Baker, 1986; Repetta and Gagosian, 1982; Tibbets et gl., 1978; Watts et gl., 1977) contain an abundance of oxygen containing caroten oids ('x anthophylls') over the less polar carotenes. Thus, extracts yielding UV/VIS spectra such as Figure 20a, are subjected first to partition between an organic solvent (ethyl ether, petroleum ether, nhex ane) and 90% (aq.) methanol in order to remove inferring xanthophylls'. Tetrapyrroles and the remaining carotenoids are then subjected to chromatographic fractionation over microcrystall ine cellulose, as detailed later.

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Oxic Surface and Deeper Anoxic Sediments 81 Figure 20b is the UV/VIS spectrum typical of the bitumen from slowly deposited oxic sediments as well as from more deeply buried sediments. The main features noted in cases such as given in Figure 20b are characteristic dihydroporphyrin (phorbides, chlorins) absorption at about 400-410 and 660-666 nm. The lack of carotenoid absorption (420-500 nm) in this type of EOM directs one to separate the pigments over cellulose without a prior organic-aqueous methanol phase extraction as given above. Maturing (Mid-diagenetic) Sediments According to the current definitions of the stages of geopigment evolution (Baker and Louda, 1983, 1986a; Louda and Baker, 1986), the defunctionalization of dihydroporphyrins (phorbides, chlorins) occurs during 'early-diagenesis.' Continued thermal stress next elicits the aromatization of these pigments to yield free-base porphyrins during 'mid-diagenesis.' The presence of free-base porphyrins in the bitumen is easily noted from examination of the UV/VIS spectrum of crude extracts. The characteristic band I (lowest energy) absorption of alkyl free-base porphyrins occurs at about 620 nm (see compounds [XXXVII]-[XL], [XLVIII]-[LII]; Appendix A). The position of this band is fortuitous since it lies between bands I and II of the dihydroporphyrins and is to lower energy (bathochromic) than the a-bands of the main geologic metalloporphyrins (NiPHs, a-550 nm; VO-PHs, a= 571 nm).

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82 Sample electronic spectra of crude extracts from which free-base porphyrins were isolated are given as Figures 20c-20e. In these cases, free-base porphyrins were found to occur with dihydroporphyrins (earlyto mid-diagenesis: Fig. 20c}, essentially alone (mid-diagenesis: Fig. 20d}, or with nickel porphyrins (mid-to late-diagenesis: Fig. 20e}. In all cases where free-base porphyrins were implicated the method of choice for their separation from other pigments was extraction into aqueous HCl (3-5% w/v: Table 3) from an ethereal solution of the bitumen. Subsequent purification steps could then include chromatography, analytical HCl (aq.)/ether partition, metallation with 63Cu and/or LPHPLC, as detailed later. Pigments which were not extractable into dilute aqueous HCl included the dihydroporphyrins (Fig. 20c) or nickel porphyrins (Fig. 20e). In these cases, chromatography over cellulose or silica, respec-tively, was indicated. Mature Sediments (late-diagenesis) and Catagenetic Bitumen (Petroleum) Figure 20f is the electronic spectrum of the crude extract of a shale which is just entering the 'oil-window Thus, the evolutionary stage may be termed latest-diagenetic to early-catagenetic (cf. Hunt, 1979; Tissot and Welte, 1978). Spectra such as these (Fig. 20f) are typical of bitumen containing both nickel (A: 390, 515, 550 nm) and vanadyl (A: 410, 536, 571 nm) porphyrins. The method of choice for the study of bitumen containing both Niand YO-porphyrins was found to be a complete and quantitative separa tion of these two populations. As detailed subsequently, much emphasis

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and effort was expended in developing reproducible and quantitative methods by which to achieve this goal. Separation-Isolation Studies 83 The previous section reveals that three main types or classes of tetrapyrrole pigments are present in sedimentary bitumen and that the type or types present relate to the stage of organic evolution. These three pigment types are the dihydroporphyrins, free-base porphyrins and metalloporphyrins. Each of these classes exhibit differen ces in physicochemical properties, in vitro stabilities and chromatographic behavior. Thus, individual separation-isolation schemes had to be tailored to each. In addition, the co-occurrence of pigment types required that any analytical scheme had to be flexible enough to allow for these intermediate cases (see Figs 20c, 20e). As a class, the funct io nalized dihydroporphyrins, including the chlorophylls per se, are the tetrapyrroles most susceptible to artifact formation during in vitro handling (Bacon and Holden, 1967; Gilman, 1956; Hynninen, 1979; Katz et gl., 1968; Scheer and Inhoffen, 1978; Seely, 1966). Thus, strict adherence to precautionary measures (see Materials and Methods) and very mild separation techniques are required. Previously, 'chlorin' {dihydroporphyrin) dominated sediments were analyzed by Baker and co-workers methyl esterification of the bitumen and a preliminary size-class fractionation through Sephadex LH-20 {GPC), with tetrahydrofuran {THF) as the mobile phase. Following the above, separation was routine ly by chromatography over silica-gel

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84 (Baker, 1970; Baker and Smith, 1973, 1975a-b; Baker et gl., 1976; Smith and Baker, 1974) or alumina (Baker et gl., 1978a-b). The above scheme was found objectionable since it e xposed t h e dihydroporphyrins to possible artifacts (CH2N2 ) and, potential l y, to peroxides (THF), as discussed previously in text (Materials and Methods). Further, the adsorbents used (silica-gel, alumina) are relatively strong and potentially catalytic. Thus, a mild and repeatable method for dealing with geologic dihydroporphyrins was sought. Chromatography over confectioners sugar, the 'classic' method for plant pigments (Strain, 1958; Tswett, 1906a-c), was tried previously (Baker and Louda, 1980a; Louda et gl., 1980) and during the present studies with various mobile phases. While the classic system of Strain (1958: 0.5 % n-propanol in petroleum ether) works well for fresh plant materials, it and other solvent systems fails to adequately handl e the diversity of pigment types and polarities present in geologic samples. Cellulose has been suggested as a replacement for s u gar (Hill, 1963; Holden, 1976). Trials with laboratory grade cellulose (ACS, suitable for chromatography) yielded poorly packed columns by either dry or slurry packing techniques. The use of microcrystalline cellulose (Merck #2331) was found to offer reasonable separation of 'geo chlorins' according to polarity class (Baker and Louda, 1981a; Louda and Baker, 1981). Thus, the refinement and standardization of microcrystalline cellulose chromatography for the rapid and reliable separation of geologic dihydroporphyrins was undertaken A wide variety of mobile phase systems were i nvestigated and stepwise gradient elution with acetone (ACE) in petroleum ether (PE)

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was found to remain (cf Louda and Baker, 1981) the most straightforward and effective. The goal of this study was to provide a rapid method for splitting geolog i c dihydroporphyrin assays into three broadly different polarity classes, each of which would then be more amenable to detailed analyses. The three 'pola rity classes' or fractions required were, in essence; (1) non-polar (phytyl esters, decarbo x ylated species, etc.); (2) mono-carboxylic acids ( pheophorbides,' etc.); and (3) very polar species (di -and tri-carboxylic acids). 85 Investigation of the chromatographic behavior of numerous standard pigments (Appendix A) lead to the following fractions being designated:* CELL-1, 3 5-5 .0% ACE/PE; CELL-2, 12.5-15.0% ACE/PE; and CELL-3, 35-50% ACE/PE, corresponding to the polarity classes given above. However, studies with 'chlorophylls-c' [XIX] and 'pheo phorbides-c' [XX] revealed that these remained on the column. Thus, a fourth fraction or 'flush' was added and consisted of elution with ACE/MeOH (1: 1, v/v. cf. Louda and Baker, 1986). Table 4 is the compilation of standard tetrapyrrole pigments (Appendix A) which were tested and the cellulose chromatographic fractions in which they eluted. Tests with 'oxy-deo xo' derivatives (900-pheophytin-a [V], 9-00-pyropheophorbide-a ME [IX], etc.) revealed that the presence of an hydroxyl moiety only slightly increased the overt chromatographic polarity of the s e pigments, relative to the precursor (keto-) pigment. Thus, the effectiveness of cellulose for the chromatographic separation of geologic dihydroporphyrins rests *cELL = cellulose chromatographic fraction

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Table 4. Representative pigments eluting in various fractions during chromatography over cellulose(fi). FRACTION SOLVENT MIXTURE AUTIIENTIC TEST COMPOUNDS ASSIGNED GEOTITRAPYRROLE ClASS Footnotes; CEIL-1" 3 -SioACE/PE Pheophytin-a[IIa Pheophytin-b[XV] Pheophorbide-a ME[VIb] 9-0D-Pheophorbide-a ME[VII] 3-MDF-9-00-PP-b ME (XVIII] 7-PDP-DOMPP-a (XIII] DOMPP-a ME (XII] Bacteriopheophytin-a[XXI] 'carotenes 'hydroxy-carotenoids' CHLORIN AND PURPURIN MEs Pheophytins, 'decarboxylated' phorbides and chlorins, alkyl porphyrins, carotenoids and (in vitro)methyl esters of carboxylic acid tetrapyrroles. "CELL-2'' 15-25ioACE/PE Pheophorbide-alVIa Pyro-PP-a(see(VIII] Purpurin-18[XXVIa] DOMPP-a(see(XII] OPE [XXXVIIa] Mono-carboxylic acid pigments "CELL-3" 35-SOioACE/PE Diand tri-carboxylic acid pigments fi Abbreviations,also found in Appendix-A,are :ME=methyl ester,OD=oxy-deoxo,MDF=methanol-desformyl, PP=pheophorbide,DOMPP=desoxymesopyropheophorbide,DPE=desoxophylloerythrin.Bracketed Roman numerals refer to the number of authentic standard pigments as listed in AP.pendixA Solvents were; ACE=acetone,PE= petroleum ether(30-60).Followin g fraction 'CELL-3" a 'FLUSHjusing acetone/methanol(1:1,v/v) was used to elute chlorophyll-c[XIX] and/or'chlorophyllides-c'[XX if present. 00 0\

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87 almost entirely upon the absence, presence and number of free carboxylic acid moieties. The extreme polarity exhibited by the 'chlorophylls-c' [XIX] can only be attributed to the presence of a free acrylic acid group, as compared to the more common propionic acid groups of chlorophyll-a [I] derivatives. Prior to the application of cellulose chromatography to dihydro porphyrin containing extracts, carotenols or free-base porphyrins are removed by partition from an organic solvent in 90% aqueous methanol or dilute aqueous HCl, respectively These procedures are covered elsewhere in text {Chapter 2). Once separated into the functional classes given above, dihydro porphyrins are amenable to more detailed analyses. Typically, the nonpolar fraction (CELL-1) is further fractionated by low-pressure high performance liquid-chromatography {LPHPLC) as discussed later, without prior derivatization. The polar fractions (CEL L-2, CELL-3), containing free carboxylic acid groups, are converted to the methyl ester derivatives (BF3/MeOH) before further c hromatographic separation. The detection of free-base porphyrins in various bitumens or fractions was detailed earlier in this section. Preliminary isolation of free-base porphyrins employs the standard technique (Materials and Methods) of extraction into aqueous HCl, subsequent neutralization and transfer into fresh organic solvents, such as ethyl ether. Specific to the present study is the development of techniques to protect, purify and classify geologic free-base porphyrins following the initial HCl/ether isolation. The methods of free-base porphyrin isolation and purification developed during these studies are shown in flow-chart style as

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Figure 21. Electronic absorption spectra (UV/VIS) representative of each stage of geologic free-base porphyrin separation, derivatization and purifi cation are shown in Figure 22. 88 The presence of free-base porphyrins in a bitumen or chromato graphic fraction is easily noted by the characteristic absorption at about 620 nm (Fig. 22a}. Initial separation of the free-base porphyrins from the non-basic components is accomplished by extraction with 5-10 % (w/v) aqueous HCl (Table 3}, neutralization and transfer of the pigments into fresh ether (Chapter 2). Thi s affords a 'crude' free-base porphyrin isolate (Fig. 22b}. At this stage, as for the example shown, there is often the presence of d i hydroporphyrin-l ike absorption as well (A = 639.5 nm: Fig. 22b}. When dihydroporphyrins were found to be present, they were separated from the more basic porphyrins by a two-stage aqueous HCl e x traction procedure. It was determined that 2.0-3.5 % HCl (aq.) would effectively remove the geologic free-base porphyrins (Fig 22c} from ethereal solution. Subsequent l y, the dihydroporphyrins, shown here i n to be homologs of 7,8-dihydro-OPEP (cf. [XIII]), can be e x tracted with 5-10% aqueous HCl (Fig. 22d}. Once freed from dihydroporphyrins, the free base porphyrins (Fig. 22c) are converted into their corresponding CU63c helates (Fi g. 22e). At this stage, these pigments are now protected from artifact formation due to unwanted, and potentially discriminatory by carbon-number (Baker and Louda, 1987}, chelation by natural isotopic abundance zinc (Louda and Baker, 1981} or copper (Quirke, J. M E., pers. comm., 1985}. That is, subsequent mass spectral analyses are simplified due to the use of monoisotopic CU63

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CRUDE EXTRACT OR LC FRACTION *UV/VIS 1< f.. =620nm) EE/t?-10 /o HCI AQUEOUS AC 10 ETHEREAL ("FREE-" BASES) (NEUTRALS) +NaHJCO or *' 3 analyze non-* EE/ H2 0 tr:ee base AQUEOUS ETHEREAL pigments discard J 1 *0 FB SPLIT 1+63Cu(ll) FIB l 63cuPHs -LC M .S. Figure 21. Analytical scheme for the isolation and purification of free-base porphyrins from sedimentary bitumen.Abbreviations:EE; ethyl ether,NaHC01 ; neutralization with sodium bicarbonate,FB; free-base porphyrins, 63-Cu indicates the formation of a metalloporphyrin derivative using 63-Cu sulfate,LC; liquid chromatography,HCl; aqueous hydrochloric acid(see Table 3). 00 1.0

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1 0 (a 0.8 0 6 0.4 0.2 QO I I I I I I I 7l 1 0 ---. (e 0 8 0 6 0 4 0 2 0 0 I I I I I I F 350 550 750 350 (b ( c (f (g ( h 550 750 350 550 750 350 550 750 WAVELENGTH(nm) Figure 22. Electronic absorption spectra representative of the various stages during the isolation and purification of geologic free-base porphyrins.(a) bitumen with freebases evident,(b)crude free-base/7,8-dihydroporphyrin isolate,(c) free-base porphyrins, (d) crude 7,8-dihydroporphyrins(HCl# > 3.0),(e) crude Cu-63 chelates of free-base porphyrins(absorption at ca.595nm due to 9-keto porphyrins),(f-g) chromatographic purification of Cu-63 porphyrins,(h) partially purified Cu-63 phylloerythrins. \0 0

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91 An additional advantage was found during the analyses of geologic free-base porphyrin arrays, as the i r Cu63-derivatives, since the presence of the decarboxylated analogs of phylloerythrin [XXXII] became evident. That is, during the routine low to medium resolution MS analyses of porphyrins, in which pseudohomologous series are separated by 14 amu, the presence of keto-substituted (+14 amu) alkyl porphyrins is masked. However, upon chelation with Cu, the presence of absorption (l = 595 nm: Fig. 22e-f) bathochromic to the a-band position (l = 560-565 nm) of Cu-alkyl porphyrins (cf. [LXXXVII], [LXXXIX], [XCIX]-[CII]) indicated the presence of a Cu-phylloerythrin [LXXX] type chromophore and revealed the need for further separation. In order to purify the crude Cu63-derivative (Fig. 22e), e xtraction of non-porphyrin bases into 10-15% aqueous HCl was used as a first step. This procedure left the Cu63-porphyrins in a state of much greater purity (Fig 22f), relative to non-porphyrin 'background. Subsequent LPHPLC separation of the Cu63-derivatives yielded the purely alkyl porphyrins (Fig. 22g) and the 9-keto-substituted analogs (Fig. 22h). Separation was achieved over methanol-deactivated silica gel (13-24 eluting with 0.5-1.0% and 2.0-3.0% acetone in petroleum ether, respectively A$ far as can be told, the present study appears to be the first to show the existence of the decarbo x ylated analogs of the phylloerythrin [LXXX] series in geologic samples. Thus, the present scheme (Fig. 21) allows sub-division of the 'classic' geologic free-base isolate (cf Baker and Palmer, 1978) into (1) dealkylated, 7,8-dihydroporphyrins (homologs of 7,8-dihydroDPEP: cf. [XIII]); (2) true free-base (alkyl) porphyrins; and (3) the decarboxylated analogs of phylloerythrin [XXXII]. The first and last of these may be.

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considered as the 7,8-dihydro-and 9-keto-precursors to the DPEPseries, respectively. 92 Sedimentary bitumen of moderate maturity (late-digenesis/ catagenesis) typically contains nickel (Ni) and vanadyl (VO) porphyrins. This statement refers to marine organic matter, as freshwater (lacustrine) bitumens lack the VO-chelates (Baker and Louda, 1986a). Further, it is now known that humic coals (cf. Palmer et gj., 1982) contain porphyrins with Fe3, Mn2+ and Ga3+ as the chelated metals (Bonnett and Czechowski, 1980, 1981). Thus, for the purposes of the present study, efforts were directed towards the development of analytical schemes which would apply to (1) the purification of Niporphyrins when they occur alone, and (2) the complete separation of Ni-and YO-populations with valid reproducible quantitation of each. This second requirement stems from both the facts that (1) the porphyrins associated with Ni versus VO are different in quality (Baker and Louda, 1983; Louda and Baker, 1981, 1986; Barwise and Park, 1983) and, (2) there is a lack of attention given to this problem by other studies (cf. Louda and Baker, 1987, 1990). During the course of these studies, the isolatio n and purification of nickel porphyrins from essentially three main types of mixtures was necessary. First, the simplest cases, were those samples in which Ni porphyrins were the only metalloporphyrins present. Samples such as these included pre-catagenetic marine sediments/shales and lacustrine samples in which the Ni porphyrins represented the bitumen fraction (extractable) tetrapyrroles characteristic of in situ digenesis (autochthonous pigments: cf. Baker and Louda, 1983, 1986a; Louda and Baker, 1987). Second are those samples of marine sediments which

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93 contain not only the autochthonous Ni porphyrins but allochthonous arrays of highly dealkylated etioporphyrins (HOEs}, complexed with both Ni-and Cu-(Baker and Louda, 1984, 1986b; Louda and Baker, 1981; 1987; cf. Palmer and Baker, 1978}. Third are the cases of marine sourced shales and bitumen (petroleum), having entered and continuing through out the 'oil-window' (catagenesis), in which both nickel and vanadyl porphyrins are present. The present section deals only with the isolation and purification of the non-polar metalloporphyrins (Nior Ni-plus Cu-/Ni-HDEs), as the separation of these from the vanadyl species is covered later. In essence, Ni-porphyrins were found to exist in two types of bitumen. Pre-catagenetic marine sediments/shales and lacustrine samples (g.g. Green River Formation) yield bitumen from which Niporphyrin s are relatively easily isolated and purified. However, the Ni-porphyrin fraction from marine sourced bitumen in the catagenetic ('oil-window') stage have proven to be extremely difficult to purify to a stage where MS analyses are 'valid.' The term 'valid' is used to imply that resultant MS represent only Ni-porphyrin ions with a high signal-to-noise ratio. Ni-porphyrin isolates from such samples were found to be best purified by combinations of chromato graphic, demetallation and derivatization techniques. Figure 23 is a composite flow-chart for the analyses of Niporphyrins as developed here. The Ni-porphyrins from pre-catagenetic sediments were found to require only two standard liquid chromatographic separation steps (silica, alumina: Fig. 23). followed by a rapid (ca. 10-20 min.) purification via LPHPLC. The LPHPLC system utilized is described later

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BITUMEN or NiPH FRACTION .----J CC /SILICA a ) nC 5-saturates b)nC5/BZ, 1 : 1 NiPHs c) BZ -"polar" Ni PHs CCI ALUMINA 3.5-5%ACE/PE Ni-alkylPHs 1 (MSA) FB-PHs:::------j .. 63Cu /// LPH PLC ( SIL/MeOH) I ba)Cu/Ni-HOEs bb) N i PHs (C#>28) 63 / CuPHs M s 94 Figure 23. Composite flow chart representing the various methods used for the isolation and purification of nickel or nickel plus copper porphyrins from marine sediment extracts. Abbreviations,CC=c o lumn chromatography,nCS= n pentane,BZ= benzene, NiPHs=Ni porphyrins,LPHPLC= low-pressure high-performance liquid-chromatography, SIL=silica gel, MeOH =methanol, HOEs= highly dealkylated etioporphyrins, ACE= acetone, PE= petroleum ether(35-60), MSA =methane sulfonic acid(i.e. demetallation step), FB-PHs = free-base porphyrins, 63Cu-= 63CuS0 4(cf.Fig.22) ,MS=mass spectrometry.

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95 in this section. In brief, the chromatographic purification of Niporphyrins involves the following three steps (Fig. 23). First, chromatography over silica gel-60 (63-200 with n-pentane allows elution of saturates while the Ni-porphyrins are retained on the column. Changing the solvent (step-gradient) to n-pentane/benzene (2: 1, v/v) then elutes the Ni-porphyrins, usually contaminated with low molecular weight heteroaromatic and aromatics. Second, separation over alumina (Grade II-III) with 2.0-3.5% acetone in petroleum ether (30-600) allows elution of the Ni-porphyrins while retaining the majority of the aromatic 'background.' Last, LPHPLC over methanol-deactivated (cf. Purcell, 1958) silica (13-24 with n-pentane/petroleum ether (1:1, v/v) as the mobile phase affords substantial purification of the Ni-porphyrins with the added advantage of absorption specific detection in order to collect the entire pigment array (Fig. 24a). In those cases when the Cu-or Cu-/Ni-HDEs are present (cf. Baker and Louda, 1984; Louda and Baker, 1981), the final step above (LPHPLC) also allows detection by chromatographic behavior and UV/VIS quantita tion of the Cu-species prior toMS analyses (Fig 24b). Further, even traces of Cu-HDEs can be detected with this technique. For example, the LPHPLC_trace given as Figure 24c reveals the presence of compounds eluting at the position of Cu-HDEs. Subsequent electronic spectral analysis confirmed their identity (A = 397.8, 526, 563 nm) and allowed quantitative assessment. In this case, the Cu-HDEs were found to constitute only 2.7% of the total (Cu-plus Ni-) porphyrins. Such a small amount of an alternate metalloporphyrin series would be lost during MS analyses if performed on the whole (Cu-/Ni-) fraction.

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a) Ill I 0.. z E I c 0 0 II ,<. WI II I I lf) z 0 0.. lf) w a: 0:: w 0 0:: 0 r--u c w ..... 0:: b) 0.. --LL If) \ Ill I Ill 0.. I -0.. z :::J I u II I 1-1 T I I I 0 10 20 Tl ME(mlnutes) c) Ill I 0.. z I Figure 24. Low-pressure high-performance liquid-chromatography(LPHPLC)separations of geologic nickel or nickel plus copper porphyrins.(a)marine anoxic shale,Sisquoc formation,California containing only nickel porphyrins, (b) DSDP/IPOD# 64-479-34-5 containing nearly equal amounts of Ni and Cu (c) DSDP/IPOD # 71-511-60 Ni porphyrins containing traces of copper highly dealkylated etioporphyrins. 1..0 0"\

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97 The nickel porphyrins present in catagenetic shales and petroleum of marine sources have proven difficult to purify. Thus, the analyses of these 'dirty' Ni-porphyrins relies upon the techniques of demetallation (cf. Baker and Palmer, 1978; Erdman, 1965; Groenning, 1953; Treibs, 1934a-b} and those developed during these studies. In these cases ('dirty' Ni-porphyrins), analysis (Fig. 23) begins as above (silica and alumina chromatography} but, prior to LPHPLC, the pigments are demetallated (methane sulfonic acid, 105C, partitioned into 2.5% HCl (Table 3}, and the resultant free-base porphyrins converted into their 63Cu-derivatives. The 63Cu-pigments (ex. Niporphyrins) are then subjected to LPHPLC, as given both above and earlier for geologic free-base porphyrins. The largest shortcoming with the above combination of chromatographic, demetallation, HCl/ether partition, derivatization and LPHPLC is that of overall yield. Shown in Table 5 are the individual stage and cumulative percent yields obtained from the analyses of fifteen oils and four shales which contained Ni-porphyrins which could not be purified by chromatography alone. Examination of these data reveals that the most drastic losses occur when Ni-porphyrins are exposed to acids. That is, the mean individual stage yields for demetallation with methane sulfonic acid (MSA} and purification with HCl/ether partition were found to be only 26% (N = 19; R = 9-42%} and 30% (N= 11; R = 16-44}, respectively. However, it has recently been reported that the addition of 1,2-ethanedithiol during the demetallation of Ni-porphyrins with MSA leads to near quantitative recovery of the pigments in free-base form (J. R. Maxwell, pers. commun. to E. W. Baker). In the future,

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Table 5. Individual step and cumulative p ercent yields obtaine d during the purifica-tion of nickel porphyrins from petroleum crudes and oil shales. OIL(O) and 1 YIELD2 I'K)l.AR PERCENT YIELD INDIVIDUAL STEP / CUMULATIVE SHALE(S) SAMPLE CODE POST LC3 g -EOM POS T MSA4 POST H C 1 5 POST 63Cu(6) POST LPHPLC7 C0-1 62 95/100 24/24 25/6 87/5 95/5 C0-2 45 95/ 100 41/41 29/12 1 07/13 95/13 C0-3 21 95/100 28/28 44/12 69/8 95/8 C04 46 95 5/100 40/40 16/6 115/7 95/7 C0-5 69 95/100 15/15 27/4 70/3 95/3 C0-6 134 95/ 1 00 13/13 23/3 119/5 95/5 C0-7 80 95/100 42/42 ----100/42 95/42 t--U-1 164 95/ 1 00 21/2b': t--U-2 66 95/100 t--U4 81 95/100 t--U-5 61 95/100 t--U8 32 95/100 t--U-9 40 95/100 22/22>'< t--U-10 32 95/100 38/38>'< t--U-19 127 95/100 12/12 25/3>': MS-6 193 95/100 23/23 19/4>'< MS-7 830 95/100 14/14 35/5> : MS-8 348 95/100 17/17 40/7>'< MS-9 675 95/100 9/9 43/4-1: FOOTNOTES: 1) Samples presented in code as Cities Service oil sample (CO-X),Mobil Oil oil sample(MO-Xl or Mobil Oil shale sample(MS-X) for proprietary reasons.All samples are of marine origin except C0-7 which is of lacustrine origin. 2)Yield calculated using E mM = 34.82 (Fuhrhop and Smith,1975)following chromatography over silica gel 'Sil-1b';Figs.23 & 27).EQM=extractable organic matter(oil or bitumen of shale). 3)Yields at each chromatographic step(Fig.23) averaged 95%. Cumulative yield at this stage represents t h e original content of the oil or shale EOM. 4)MSA = methane sulfonic acid demetallation as given in text and Figure 23. S)HCl = Aqueous HCl/ethyl ether partition of freebase porphyrins as given in text(cf.Figure 21). 6) 63eu =in vitro metallation of free-base porphyrins(ex,NiPHs via MSA)with 63cu Z+.(see Figs.21-22) 7) LPHPLC = low-pressure high-performance liquidchromatography,as given in text and Figure 37. >'<) Asterisk indicates that the purification was stopped at this stage as the isolate so derived was considered pure enough for valid mass spectral analyses. \.()

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99 refinement of the present scheme (Fig. 23) to include 1,2-ethanedithiol will certainly increase the precision of these studies. The introduction of sulfonic acid derivatized silica for the purification of vanadyl porphyrins by Barwise and Whitehead (1980), as detailed later, prompted investigation of these media for the purification of Ni-porphyrins as well. Given as Figure 25 are the percent yield and resultant mass spectral distributions for aliquotes of a Niporphyrin isolate {"A" in Fig. 25) from a sample of Green River oil shale (Eocene: Colorado, U.S.A.). The initial isolate was obtained by chromatographic purification alone (Fig. 23: Fraction Sil-1b/Al-2/ LPHPLC) and represents 95 8% of the total estimated Ni-porphyrins in the original bitumen. Aliquots of this isolate were either chromate graphed over sulfonic acid derivatized silicas ("BP"; J. T. Baker aliphatic ["ALI"] or aromatic ["AROM"]) or demetallated with methane sulfonic acid {"MSA": Fig. 25). In each case where Ni-porphyrins were exposed to acidic media, the percent-DPEP {0/E-value) was found to increase This, of course, implies selective destruction of the ETIOseries. Further, the C-30 member of the DPEP-series, repeatedly shown to be dominant in this sample, was found to have been preferentially destroyed (Fig. 25). Even though recoveries from the sulfonic acid derivatized silicas were high {81-95%), elution of the columns with polar solvents (methanol, acetone, tetrahydrofuran) recovered small amounts of free-base porphyrins. Thus, alteration of Ni-porphyrin homologies upon exposure to acidic media appears to function by selective demetallation of certain members of both DPEP-and ETIO-series. As given later, demetallation on the acidic silicas was found not to occur with the more stable vanadyl pigments. Going on the above,

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a) b) c), .-r& d) e) I I I I I 24 26 28 30 32 34 36 CARBON NUMBER 1.16 30.53 28.90 95 + 1.90 30.60 29.04 81/o(a ) 1.67 30.60 28.82 95/.l_a) 1. 25 3061 28.46 94 /..( a) 1.25 30.57 2a95 350fo(a) DIE 1 c0 1 CE 1RECOV 99 Figure 25. Mass spectral distributions obtained for aliquots of a nickel porphyrin array which were exposed to various acidic media. (a) nickel porphyrins as isolated from Green River oil shale by LC alone,(b) sample of a passed over sulfonic acid silica from British Petroleum Ltd. ,(c) sample of "a" passed over aliphatic sulfonic acid derivatized silica(J.T.Baker Co.), (d) sample of "a" passed over aromatic sulfonic acid derivatized silica(J.T. Baker Co.), (e) "a",as free-base porphyrins,following demetallation with methane sulfonic acid. D/E= DPEP-toETIO ratio, c0 and CE are the average carbon n umber s of the DPEP and ETIO series,respectively(see Table 23).Percent recoveries for b through e cal culated using "a" equal to 100 percent.

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101 sulfonic acid derivatized silicas were not used during the purification of geologic Ni-porphyrins. Previously, it was reported (Louda and Baker, 1981) that even such mild techniques as gel permeation chromatography (GPC) through Sephadex LH-20 can lead to discrimination among the series and carbon-numbers of Ni-porphyrin arrays. Figure 26 contains the mass spectral distributions of the main-body (b) and the 'tail' (a) of aNi-porphyrin isolate as eluted from a Sephadex LH-20 (GPC) column. Although the late eluting ('tail': Fig 26a) material was only about 5% of the total, severe discrimination by carbon number was found. Collection of porphyrin fractions by visual examination of columns ('bands') or eluates alone is concluded as being invalid and electronic spectral assay of all is required. During the initial stages of the development of a chromatographic system with which to separate the metalloporphyrins (Ni-, VO-) of bitumen, it was decided that the overall process must meet the following criteria: (1) Ni-and VO-porphyrins must be able to be completely separated, one from the other; (2) valid and reproducible quantitation of each species, as the native metallo-chelate, should be possible at an early stage of the analysis; (3) each technique and the overall process should be maximized for yie l d and minimized as to porphyrin class/carbon-number discrimination; and (4) the final isolate should be sufficiently pure so as to yield 'clean' (high signal-to-noise [S/N] ratio) reproducible mass spectra and/or HPLC analyses. To date, analyses of the metalloporphyrins of shales and petroleum have employed either demetallation (Alturki et gj., 1972; Baker, 1966, 1969; Baker et gj., 1967; Eglinton et gj., 1984; Groennings, 1953;

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a) I b) c) 22 24 26 28 30 32 CARBON NUMBER 102 Figure 26. Mass spectral distribution for gel permeation fraction collected during the of nickel porphyrins. (a) late eluting ('tail) material, (b) main body of the red-brown band ,(c) total reconstitued isolate. Sample, DSDP/IPOD # 63-467-110-J(see Louda and Baker,1981).

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103 Hodgson et gl., 1972; Mackenzie et gl., 1980) or various chromatographic methods without (reported) estimates as to the fraction of the original pigment complement which the final isolate represents (cf Barwise and Roberts, 1984; Barwise and Whitehead, 1980; Hajibrahim et gl., 1978; Mackenzie et gJ., 1980, Sundararaman, 1985). Existing data also reveals that, for the vanadyl porphyrins, demetallation is destructive, discriminatory (Ekstrom et gl., 1983a; Gallegos et gl., 1983; Roberts and Scammells, 1983) and can also lead to alkyl rearrangement at the porphyrin periphery (Quirke J.M.E., 1987: pers. commun.). Severe loss of pigment during demetallation, as discussed earlier for the Ni-porphyrins, has been known for some time (Sugihara and Bean, 1962). The report of alkyl sulfonic acid derivatized silicas (Hancock and Kirk, 1981) and the application of same to the purification of VOporphyrins, as the native metallo-chelates (Barwise and Whitehead, 1980), represents a milestone in 'petroporphyrin' analyses. Based on the report of Barwise and Whitehead (1980) the present study was undertaken to assess the reported techniques as a 'routine' method for VO-porphyrin isolation and purification without the need for demetallation. The method reported by Barwise and Whitehead (1980) was as follows: topped (500-650C) oi l was fractionated over silica gel-60 using heptane, toluene and ethyl acetate for the elution of saturates, aromatics plus Ni-porphyrins and resins plus VO-porphyrins, respectively. Next, the resin plus VO-porphyrin fraction was chromatographed through a column of functionalized silica with 2 % ethyl acetate in toluene as the mobil phase (93% recovery reported).

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104 Trials with the above system using petroleum crudes revealed several shortcomings. exposure to high temperatures for and the evaporation of toluene solvent was objectionable. the application of a petroleum crude or shale bitumen to the first chromatographic environment in n-heptane, or n-pentane as desired here, invariably lead to asphaltene precipitation and considerable column Third, examination of the Ni-porphyrin fractions (toluene eluate) obtained with this method revealed the presence of considerable amounts of 'non-polar' (higher m.w.) VO-porphyrins. Starting with the system of Barwise and Whitehead (1980), given above, numerous alterations of solvents and chromatographic media were tested in an attempt to develop one chromatographic regime which would be applicable to the analyses of Ni-and VO-porphyrins in all bitumen samples. The result of these studies is given as a flow-diagram in Figure 27. Crude oil or total bitumen (EOM) is applied to a column of silica gel-60 (63-200 Merck in benzene solvent. Sequential elution with benzene, benzene:ethyl acetate (2:1, v/v) and ethyl acetate affords three fractions (Sil-l, Sil-2, Sil-3: Fig. 27). Fraction 'Sil-l' contains saturates, aromatics, low mw heteroaroNi-porphyrins and 'non-polar' (high mw) VO-porphyrins. Fraction 'Sil-2' contains most of the heteroaromatics, low m.w. 'resins' and the bulk of the VO-porphyrin population. Fraction 'Sil-3' is considered as a flush to ensure the total elution of VO-porphyrins. In cases where VO-porphyrins are evidenced by electronic spectral assay, 'Sil-3' is pooled with 'Sil-2', otherwise it is discarded. The next step in the analysis is the chromatography of 'Sil-l' (Fig. 27, cf. Fig. 23) over silica gel-60 made up inn-pentane.

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/BITUMEN, EOM,OIL(acc.wt.) U V / VIS BZ (QUAL= ;QUANT= 0 ) t t EA BZ \ BZ /EA( 2 :1) SATS-!-1a-nC5 1b-nC5/8Z( 1 :1) \ 1 c-8Z ---IF<) 1d-8Z/ EA(2:1 ).!..llfor VOPHs 1e-EA --IF( ) disc.) (1b)CRUOE N i PHs J AI-CC/PE('care') \ J .I tor VO PHs IF() IF(-) S03-SILI BZ(fast) AI-CC/PE('quick' ) .t diSC. 1-25"/oACE/PE \ OTHER -rp-hplc ., 13 \ ( hplc,Cr03,'b C,etc.) FB-PHs l63Cu 63cu PHs h p lc NICKEL P ORPHYRINS \ \ VANAOYL PORPHYRINS 105 Figure 27. Flow chart for the separation and purification of nickel and vanadyl porphyrins from geologic b itumen. Abbreviations:dis= dissolve,BZ=benzene, CC=column chromatography SIL=silica gel, EA=ethyl acetate,nCS= n-pentane, Al =alumina, ACE=acetone, PE=petroleum ether(35-60), S03-SIL = aliphatic sulfonic acid silica, NiPHs= nickel porphyrins, VO-PHs = vanadyl porphyrins.(See Fig.23 re lower left)

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106 Following the elution of Ni-porphyrins with n-pentane benzene (2:1, v/v), any 'non-polar' (high m.w.) VO-porphyrins are eluted with benzene or benzene:ethyl acetate (2:1, v/v) and pooled with '5il-2.' The combination of '5il-2' and '5il-lc/1d' (Fig. 27) represents the crude VO-porphyrins, free of Ni-porphyrins ('5il-1b'). Quantitation of the VO-('5il-2+1c/1d') and Ni-porphyrins ('5il-1b') is performed at this stage using electronic absorption techniques given in a subsequent section. Further purification of the 'crude' VO-porphyrins involves three chromatographic operations, each being performed rapidly (2-5 min./ ea.). Fraction '5il-2+1c/1d' is dissolved with 2% ethyl acetate in benzene and ran through a short (0.8x8 em, bed dimension) column of aliphatic sulfonic acid bonded silica gel (40 J. T. Baker #7045)* made up in the same solvent. Typically, the total eluate is collected and labelled '503-5IL-A' (Fig. 27). Following evaporation of the solvent, the '503-5IL-A' fraction is dissolved with minimal benzene and placed upon a column of aluminum oxide (Merck #1077: 63-200 adjusted to Brockman activity II-III (3.5% H20; v/w), made up in petroleum ether. VO-porphyrins are eluted with 15-25% acetone in petroleum ether, following previous development of the column with 3-7% acetone in petroleum ether. The last step involves re-chromatography of this fraction ('Al': Fig 27) over a fresh bed of aliphatic sulfonic acid bonded silica gel as given above. The final isolate ('503-5IL-B': Fig. 27) is of sufficient purity for mass spectral and/or HPLC *rests (N=3) with standard pigments yielded the following average recoveries: VO-DPEP [LXXXVIII] 99 2%; VO-OEP [VO-L] 95 2%; VObenzoetio [CXVIII] 100 2%.

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characterization, as is also evidenced by the quality of resultant electronic spectra (Fig. 28). 107 In order for any isolation scheme to be effective in geochemistry it must be of high yield and non-discriminatory. Therefore, following development using a single oil sample, the finalized method (Fig. 27) was monitored for both individual step and overall (cumulative) yield. These data are given in Table 6. The four samples listed as 'Group-1' in Table 6 were cases in which emphasis was placed upon the total transfer of each fraction onto the next chromatographic system. Overall, it can be stated that total recovery (ca. 100% yield) of the originally YO-porphyrins is attained. Variations in the amount of VO-porphyrins estimated by electronic spectroscopy at each step (Table 6) appear to derive primarily in the way one estimates the non-porphyrin background (best fit curve), as is detailed later. The "100%" yield claimed for the isolates following silica-gel chromatography derives from two facts. First, recovery tests performed with VO-DPEP [LXXXVIII], VO-ETIO-I [CVII], VO-ETIO-III [CVIII], VO-OEP [VO-XLIX], and VO-benzoetioporphyrin [CXVIII] chromatographed over silica gel developed with benzene:ethyl acetate solvents (Fig 27) provided 100 3% yields. Second, it is only after the total separa tion of the Ni-and YO-porphyrins that valid estimates of both can be attained. This, of course, is due to partial overlaps of the corresponding a-band absorptions, with regards to half -band widths (see "Electronic Spectroscopy"). In practice, the application of the present scheme for the isolation and purification of Ni-and YO-porphyrins (Figs. 23, 27) does

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1.0-.---:----------08 w u z 0 6
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Table 6 Recovery of vanadyl porphyrins during chrom-atographic analysis: Individual step and cumulative yield data. SAMPLE( 1 ) PERCFNT YIELD ( 2 ) GROUP SAMPLE(1 ) INDIVIDUAL STEP/CUMULATIVE(J) BITUMEN 'IYPE CODE SIL SOJA A l 2o3 S03B (MFAN) GROUP#1 OIL KEliO 100 98/98 121/118 95/112 OIL NESS 100 108/108 94/102 98/ 100 TAR MJ-19 100 94/94 99/93 98/91 SHALE MS-5 100 98/98 88/86 106/91 (98) GROUP#2 OIL MJ-11 100 93/93 80/74 94/70 OIL MJ-12 100 68/68 102/.69 104/74 OIL MJ-13 100 96/96 104/100 77/77 O IL MJ-14 100 77/77 77/59 98/58 OIL MJ-15 100 82/82 89/73 86/63 OIL MJ-16 100 66/66 75/50 77/39 OIL MJ-17 100 70/70 91/64 75/48 OIL MJ-18 100 82/82 85/70 91/64 OIL CLC 100 92/92 112/103 88/91 TAR SHGS 100 89/89 103/92 90/83 TAR SBS 100 1 01/10 1 103/104 87/90 TAR PRC 100 91/9 1 106/96 77/74 SHALE Bl.M 100 skipped 67/67 87/58 SHALE MS-6 100 96/96 94/90 92/83 SHALE MS8 100 85/85 95/81 80/65 SHALE MS-9 100 79/79 108/85 79/67 SHALE B6486 100 89/89 96/85 93/79 (70) FOOTNarES: ( 1) GROUP#1 represents samples for which a total transfer of material ont o each column was attempted.GROUP#2 represents samples for which minimization of the width of the loaded zone on the top o f the column was stressed. SAMPLE IDENTITY taken here as unimportant and are given in codified form.All of moderate to high sulfur content(1-6%S) and were of marine origin. (2) Calculated from the Beer-Lambert relationship using2:mM= 26.14 at 571.5nm(Falk,1964) ,followin g correction for non-porphyrin background,as shown in Figure 60. (3) Chromatographic media abbreviations as used in Figure 27.SIL= silica gel-60,S03A and S03B = first and second pass over alkyl sulfonic acid derivatized silica, and Al2 0 3 = a lumina (grade II-III). 109

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110 not require the total transfer of each fraction to each subsequent chromatography. This derives from the quantitat ion of the metal l o porphyrins at an early stage i n the analysis and that, i n solution, the laws of diffusion dictate that each aliquot is a representative of the whole. However, to assay the yields which can be e x pected duri n g 'routine' metalloporphyrin analyses, records were l ik ewise k ept ( Table 6). The oils, tars and shale bitumens g iven as 'Group 2' are r epresentative of such 'routine' analyses. That is, fol lowing dissoluti on, emphasis is placed upon minimizing the width of t h e starting zone for each chromatography, rather than upon affectin g a complete trans fer. Thus, a certain residue remains behind at eac h t ransfer and is evidenced by material on the walls of flasks and transfer pipets. In these 'routine' cases, a mean of 70% was found f o r the overall cumulative yield. However, for the r easons given above, i t i s fe l t tha t t h e q ualit y of the final isolate does reflect that of the orig inal whol e. On a theoretical or speculative basis, if one consi ders a 90% transfer efficiency for each of three successive transfers, then an overall yie l d of 73% (0.9 x 0.9 x 0.9) could be e x pected. T h i s appears to be the case during the 'routine' application (Table 6) of the finalized analytical scheme (Fig 27). The main focus of develop ing the analytica l scheme given a bove (Figs. 23, 27) was to provide a quantitative and non-di scriminator y method for the complete separation of metallo(Ni-, VO-) porphyrin arrays. Emphasis and effort were e xpended here i n qrder to assure the most valid intercompar ison of samples possible. To date, n o such studies can be located in the literature. Further, standardizat ion in

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111 the specialty of porphyrin geochemistry is likewise lacking and this problem has only recently been addressed (Louda and Baker, 1987, 1990). Aside from determining that all porphyrin series and carbon numbers could be isolated and characterized in a non-discriminatory manner, it was also of interest to investigate the chromatographic behavior of the VO-porphyrins This interest derived from two fronts. First, it was necessary to determine the effect of chromatographic 'heart cutting* on the quality of the final porphyrin assay Second, it was wished to investigate the potential discriminatory power of liquid chromatography in order to subdivide porphyrin arrays prior to more detailed (HPLC) separation and structural studies. In order to answer both of these questions, 'elution analysis** of VO-porphyrins was performed In essence, this study was a detailed examination of the scheme (Fig. 27) developed for the 'routine' analyses of VO-porphyrin arrays. In the present case (Fig. 29), the fractions obtained by the first chromatographic separation ('Sil.l': Fig. 27) were not pooled. Rather, those VO-pigments which exhibited reduced chromatographic polarity, relative to the bulk of the array, were kept separate. Specifically, the late ('tail') eluting material of 'Sil-l' and those VO-porphyrins recoverable from the main body of 'Sil-l' ('Sil-lc/d': Fig. 29) were pooled. This subset of the VO-porphyrins, as will be *Heart cutting': Collecting only the center, or 'heart,' of a chromatographic band (zone, fraction) while discarding early ('head') and late ('tail') eluting material **'Elution analysis': Subdivision of an eluate in order to analyze the exact makeup of the fraction, from early to late eluting material

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BOSCAN ASPHALTENE I LC SI LICA 0H (SIL-1) LC-SILICA + 1a-nC5 1b -nC5/0H--NiPHs 1c-.e5H --VQ-BENZ (enriched: BE C34) 5 /o(mol) + LC-S03:SIL LC-ALUIVIINA 15%CH2CI210H BEE,AI-1(34 80) BEE,AI-2(32-46 ED ;E36 &32-3880) LPHPLC: SILICA LC -A(33-46 BE SO) LC-8(32-46 ED) HIGH CARBON No. VO-BE & VO-E 0H/EA, 1:2 (SIL-2) --, VO-PHs ("MAIN") 9 5/o(mol) LC-S03:SIL 4 LC-ALUIVIINA .e)H OfoCH2CI2 f Al1 BEt\ i Al-2 Al3 '=' ;:.: AI 4 '"' I AI AI -7 1 ?' : Al8 t Al9 o 80 Al-10 : o V MAIN A L KYL VO-PHs 112 Figure 29 Chromatographic flow chart for a study of the 'early-eluting' or 'non-polar' vanadyl porphyrins present in maturing oil shales and petroleum crudes. Abbreviations: Refer to caption for Figure 27, asterisks indicate the molar percentage of each fraction,CH2Cl2= methylene dichloride,BEE= benzoetio-enriched,BE=oenzoetio,E=ETIO,D= DPEP,BD=benzo-DPEP, H= benzene.

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113 shown, was enriched in the high-carbon number (C33-C46+) members of the benzo-ETIO, DPEP-and ETIO-series Collectivel y th i s fraction was labelled 'BEE,' for This contrasts with the 'MAIN' bulk of the VO-porphyrins which primari ly c onta ined t h e DPEPETIO-and benzo-DPEP-series of a lower carbon number range (C26-C40). Prior to mass spectral anal y ses, the enrichment of the benzo-type pigments in the less polar fractions was a lso evidenced b y t h e electronic spectrum of this fraction (Fig. 30a), relative to that of the main portion (Fig. 30b). That is, the low energy visibl e (a) band of the VO-benzo-porphyrins is bathochromic (Aa = 591 nm [CX VIII]) to that of the VO-alkyl porphyrins, such as VO-DPEP (Aa = 574 nm [LXXXVIII]) or YO-etioporphyrin-III (Aa = 571 nm [CVIII]), and is clearly evidenced by a large shoulder in Figure 30a. Second derivative electronic spectroscopy (dashed trace, Fig. 30a) of this fraction ('BEE' : Fig. 29) revealed the exact position of maximal absorption responsible for this shoulder to be at 592 nm, close l y m atching that of VO-benzoetioporphyrin [CXVIII]. Subsequent analysis of the benz-enriched VO-porphy r ins ( [BEE] Fig. 29) included passage over sulfonic acid derivatized silica, without fractionation, and chromatography over alumi n a, developed with methylene dichloride in benzene* (15:85, v/v: Fig. 29) D uring chromatography over alumina, visual inspection of the column revealed that the early eluting material was red while the tail of this fraction had a decidedly green hue. Two fractions were collected and labelled 'BEE'/Al-1 and -2, r espectively. The electronic absorption spectra o f *Identical results were obtained using the same percentages of acetone in petroleum ether (30-60C).

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1 0-,.-----------, w 0.8 u z <( ro 0 6 0.:::: 0 lf) (]) 04 <{ (a 0 0 I I I I I) I '9 350 550 750 350 WAVELENGTH(nm) ( b 550 750 Figure 30. Electronic absorption spectra of (a) a benzoporphyrin enriched and (b) the main fracti on of vanadyl porphyrins from Boscan asphaltenes.Solvent= benzene.Dashed trace in "a" is t h e second derivative indicating the benzoporphyrin absorption maximum at 690 nm. f-" f-" .p-

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115 these two fractions (Fig. 31) reveal a further increase in the amount of VO-benzoporphyrins in the later eluting material (Fig. 31b). Fraction 'BEE'/Al-2 (Fig. 29) was next subjected to LPHPLC over methanol-deactivated silica gel (13-24 with n-pentane:benzene (2:1, v/v) as the solvent. In this case, the majority of the VO-benzoporphyrins (Fig. 32a) were found to elute prior to the VO-alk ylporphyrins (Fig. 32b). Thus, by the alternation of silica-gel and alumina it was possible to obtain an isolate containing only VO-benzoporphyrins. The e l ectronic spectrum (Fig. 32a) of this fraction ('BEE'/Al-2/LC-A: Fig. 29) matches very that of standard VO-benzoetioporphyrin [CXVIII]. The average mass spectrum of the final VO-benzoporphyrin isolate ('BEE'/Al-2/LC-A) is given as Figure 33. The carbon number range for both the VO-benzo-DPEP (VO-BD) and VO-benzo-ETIO (VO-BE) series was found to be C33 to C46, with C39 and C36/C38 dominancy, respectively. The VO-BE series made up 71% (V0-80/VO-BE = 0.41) of this fraction and the DPEP-and ETIO-series were absent. The later eluting material from the last stage of the benzopor phyrin enriched split ('BEE/Al-2/LC-B': Figs. 29, 32b) was found to contain VO-DPEP-, ETIO-and benzo ETIO-series in the same carbon number range (C33-C46) as the above fraction. As a result of the above analysis of the early eluting material in VO-porphyrin arrays, it can now be stated that a failure to adequate l y e xamine the Ni-porphyrin fractions obtained from bitumen ('Sil.l': Fig. 29) will alter the final arrays found for both the Ni-and VOspecies in several ways. First, these higher molecular species will be included with the porphyrins assigned to the Ni-species. Second, the

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1 0 (a 0 8 w u z <{ 0.6 rn 0:: 0 0 4 <{ 0 0 I I I 1_, ? I 350 550 750 350. WAVELENGTH(nm) (b 550 750 Figure 31. Electronic absorption spectra of fractions "Al-l" (a) and "Al-2" (b) obtained from chromatography of a 'benzoporphyrin "BEE" in Fig.29) vanadyl porphyrin isolate over alumina.Solvent = benzene. ...... ,...... (j\

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0.8 w u z <{ 0 6 ro 0:: 0 <{ 0.2 b) 0.0 I I I I I I T I 350 550 750 350 WAVELENGTH (nanometers) 550 750 Figure 32. Electronic absorption spectra of the early (a) and late (b) eluting van9dyl benzoporphyrins obtained during the LPHPLC of fraction BEE/Al-2(see Fig.29) over methanol deactivated silica. Solvent= benzene. f-L f-L -....!

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30 32 / I 34 36 38 CARBON \ \ 40 42 44 NUMBER 46 48 Figure 33. Mass spectral histogram of the vanadyl benzoporphyrins(Fraction BEE/Al-2/ LC-A:See Fig.29) isolated from the early eluting portion of the pigments in Boscan asphaltene.Be nzo-DPEP and Benzo-ETIO series are by solid and dashed lines, respectively. ....... ....... 00

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119 presence of the {VO-) benzoporphyrins in the non-polar fractions may alter the interpretation of Ni-porphyrin geochemistries. These first two points, of course, assume demetallation of the Ni-porphyrin fraction(s) prior to MS or HPLC characterization. Last, is the fact that certain VO-porphyrin species are of sufficiently low polarity so as to not be retained with the molar bulk of these pigments during 'routine' chromatographic splits of Ni-/VO-chelates. This then implies that, unless safeguards are added (Fig 27), the true extent of VO-porphyrin alkylation will be underestimated. non-polar high carbon number portion ('BEE': That is, even though this Fig. 29) is minor (ca. 46 % mol), the calculation of alkylation indices (AI: see section on Mass Spectrometry) will suffer inordinately due to the loss of these high mass 'lever-arm' components. The bulk of the vanadyl porphyrin array from Boscan crude oil ('MAIN,' Fig. 29: 95% mol) was amenable to very detai led analysis, simply due to the larger amount of material present. The following 'elution analysis' was designed to reveal three results. First, the order of the elution of VO-porphyrin series and the carbon numbers within each series should be known. Second, the effects of chromato graphic 'heart cutting' upon final porphyrin isolates were, to date, unknown. Third, any potential alteration of overall or average {MS) porphyrin distributions following reconstitution from individual sub fractions needed to be shown. The main fraction ('MAIN,' Fig. 29) of the VO-porphyrins from Boscan crude oil was passed rapidly over alkyl sulfonic acid derivatized silica (Fig. 29, f. Table 6) and the entire eluate, following evaporation, was subjected to chromatography over a lumina. Development

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120 of the column with benzene:methylene dichloride or petroleum ether: acetone (85:15, v/v) yielded a near Gaussian distribution of YOporphyrins, by concentration, along the column {Fig. 34a). During the elution of pigment, ten fractions were collected and analyzed by electronic and mass spectral methods. The first three fractions, collectively 6% of the whole, were repooled to provide sufficient material forMS analysis.* The mass spectra of these eight fractions {Al-l, -2, -3 pooled: Fig 29) are given both in tabular {Table 7) and histogram {Fig. 35) forms. Various indices were calculated {Table 8) from these raw data and include: the weighted average mass (X) and average carbon-number (C) for each series as well as the DPEP-plus ETIO-series together {X); the percent benzo-porphyrins in each fraction; and the ratio of the DPEP-to ETIO-type nuclei in both the benzo-{80/BE, %80) and alkyl-series porphyrins (0/E, %0). Selected parameters are plotted versus elution volume in Figure 34. Examination of this figure, as well as the numerical {Table 8) or histogram (Fig. 35) MS data, reveals several patterns within the chromatographic behavior of VOporphyrins. First, the higher molecular weight members of each series are less polar than and elute prior (Fig. 34b) to the lower molecular weight species. This behavior is attributed to two structural features of 'petroporphyrins.' High molecular weight geologic VO-porphyrins are known to possess extended n-alkyl substitution (Baker and Palmer, 1978; *Determination of mass spectra and the indices or parameters calculated from same are detailed in a subsequent section ("Mass Spectrometry").

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w 0.0 ) a 3 o,, ) Frs.'l+ 2 J ...,. 0.2 ,.""' \ __.J 0 w 0.6 I w 0 / 4: 0 __.J I w 0 0 0 1.0 2 0 3.0 (a) }19 VO-PH/ml Eluate X(*) 500 550 0/o BENZ ( *) 0 5 10 1 5 _L I I I I I I I I I b) 1-c) / / I 0. 0 /I I 0 0 5/o 7.50fo 15/o \/ II o fJ o. L I 'I Q) /'\._ I ,. 0 ... v w '\. \.. m I '\_ 1', cLt 11 0\ o, -0 a_ I \'-_0JC o 'o U I \ "-._ ......_ NQJ I u u 40 60 80 100 0 2 4 6 (b) OfoDPEP(0 ) (c) BD/ BE () w 2 a_ -0 I[) ('. Figure 34. Quantitative and qualitative analyses of the elution of vanadyl porphyrins from columns of alumina. (a) Concentration versus fraction,by elution volume. (b)Coplot of percenet DPEP or weighted average mass(X) versus elution volume.(c)Co-plot of percent benzoporphyrins or the benzo-DPEP to benzo-ETIO ratio versus elution volume.Mass spectral indices given in Table 23.Qunatitation as given in Table 16. 1-" N 1-"

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122 Table 7 Isotopically corrected, averaged and no rmalized low voltage (8-12 eV) mass spectr a for chromatographic fraction s o f t h e 'main' vanadyl porphyrins from Boscan asphaltenes(1). (2) :t of(3 ) (4) CWDI INm1SnY( 5 ) nv.cnoo 1Ul'AL SERIES C-25 C-26 C-27 C-28 C -29 C-JO C-31 G-32 C-33 C-34 C-35 C-36 C-37 C-38 C-39 C-40 C-41 Al-1/3 4 DPEP 0.7 3.0 12.5 22.7 39.7 45.6 44 0 51.4 63.8 90.0 100.0 90 8 39.4 17.1 7.3 0.3 0 6 ( N-18) ETIO 1.6 6 0 9.2 12.8 18.1 28.0 42. 2 52.9 36.5 19. 7 J.S.3 7.3 2.8 1.1 80 1.2 1.3 2 0 3.2 4.2 4.8 3 4 3 7 2.2 5.1 6 2 3. 7 tr tr BE 0.5 2.8 5 8 5.3 8.5 6.1 3.9 5.6 6.3 7.8 12.5 8.7 tr tr Al-4 4 OPEP 9.2 19.9 34.6 56.1 52.2 100.0 72.7 50.7 J.S.9 11.4 tr tr tr tr ( N-10) rno 7.1 12.8 .x>.9 50.9 79.9 63.4 38. 7 20.9 8.6 2.4 80 8.1 2.9 3.8 BE 2.2 3.3 5 8 9.3 5.2 2 4 Al-5 21 D PEP 0.8 5.6 16.2 36.6 43.6 45.5 59.8 82.6 100.0 78.7 40.0 23.5 14 4 11.1 6.3 2.9 1.7 (N 24) ETIO 3.2 12.5 26.6 48.5 86.0 95.2 65.6 34.7 19.4 9.3 4.3 l.l 0 .7 0.4 80 0.6 1.8 1.5 0.9 1.5 1 0 1.3 0.7 0.4 0.3 BE 1.3 2.5 2.1 2.0 l.l 1.7 1.3 1.2 l.O 0.2 Al-6 25 DPEP 0.9 4.3 8.2 16.2 27.9 39.5 53.1 100. 0 55.3 31.3 12. 2 3 4 0.9 0.3 0.2 (N 16) ETIO 0.6 2.6 8. 7 18. 5 32.3 35.0 23.3 8.0 3.5 1.6 1 0 BD 0.3 2.3 1.0 BE 1.1 1.1 1.9 1.3 Al-7 21 DPEP 1.5 4.2 21.8 27.3 59.7 77.5 100.0 31.0 8.9 0.7 (N-18) ETIO 1.9 4.9 l2.B 13.1 12.6 1.9 80 1.9 5.4 0.3 BE 1.7 1.4 0.2 Al-8 13 DPEP 0.1 0.9 3 8 24.6 35.9 51.6 100.0 45.2 12.2 2.6 (t+-26) ETIO 0 2 l.O 4.0 6 1 9.2 2.4 80 0.3 2.6 9 1 1.2 BE 0 1 0.8 1.1 0.6 Al-9 6 DPEP 8.6 53 9 99.5 99.5 100.0 55.7 11.7 ( N-23 ) ETIO 8.5 7.6 11.1 1\1) -U.2 16.2 u.s BE Al-10 6 DPEP 0.5 6.8 42.8 68. 4 100. 0 80.2 25.0 9.0 2.1 (N-47) mo 0.2 2.6 6.5 3 4 1 1 0 9 0.6 0.4 0.1 BD 3 5 8 7 18. 6 12.6 5.9 2.7 8E 2.1 2.0 2.1 1.6 Al-(4-9) 90 OPEP tr 0.5 3.2 19.4 47.6 89.6 100 0 48.8 26.8 12. 0 4.9 2 4 tr POOLED rno 2.0 8 3 17.1 29.3 32.8 23.7 14. 2 6.8 2 9 0.7 (N-36) BD 0.8 3.0 7.2 2.6 1.6 0 4 tr BE Al-( 4) 90 DPEP 0.6 3.7 10.0 31.1 44.8 61.6 84.4 100.0 64 6 40.2 18. 7 9.0 5.2 3 4 2.1 0.9 0.4 CALC. ETIO 0.2 2.5 9.3 19.4 31.7 42.6 39.0 26.1 11.6 9.9 4.1 1.8 0.4 0.1 0.1 BD 2.4 6.0 2.2 0.3 0.4 0 4 0.4 0.1 0.1 0 1 BE 0 4 1.6 2 4 1.9 1).6 0.3 0.5 0.4 0.3 0.3 fCXJ'lN)'IU : or cu t ' represents the vanadyl porphyrins \lhich did no t elut e frao silica ge l vith benzene alone but rather required benzene/ethyl ac:.etate(3:1, v/v) for elution .Ear1y eluti" YaMdyl porphyrins(re silica cohmu)...,re enriched in the benzoporphyrin series( ca. 23.4:t,wt.) relative to fraction(ca.5.21,wt.). (See text). 2) Fractions eluted froo neutral alunina(lloelm Grade II-III) vith incrrasing percentages of awthylene chloride i n benzene(See text). Parenthesized value of "N" ref us to the nuobu' of mass S!*'tral scans collec:ted and .....,r13ed for each fraction. "Al-(4-9) POOLED" irdic.ates a sample made up of fractions Al-4 through Al9,,following the physical (re-)pooling o f 5.1f!le. "Al-(4-9)CALC." refers to a calculated distribution arrived a t by the ""i&thed addition of the irdividual mass spectra for these sePI'rate frxtions. ''Veighting" ,.... porformd via W/VIS usingt.M 26.14 at 572tn 3) Percent of total was c:aleulated using W/VIS ,as &ivan in footnote #2. 4 ) Series ablxeviations are as follows: DPEP-deoxophy lloerythroetiopolllhyrin(alt.CAP-o cyeloalkanoporphyrln.c:.f. et al. 1989),ETIQo etioporphyrin,BD-benzoETIO(See text,c:.f.Ba u r ll al.,1967) ---5) Normalized intensities of mass spectral peaks are reported ......,.t 'IOn,isotoplc:. correction ard nonnalization,vith the rrost intense peak equal to 100.0. Carbon nunber derived froo nc8inal mas s s eries for e ach oortlhvrln tYCe.

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. TOTAL 4 4 21 25 21 13 6 FRACTION t.FRAC .4 .. A .A. .A. .A ,.,A.,,,.,.,, 24 28 32 36 40 44 CARBON NUMBER(D) ALKYL PORPHYRINS 4 5 7 8 = g ,.......,......, I (,-, -,-., --.-6 rill II A II II ffi I ,.,.,,,,,,.,.,,, 24 28 32 36 40 44 CARBON NUMBER( BD) BENZ-PORPHYRINS 5 123 Figure 35. Mass spectra of Boscan asphaltene vanadyl porphyrins as separated into ten fractions by chromatography over alumina(See Fig. 29, 'MAIN").Benzo-porphyrins (right hand series)were normalized alone for clarity. (See T!'lble 7).

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Table 8 Mass spectral indices derived for chromatographic fractions of vanadyl porphyrins from Boscan asphaltenes. MASS SPECTRAL INDEX(J) <2> x0 1 XE I <1; xBol CBo XBE" CBE x(D+E) %BENZ(4)BDIBE 7. Bo(5 ) D IE(6 ) 7.DPEP(6 ) A l Al-1/3 4 558.2/33.23 550.1/32.51 570.6/34.54 575.6/34.76 555.9 11.5 0.56 35.9 2 .48 71.3 Al-4 4 550.8/32 .70 546.1/32.22 565.3134.16 548.9/32.85 548.8 5.5 0.53 34.6 1.34 57.3 Al-5 21 544.7132.26 5 26.9130.85 586. 9135.71 587.2135 .59 537.3 2.4 0.69 40.8 1.40 58. 3 Al6 25 529.9131.2 1 510.1/29.65 551.7/33. 1 9 545. 8/32.63 524.4 1.8 0.67 40.1 2.62 72.4 Al-7 21 526.5130.96 497.5128.75 546.0132.79 544. 6132.54 522.9 2.8 2.30 69.7 7 .OS 87.6 Al-8 13 519. 9/30.49 491. 5128.32 546.9 132.85 548.8132 .84 517.7 5.0 5 .08 83.6 12.09 92. 1 Al9 6 513. 4130.03 488.3128.09 558. 7133.69 ----nd-----511.9 8.1 .s. .S.100.0 15.77 94.0 Al-10 6 512.6129 .97 496.1128.65 542.3 1 28.65 528. 7134.41 511.8 1 5 1 6.97 87.5 21.19 95.5 Al-4/9( 7) 90 537.9131.78 513.9/29.92 551.8/33.20 ----nd-----531.3 3.1 < <100.0 2.67 72.8 (pooled) --Al-419( 8) 90 (calc) 534.2/31.58 521.5/30.46 552.0/33.21 556.9/33.42 530.5 3.0 1.45 59.2 2.42 70.8 FOOTNOTES: 1) F ractions as collected from chromatography over n eutral alumina(Woel m grade II-III)and more fully described in Table VII. 2) Percent of total refers a total of 100.07. for fractions 1 through 10,inclusive(see Table VII). 3) Mass spectral indices calculated as described latter in Table XXIII. 4) Percent Benzoporphyrins refers t o the percent Benzoporphyrins in the fractio n in question. 5) Percent benzo-DPEP series and BD/BE were calculated using BD+BEc100. 0. 6) DPEPto-ETIO ratio,percent DPEP and alkylation index were calculated using only the DPEP and ETIO series,That is, the benzo-porphyrins were not factore d into these indices. 7) Data pre sen ted represents that determined on a physically (re-)poo led isolate including fractions 4 through 9,inclusive. 8) Data presented represents a mathematically combine d or 'expec t ed' i so late derived by the weig hted addition o f fractions 4 throngh 9 using lN/Vl S( 26. 14) f o r the calculation o f quantity('welghting'). 1. 1. 1 0.1 o.: 0. 0.1 0.1 o.c o .:: 1-6 N

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125 Quirke et g}., 1980a) and this alkyl nature most likely masks the polarity imparted by the vanadyl oxygen. Further, lower carbon number species, most likely arising by dealkylation mechanisms (Baker and Louda, 1983, 1986a-b), are known to contain open-B positions (see Fig. 3) and to exhibit enhanced polarity upon chromatography (Hajibrahim et gj., 1981; Louda and Baker, 1981; Quirke et gj., 1979, 1982). Second, the OPEP-series exhibits higher polarity and elutes after the ETIO-series. This fact has been shown previously for silica based chromatographic media (Alturki et gj., 1972; Hajibrahim et gj., 1978, 1981; Quirke et gj., 1979, 1982) and is reported here for alumina (Figs. 34b, 35; Table 8). Third, of the four main porphyrin series (0, E, BO, BE: Table 8), the VO-benzo-OPEP series exhibits the highest chromatographic polarity on alumina. It has been reported that the benzo-porphyrins (''rhodes") are the least polar species when chromatographed as free-base derivatives over silica (Barwise and Whitehead, 1980; Hajlbrahim et gj. 1981). As shown herein (Figs. 29-33), and indicated in one previous report (Barwise and Whitehead, 1980), the least polar VO-porphyrins are those of the benzo-ETIO .series, allowing for alkylation differences among the various series. Finding that the VO-benzo-ETIO series is relatively non-polar upon silica or alumina chromatography, while the corresponding benzo-OPEP series exhibits enhanced retention on alumina, allows an effective split of these two types to be made. Examination of Figure 34c clarifies this statement. That is, the early eluting fractions are enriched in the VO-benzo-ETIO series while the VO-benzoOPEP pigments are retained and elute last.

PAGE 154

The order of vanadyl porphyrin series elution from alumina was found to be: benzo-ETIO, ETIO, DPEP, benzo-DPEP. However, this elution order does not yield complete separat ion of each series from the others as overlap does occur along this pattern. As discussed above, differences in both the degree and extent of peripheral alkylation and the presence of open B-positions serve to modify the chromatographic mobility of the various porphyrin skeletal series. 126 The patterns of chromatographic mobility found for geologic VOporphyrins clearly reveal that there is a large potential for the analytical truncation of these naturally heterogenous arrays. Previous discussion, therefore, mandates that for each individual analysis, all eluates must be assayed for both the quality (Nivs. VO-) and quantity of metalloporphyrins present. Further, only by the pooling of all pigment containing fractions, after a complete N i -/VO-porphyrin split, can the true nature of each array be ascerta in ed. These results and c onclus ions were incorporated into the finalized scheme (Fig. 27) for the analysis of Ni-and VO-porphyrins i n bitumen. Aside from obtaining a single final isolate ('pool') for a certain metallo-porphyrin (Nior VO-) it is theoretically possible to reconstitute an overall array from individual fractions. In o r der to test the validity of such a practice, fractions Al-4 through Al-9 from the Boscan VO-porphyrin study (Figs. 29, 35; Table 8) were pooled, yielding 'Al-4/9 POOL.' The averaged mass spectrum of this 'POOL' is given as Figure 36a. In addition, a reconstituted overall MS was calculated (Fig. 36b) using the quantitative electronic spectra l data (Tables 7-8) as the weighting factor for the summation of the individual mass spectra of fractions Al-4 through Al-9 (Tables 7 8). Fractions Al-l

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a) b) c) d) -I / / fj /i 127 ----Figure 36. Comparison of mass spectra obtained on repooled vanadyl porphyrin chromatographic fractions(a) and that obtained by the weighted reconstitution of same(b) using the individual mass spectra for each fraction(See Figure 35 and Table 7). Solid line= DPEP series,Dashed line= ETIO series. (c)Overlay of DPEP series from a '(solid) and 'b' (dashed). (d) Overlay of ETIO series from 'a'(solid) and 'b'(dashed). Fractions were via quantitation as given in Table 16 and mass spectral data given in Table 7.

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through Al-3 (Al-1/3) and Al-10 were not added to this pool, since future studies on these chromatographic end-members are planned. 128 Comparison of the resultant MS for the VO-porphyrin pool and that calculated from its composite chromatographic fractions can be performed both graphically (Fig. 36) and numericall y (Tables 7-8). In all cases, the reconstituted MS (Fig. 36b) yields an augmented presence of the non-major components. For example, the higher carbon-number members (C39+) of both the DPEP-and ETIO-series, enriched in fractions Al-4/5, were not detected in the 'POOL' (Al-4/9: Table 7, Fig. 36a). This is attributed to a dilution of their abundance (importance ?) in the to such an extent that these s ignal s were lost i n the mass spectrometer background. The presence of C38-C41 members in the DPEP-and ETIO-series of fractions Al-4 through Al-6, and their absence in the pool of Al-4/9 (Table 7), also lead to a higher alkylation inde x (AI: Table 8) for the reconstituted MS, relative to the pooled array. Comparing the histogram outlines for both the DPEP-(Fig. 36c) and ETIO-series (Fig. 36d), this enhancement of the importance of minor components resulting from the reconstitution of fractions i s a lso evident All of the above data and discussion lead to only one conclusion regarding the isolation of VO-porphyrins from geologic samples. That is, the entire array of vanadyl pigments must be isolated and 'fingerprinted' (MS) as such. If this is not done, then 'heart cutting' and/or reconstitution from sub-fractions can only lead to erroneous analytical results. This becomes even more important when dealing with samples and sample-suites for which valid intercomparisons are required. The need for a standard technique for both the isolation and

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129 'fingerprinting' of geologic porphyrin arrays has also been addressed elsewhere (Louda and Baker, 1987, 1990). LPHPLC LPHPLC, low-pressure high-performance liquid chromatography, was investigated as a potential method for the separation of the myriad of chlorophyll derivatives encountered in earlyto mid-diagenetic sediments. The main goal of this facet of the present studies was to develop techniques which would allow the separation/purification of dihydroporphyrins in sufficient quantities such that identification, if only tentative (g.g. chromophore typing), by a battery of non-destructive and destructive physicochemical tests could easily follow. In addition, given the lack of conventional HPLC for the present study, a chromatographic system with higher resolving power and lower detection limits than column chromatography for use with porphyrins and metallo porphyrins was needed. LPHPLC, then, was designed to follow previous chromatographic separation of tetrapyrroles by gross polarity differences. In this manner the potential resolving power of LPHPLC could be optimized for subtle rather than strong structural differences. The LPHPLC system employed is shown diagrammatically in Figure 37. Solvent is delivered {50-200 psig:300 psig max) via an explosion-proof {Fluid Metering Inc., Model-FXP) pump. Columns and column-fittings were 8 x 250 mm (ID) glass and Teflon, respectively, Michel-Miller style {Ace Glass) with a septum port allowing injection directly onto the head of the adsorbent bed. Eluates were monitored with a variable {190-700 nm) wavelength detector {Laboratory Data Control, Spectra monitor III) coupled to a strip chart recorder (Houston Instruments,

PAGE 158

PUMP PRESSURE GAUGE SYRINGE SHIELDS COLUMN 8x 250mm 13-24um SILICA Figure 37. Schematic of the low-pressure high-performance liquid-chromatographic (LPHPLC) system used in the present studies. PUMP= Fluid Metering #EXP-l,COLUMNS= Ace Glass Inc. Michell-Miller system,DETECTOR= LDC Spectromonitor-II,RECORDER= Houston Instruments 'Omniscribe'. .... w 0

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131 Omniscribe). Columns were packed with 13-24 silica gel in regular (Whatman stock #LPS-1) or C-18 reverse (Whatman stock #LRP-1) phase at 220-250 psig until further compaction of the bed ceased. Early trials using 175 alumina, while affording an easily monitored system (Louda and Baker, 1981), were not continued, as separations were poor. The LPHPLC system described above (Fig. 37) was used only in isochratic and step-gradient modes. Trials with continual gradient evolution, such as solvent B flowing from the bottom of an Erlenmeyer flask into solvent A in a straight sided bottle (i.g. concave) for delivery, were soon discontinued as repeatability was lacking. The LPHPLC system developed and usedduring the present investiga tions was run in essentially three ways. First, activated silica gel yielded regular, alternately 'normal', phase separations. Second, storage of silica gel columns in solvent mixtures with high volume percentages of methanol (g.g. acetone:methanol, 1:1, v/v) yielded "methanol deactivated silica" (cf. Purcell, 1958). This, essentially regular phase, column adds the dimension of liquid:liquid partition to strightforward adsorptive chromatography and, as will be shown, was most useful in the dissection of non-polar tetrapyrroles (g.g. cellulose fraction 1:"CELL-1'' in Table 4 and text). Third, octadecyl silane (C-18) bonded silica afforded reverse phase (aqueous acteone and/or methanol solvents) and non-aqueous reverse phase (NARP: solvent regimes without water) separations. Though numerous test mixtures of authentic pigments (Appendix A) and solvents were screened only a few shall be reviewed here. LPHPLC was also a primary tool used in determining the purity of standard tetrapyrroles (g.g. Figs. A3, A10, A17: Appendix A). The following

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132 LPHPLC experiments were chosen to serve as examples both of the separation of a variety of structurally different known tetrapyrroles and certain analytical data gathered during studies on marine algae and sediments, as detailed later (Chapter 4). During senescence, cell death and sedimentation it is well known that the chlorophylls -a [I] and -b [XIV] rapidly loose Mg to yield the corresponding pheophytins [IIa, XV] (Daley and Brown, 1973; Hendry et gl., 1987; Jeffrey, 1974; Louda and Baker, 1986; Shuman and Lorenzen, 1975). Further, herbivory of phytoplankton has been reported to involve phytyl ester cleavage and to yield almost solely pheophor bides as the chlorophyll derivatives in zooplankton fecal pellets destined for subsequent incorporation into surface sediments (Brown et gl., 1977; Currie, 1962; Daley, 1973; Hallegraeff, 1981; Lorenzen. 1967 a-b; Newton-Downs and Lorenzen, 1985; Schuman and Lorenzen, 1975). Thus, in the course of study on early chlorophyll diagenesis one can quite reasonably expect the presence of, at least, the following pigments: pheophytin-a [IIa], pheophytin-b [XV], pheophorbide-a [VIa], a variety of interferring carotenoids (g.g. Baker and Louda, 1982; Repetta and Gagosian, 1982) and several analogs of the above tetrapyrroles which have lost the C-10 carbomethoxy moiety (g.g. [III, VIII, X] i g. pyro-dihyroporphyins. Fig. 3 and text: see Baker and Louda, 1982; Keely and Brereton, 1986; Louda and Baker, 1986). These and selected other standards were therefore used as chromatographic probes to refine the LPHPLC system for use in the investigation of senescence and early chlorophyll diagenesis Several HPLC methodolgies have been reported for the 1-step separation of chlorophylls (-a [I], -c [XIX]). pheophytins (-a [IIa]).

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133 pheophorbides (-a [VIa], -c [XX]) and coincident carotenoids {Abaychi and Riley, 1979; Bidigare et gJ, 1985; Brauman and Grimme, 1979; Brown et gl., 1981; Eskins et gl., 1977; Furlong and Carpenter, 1988; Schoch et gl., 1978; Vernet and Lorenzen, 1987). While these represent great strides in the study of mixed layer and overall water column chloro phyll dynamics, it is the opinion of this author that no single step chromatography can possibly handle the myriad of structures encountered during early diagenesis. Though the analytical attack on early diagenesis has just begun, certain inroads have been made. As detailed early diagentic chlorophyll derivatives are easily split {Table 4) into; {CELL-1) phytyl ester (''pheophytins") and non-polar (i.l. decarboxylated) species, (CELL-2) mono-carboxylic acid dihydroporphyrins, and (CELL-3) di-/tri-carboxylated pigments plus chlorophyll{-ide)-c [XIX, XX]. Reverse-phase purification of pheophytin-a [IIa] is detailed in Appendix A and can even discriminate between the number of double bonds in the diterpenoid ester moiety (Fig. A3). Given that chlorophyll{s)-c [XIX] and carboxylated chlorophyll derivatives {pheophorbides, chlorin and purpurins) will be found in the CELL-2 and CELL-3 fractions, then the least complex mixture of pigments one might encounter in CELL-1 would include pheophytin-a [IIa] and pheophytin-b [XV]. In addition to these dihyroporphyrins, the interferring presence of certain carotenoids, all of which co-elute in CELL-1, must be dealt with This first and most simple case was studied with a mixture of pheophytin-a [IIa], pheophytin-b [XV], carotene, echinenone, zeaxanthin/lutein and a small group of oxidation products (mutatochromes: see Baker and Louda, 1982} found in

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134 commercial (Roth, F. R. G.) a-carotene. Numerous solvent mixtures and step-gradients were tried with this mixture until repeatability was achieved for a run lasting less than one hour, an arbitrary maximum decided upon during these studies. Figure 38 is a sample LPHPLC separation of the above test mixture. Time and band widths could be appreciably shortened without loss of resolution with other solvent regimes. However, as will be shown, by allowing considerable free space on the column, the presence of other pigments in more complicated geochemical mixtures is more easily noted Following development of a workable LPHPLC system, studies on algae and sediments, the results of which are covered in the next section (Chapter 4), followed. During studies on the CELL-1 fraction from extracts of algae (phytoplankton), sediment trap or sediment samples, it was often necessary to make two LPHPLC runs. The first, or trial, run was as given above. The results from this trial gave an indication of the pigment diversity and guided minor changes in the solvent system needed to affect better separation. Mild acid (HCl) treatment of the extracts of diatoms (Fig. 39a) and unicellular green algae (Fig. 39b) resulted in CELL-1 fractions yielding LPHPLC chromatograms essentially the same, but lacking pheophytin-b [XV] as present in the authentic test mixture (Fig. 38). The lack of pheophytin-b [XV] in the case of the green phytoplankter is apparently a case of species specificity, as chlorophyll-a [I] to -b [XIV] ratios for unicellular members of the Chlorophyceae fluctuate widely (Jeffrey, 1976, 1980).

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w (f) z We O::LO N 0 II r-t< u w r-w 0 0 10 20 30 40 TIME, min. I 5/oACE/ PE Figure 38. Chromatogram obtained during a test of the LPHPLC system using authentic Bcarotene(l), pheophytin-a[Ila]-(2) and pheophytin-b[XV]-(3) to simulate the range of 'non-polar' pigments obtained as Fraction #1 from chromatography over cellulose(See Table 4). f-" w V1

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0.64 I o:osl 0.32 AUFS 0 .161 0.08 I 0.32 I 0.16 (a) I E ( b) 3 Cl 0 2 1 ..-II r< ......., I 3 z l/) w l 1 0:: 2 5 0:: I I 4 0:: 0 u 4 w 0:: TIME,minutes r1 1 2 3. 5 2 3. 5 5 7. 5 1 0 ACE TONE(%, v) IN PETROLEUM ETHE R(30-60) GRADIENT STEPS Figure 39 LPHPLC Chromatograms for cellulose fraction-l(See Table 4) pigments obtained from acidified extracts of fresh viable axenic cultures of (a) the diatom Synedra sp. and (b) the unicellular green alga Closterium sp .. Pigment code:l= 8 -carotene, 2= dehydro-and retro-dehydro-carotenes, 3= pheophytin-a[Ila], 4= monohydroxy carotenoid, 5= lutein/zeaxanthin. f-0 w (j\

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137 Utilization of the present LPHPLC system with early diagenetic sediments militated not only minor solvent changes but alteration of extract treatment as well. As detailed previously in text, examination of the electronic spectra of crude extracts (Fig. 20 and text) and experience with such data are valuable tools in choosing subsequent separation schemes. Thus, while the LPHPLC studies of CELL-1 fractions from extracts of the two deep sea sediment samples discussed below are still comparable to the known pigment {Fig. 38) and algae extracts (Fig. 39) on the basis of dihydroporphyrins, the entire pigment complements of CELL-1 are not. Figures 40a and b are the LPLHLC chromatograms from sediments recovered from 15.0 and 180.6 meters sub bottom, respectively, within the oxygen minimum zone of the Guaymas Basin, Gulf of California (z = 747 m; DSDP/IPOD Leg 64: see Baker and Louda, 1982; Curray, Moore et gJ., 1982). Pretreatment of sample 64479-3-2 (Fig. 40a) involved extraction of the crude extract in benzene/ petroleum ether (ca. 1 : 5, v/v) with 90% aqueous methanol (Fig. 17 and text) to remove the majority of carotenols. Pretreatment of sample 64479-13-1, found to contain free-base porphyrins during examination of the UV/VIS spectrum of the crude extract (cf Fig. 20c), included three extractions of the extract in ethyl ether solution with 2.5% HCl (w/v, aqueous: Fig. 21, Table 3). Peak identifications are included in the legend of Figure 40 and, as this section deals with the LPHPLC technique, geochemical implica tions are discussed in the following chapter {Chapter 4). Sample 64-479-3-2, 15.0 meters sub-bottom, yielded a LPHPLC chromatogram {Fig. 40a) very similar to what one would expect for an extremely immature marine sediment of diatomaceous, plus assorted herbivore (g.g.

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E c: 0 .< w tn z 0 0.. Vl w a: cr w 0 cr 0 u w a: -a: 0 '"" u w '"" w 0 1 2 3 7 5 12/. j3.05/o I 15 1 12 ,1.1, 10 0 I 0 5"/o 1 1 "/o I 2 / o j3 .5"/o I I I I I I I j 7 5/o I I I 70 90 4 '10 30 50 TIME, minutes X "I = "I ACETONE IN PETROLEUM ETHER SOLVE NT I 110 ( a ) ( b ) 1 38 Figure 40. LPHPLC chromatograms of the cellulose fraction -1 pi&ments from extracts of marine sediments recovered at 15.0 (a) and 180.6 (b) meters sub-bottom in the Guaymas Basin(DSDP/IPOD Le g 64: Se e Baker and Louda,1982; Curray, Moore et al.,1982). Peak identifications; 1= B-carotene, 2= peryYene, 3= dehydro-carotenes, 4=pheophytin-a[IIa], 5= pyropheophytin-a[III], 6= phytylated purpurin-18(see[XXVI]), 7=fucoxanthinols,8= 5,6-/5,8-carotenoid epoxides,9=copper porphyrins,10= decarboxylated DOMPP-a(see XII]), 11= functionalized perylenes(see Louda and Baker,1984),1 2=decarboxylated nickel phylloeyrthrin[LXXVIII],13=meso-and pyrrorhodin-like pigments,14=decarboxylated mesopyropheophorbide -a(see[X]),15=unidentified 9-keto-phorbide.

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139 zooplankton), organic input. In this case, operation of the LPHPLC system was able to include a sol vent profile similar to that used with known m ixture (Fig. 38) and unispecific phy top lank ton (Fig. 39) test runs. Once early chlorophyll diagenesis has progressed to a stage i n which free-base porphyrins are just beginning to f o rm, via a r o matization of dihydroporphyrin precursors, pigment diversi ty is found to b e e x ceedingly high. Figure 40b, the LPHPLC separation of C E LL-1 f rom a diatomaceous sediment of 180.6 meters burial d epth, reveals this complexity. E x amination of Figure 40b, not ing d i f ferences in solvent changes when compared to Figure 40a, serves an e xample o f how and why a single chromatograph i c regime cannot handle a l l cases. I n the present case (Fig. 40b), increases in solvent pola rity ( v i z. per centage of acetone) needed to be m i lder and temporally l e n g thened. O f the 20 or so compounds ('peaks') i n this chromatogram, at l east 1 5 are tetr apyrrole pigments and these were just those present in the non-polar (phytylated or decarbo x ylated: CELL-1, Table 4 and tex t ) fraction. As inferred above, the complement of d i h ydroporp h y r i ns, and in some cases porphyrins, in a sediment includes polar (carbo xyl ated) species as well as the non-polar phytyl ester or decarbo x ylated forms. The tetrapyrrole carbo xylic acids, isolated either as CELL-2/-3 fractions (Table 4) or from chromatography over s i l ica (Fig. 27), are converted to their methyl ester form with BF3-methan ol ( Chapter 2) prior to LPHPLC separations. In the case of tetrapyrrole acid methyl esters, both regular and reversed phase separat ions were devel oped. Figure 41 contains LPHPLC chromatograms obtai ned over 'regular' phase silica.

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........... (a ) E c 0 II t< .........., w (/) z 0 0.. (/) w 0::: wW 0::: ( b) w CllW a.. a.. 0 a..o 0::: 2 0 0 f:d 0 0::: 0 10 w 2 Cll a.. a.. a. E 40 w w2 2 Cll Cll a.. a_ a.. a.. a. w 2 .0 a.. a.. 20 TIME, min. 140 60 80 30 40 Figure 41 LPHPLC chromatograms of porphyrin/dihydroporphyrin test mixtures using normal phase(i.e.adsorptive)silica gel.(a) Solvent= isochratic 3.5% acetone in petroleum ether.(b) Solvent= hexanes/methylene dichloride, 3:1,v/v.Pigment abbreviations given in text and Appendix A

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141 In the first case, Figure 4la, relatively good separation of this simple three component mixture was achieved with isochratic acetone (3.5%, v/v) in petroleum ether. Ignoring pyropheophorbide-a ME [VIII] for now, this system was developed for the separation of deoxomesopyropheophorbide-a [XII] and deoxophylloerythrin [XXXVIIb] methyl esters. In geologic samples these latter two pigments, as well as their decarboxylated forms (7,8-dihydro -DPEP and DPEP [XXXVIII], respectively), usually co-exist and are difficult to fully separate by aqueous HCl-ethyl ether partition. Thus, an LPHPLC method specific to the study of the aromatization reaction in tetrapyrrole diagenesis was needed and obtained. The run shown (Fig. 4la) was performed with a larger scale column (22 x 130 mm) than used in purely analytical (8 x 250 mm) separations and was designed for in vitro aromatization studies. In the second example (Fig. 41b) a more complex mixture of standard tetrapyrrole acid methyl esters was assembled for separation trials. Here the six pigments, in order of elution, were deoxophyl loerythrin [XXXVIIb], deoxomesopyropheophorbide-a [XII], meopyropheophorbide-a [X], pyropheophorbide-a [VIII], pheophorbide-a [VIb] and pheophorbide-b [XVII], all as the methyl esters. The study of fraction CELL-2 pigments (Table 4) is still underway for geochemical isolates and the above data shall be used in initiating an HPLC study of geologic dihydroporphrin acids. It should be noted that the order of elution of OPE-ME [XXXVIIb] and DOMPP-a-ME [XII] from methanol deac tivated silica switches between the run with acetone/petroleum ether solvent (Fig. 41a) and that with methylene dichloride/hexanes (Fig.

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41b). This fact will also aid future HPLC studies dealing with the aromatization reaction. 142 One reaction believed to be of great importance in the diagenesis of chlorophyll is the so-called 'pyro' reaction (Louda and Baker, 1986; Treibs, 1936). That is, the loss of the 'carbomethoxy' (methyl formate) moiety from the C-10 position on the exocyclic ring (see Fig. 3). Thus, a LPHPLC method was developed to study this reaction as well. In this case, Figure 42, development of a separation through alteration of the amount of solvent (acetone) modifiers (methanol/ water) is demonstrated. In the last case (Fig. 42b), doubling the column length did afford complete separation but also quadrupled analysis time. Thus, for 'routine' assessment, the system shown as Figure 42c is more than adequate During all of the above, and other, LPHPLC experimentation, it was found that chromatography of phytylated pigments (pheophytins [IIa-b], [XV]) over reverse phase silica with 3-5%+ water as a solvent modifier lead to excessive (>60 min.) retention times. This fact was incor porated into the present analyses as a test for the presence ('retention') of the phytyl moiety. The preceding has dealt with the LPHPLC of dihydroporphyrins, emphasizing fraction CELL-1 (Table 4) pigments. LPHPLC was also examined as a tool for the separation, isolation and/or purification of porphyrins per se Fig. Al7). The majority of authentic standards given in Appendix A were either purified or checked for purity via LPHPLC analysis. In dealing with freebase or metallo-porphyrins it was found, as given for HPLC

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El 2 c 0 (a) II .< w Vl z f2 Vl w a:: a:: w 0 a:: 0 u w a:: 0 5 10 2 (b) (c) 2 0 5 10 15 TIME,mln. (d) 2 ..,--,,--.,.--y--,---, 0 10 20 30 40 50 60 Figure 42. Reversed phase LPHPLC chromatograms obtained during the development of a system with which to separate pheophorbide-a ME(peak#l [VIb]) and pyro-pheophorbide-a ME(peak #2 [VIII]).Column = 8 x 250 mm,C18 silica(Whatman LRP-1,13-24 um).Solvents: (a) Methanol/acetone, 95:5 ; (b) Methanol/acetone, 90:10 ; (c) methanol/acetone/water, 90:5:5 ; (d) as 'c' but with 8 x 870 mm column length. ........ .j:'w

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144 (Hajibrahim et gl., 1978, 1981; Sundararaman, 1985), that reverse-phase chromatography is superior to 'normal' adsorption techniques. Table 9 contains the retention times for selected free-base, nickel and vanadyl porphyrin standards. As given in the legend of Table 9, all of these were run under identical conditions such that elution trends would be comparable between authentic standards and geochemical isolates. Trends which emerge from the data in Table 9 follow here. Comparing free-base porphyrins, whether alkyl (g.g. [L], [LI]) or acid methyl esters ([XXXVIIb]), with nickel ([LXXXVIb], [CXa] ) and vanadyl ([CVII]) chelates, it is found that complexation with nickel greatly enhances the lipophilicity or alkyl nature of the ligand, while the reverse is true upon insertion of the vanadyl radical. Examination of these data for a suite of free-base ETIO-type (Fig. 2) porphyrins ([L], [LI], [LII], [LVII] Table 9) reinforces the fact that the more highly alkylated homologs within a porphyrin series exhibit increased retention on reversed phase (octadecyl silane bonded silica) media. Among the various porphyrin skeletal types (Fig. 2), using the known vanadyl pigments ([CXVIII], [CVII], [LXXXVIII]: Table 9) as a guide, the order of reverse phase chromatographic retention is found to be: BENZO < ETIO < DPEP. This follows the order given by others (Barwise and Park, 1983; Hajibrahim et gl., 1978, 1981; Quirke et gl., 1982; Sundararaman, 1985) for free-base and vanadyl chelates alone. As true HPLC techniques for the analysis of vanadyl porphyrins, either as the native pigments (Sundararaman, 1985) or as demetallated (free-base) derivatives (Kaur et gl., 1986; Verne-Mismer et gl., 1986), were appearing during the course of these studies, the present LPHPLC

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Table 9. Retention behavior of free-base, nickel and vanadyl porphyrins during reverse phase LPHPLC(1). COMPOUND2 CODE [XXXVIIb] see[LVII] [LVII] [LI] [LII] [L] [CXVIII] [CVII] [LXXXVIII] [LXXXVIb] [XC] [CXa] FOOTNOTES: Deoxophylloerythrin-ME Tetramethylporphyrin Deuteroetioporphyrin-IX Etioporphyrin-! Etioporphyrin-III Octaethylporphyrin VO-'benzoetioporphyrin' VO-Etioporphyrin-I VO-Deoxophylloerythroetioporphyrin Ni-Deoxophylloerythrin-ME Ni-Deoxophylloerythroetioporphyrin Ni-octaethylporphyrin 7.4 4.2 7.0 10.4 10.3 12.3 5.4 7.2 8.0 19.0 24.3 23.3 145 1)Low Pressure High Performance Liquid Chromatography(RP-LPHPLC) system diagramed in Figure 37.COLUMN 8 x 250mm;PACKING,Whatman #LRP-1,C-18(0DS)silica gel(12%C acetone/methanol/water(90/5/5 v/v/v) at 5.1 mLmin (150 psig); INJECTION;Sample dissolved in minimal acetone,diluted with acetone/ methanol(1:9,v/v) and injected(150 uL)with system at 25-35psig 2) See Appendix-A. 3)Rt = retention time in minutes.DETECTION at 400.0nm.Typical halfband widths(viz.time)equaled 2 minutes.

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146 system was studied primarily as a method for the concentration of structural types prior to detailed HPLC analyses Figure 43 is the LPHPLC chromatogram of the vanadyl porphyrins from a late Cretaceous black shale (see Baker et gl. 1977). While this 'separation' is far from spectacular, given recent advances in true HPLC (Sundararaman, 1985), two important results were obtained. First, considerable cleanup of the overall vanadyl porphyrin assemblage did occur That is, non-porphyrin 'background' was lowered, as seen by electronic spec troscopy. Second, the elution trends found for known pigments (Table 9) were maintained with geologic isolates on this system. Thus, during future studies it shall be relatively easy to concentrate a structural type (g.g. BENZ, DPEP or ETIO: Fig. 2) prior to detailed and correspondingly simplified HPLC analysis. The same elution trends were found for geologic nickel and freebase porphyrins Nickel porphyrins, however, were found to be only slightly purified by RP-LPHPLC as the majority of background interferring components co-eluted. Thus, Ni-porphyrins from maturing shales and petroleum crudes continue to be isolated and purified by the demetallation/partition/re-metallation (63Cu) techniques described earlier (Figs. 23, 27). As an e x ample, the nickel porphyrins isolated from a lacustrine oil and which could only be purified to a point where subsequent mass spectra had a signal-to-noise (S/N) ratio of about 23:1 were demetallated The resultant free-base porphyrins were subjected to HCl/ether partition and then reacted with mono-isotopic copper. The Cu63-derivatives were subsequently purified via RP-LPHPLC (Figure 44) and yielded easily interpretable mass spectra with S/N values on the order of 10:1.

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,..--.. E c:: 0 II ..._., w (/) z 0 Q_ (/) w 0::: 0::: 0 I II /11 2 1 314 tJ w I-w 0 I --T 0 10 20 30 40 TIM E(min.) MeOH MeO H I H 2 0 J 9:1 Figure 43. Reverse phase LPHPLC chromatographic trial with vanadyl petroporphyrins from DSDP sample 41-368-63-2(See Baker et al.,1977).Fractions characterized as: (l)VO-B > VO-D, (2) VO-B = VO-D, (3) VO-D (4)VO-D = VO-E, (5) VOD < VO-E. Abbreviations: B = benzoporphyrins,D= DPEP series, E= ETIO series. .._. -......!

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On the basis of polarity classes, or alternately, the presence/ absence of oxygen containing functional groups, the fossilization of tetrapyrrole pigments (esp. chlorophyll[s]) is found to include a relatively simple mixture in source organisms, evolve through an 148 overwhelmingly complex melange during early diagenesis and end in a product mixture of alkyl porphyrin chelates which are easi l y separated by the nature of the metal atom (Ni2+) or radica l (V02+). Therefore, during the present geochemical study, LPHPLC was employed primari l y for the study of early diagenesis (dihydroporphyrins: see Table 4). Freebase and metalloporphyrins were mainly analyzed using modifications (Figs. 21, 23, 27) of more 'classic' techniques. Electronic Absorption Spectroscopy and Chromophore Identification Pigments, by definition, are colored substances and therefore must interact with 'light' so as to transmit or absorb incident radiation. These interactions, most notably the absorption of phota in discrete energy units or quanta, occur in very precise and wel l defined manners. So consistent is the absorption spectrum of a given compound that not only its identity ('quality') but also its apundance ('quantity') is discernable. The majority of undergraduate chemistry texts today cover, in more or less detail, the basic principles of electronic absorption spectroscopy. Review of basic principles and source of various definitions for the present study was mainly from the texts of Rao (1967) and Meites and Thomas (1958). The application of absorption spectroscopy to the study of chlorophyll, heme and their derivatives began in earnest with the studies of Willstater and Stoll (1913), in which each phase and product

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149 ,-.... E c 0 0 II t<. Es Ds ...__, w (/) z 0 a.. (/) w 0:: '63 Figure 44. Reverse phase LPHPLC chromatogram of the Cu -porphyrins derived via in vitro treatment of petroleum nickel porphyrins with methane sulfonic acid('demettalation') and mono-isotopic copper-63 sulfate.

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150 of chemical manipulation was 'spectroscopically' monitored. Aside from describing chlorophyll and its in vitro chemistry, a variety of spectral types and methods of quantitation were introduced. As cited in Willstater and Stoll (1913), the premier spectral examination of chlorophyll appears to have been that of N. A. Monteverde (Act. Hart: Petropol, 13, no. 123) in 1893. The monumental studies of the group under Hans Fischer, primari ly Adolf Stern, not only gave us modern pyrrole chemistry but defined tetrapyrrole spectroscopy in quantitative terms (Fischer and Orth, 1937; Fischer and Stern, 1940; Pruckner and Stern, 1936; Stern et . 1934-1937c). The spectral "types" of tetrapyrrole pigments, used to date in descriptive discussion, were first presented in comprehensive text by Stern and Wenderlein (1936b). Tetrapyrrole spectra have been examined and reviewed on the bases of empirical data alone (Gurinovich et . 1968; Marks, 1969; Smith, 1975), relation of Kekule-type extended HOckel chromophore formulations to empirical data (Treibs, 1973) and detailed molecular orbital (MO, Pariser-Parr-Pople) calculations aimed at complete understanding and prediction of the underlying physics (Buchler, 1978; Gouterman, 1978; Scheer and Inhoffen, 1978; Weiss, 1978). As the present treatise is directly primari ly at the gathering of empirical electronic absorption data and auxochrome manipulation for use as 'tools' in chromophore identifications with geologic tetrapyrroles, treatment of theory shall be minimized. The porphyrin chromophore has been elegantly described by Gouterman (1978) as follows:

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151 ... the electronic 'heart' of a porphyrin is the inner 16-membered ring with 18 rr electrons .... This electronic 'heart' is responsible for porphyrin-type optical spectra, which are then 'pertubated' to a greater or less extent by various chemical modifications to the basic structure." Spectral Types For the most part, each of the macrocyclic tetrapyrrole nuclei given earlier (Fig. 2) in text exhibit separate 'cardiologies' in that their "electronic hearts" are different. These core structures yield absorption spectral types which are classified on the basis of band order, extinction and relation to known structure. Figure 45 contains the four basic spectral types considered during the present work. Additional spectral types, given be1ow, follow the lead of Stern and Wenderlein (1936b: cf. Baker and Palmer, 1978) and represent a fine tuning of spectral-type nomenclature. The four sample spectra (Fig. 45) were chosen to represent the degrees of reduction (viz. hydrogena tion) and central atoms, hydrogen or metal, most common to geologic tetrapyrrole pigments. These spectra also serve as points of reference for the nomenclature specific to the various absorption bands and analytical ratios. In essence, there are two main regions of a porphyrin-type electronic spectrum, the near ultra-violet (UV) and (VIS). In the near ultra-violet to violet (350-425 nm) is the extremely intense (e 105-106M-1 cm-1 ) so-called Soret band, named_after its discoverer (Soret, 1883). The Soret band has also been classified as a strongly allowed excited (rr-rr*) "B" band by Platt (1956) and is a highly characteristic absorption feature in the spectra of macrocyclic tetrapyrroles with an uninterrupted conjugation pathway (Gouterman,

PAGE 180

152 N 0 0 -t--r--r--r--r--r--r---r--=:::j 350 550 750 350 550 750 WAVELENGTH(nanometers ) Figure 45. Main electronic absorption S?ectral types of concern in tetrapyrrole geochemistry. (b) free-base porphyrin, (c.) phorbide(alt. 'chlorin ) (d) bacteriophorbide.Band identifications discussed in text.

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153 1978; Scheer and Inhoffen, 1978). All four sample spectra (Fig. 45a-d) contain Soret band absorption and this band is to be designated by 'S' throughout this text. In cases such as bacteriopheophytin-a ([XXI] Fig. 45d), where more than one well defined Soret region maximum occurs, the present study uses arabic script for sequential numeration beginning with S1 for the peak at lowest energy (longest wavelength). Therefore, as seen in Figure 45d, S1 = 388 nm and S2 = 362 nm. However, in all cases, ratios of Soret (UV) to visible (VIS) band absorption intensities employ the band of highest extinction. In the present example, highest UV absorption is due to the S2 peak (Fig. 45d). The visible plus nearest infra-red (420-850 nm) portion of the electronic spectra of tetrapyrrole pigments contains absorption bands more highly characteristic of individual porphyrin species and can therefore be considered as a 'fingerprint' region Bands in the visible have been classified as Q-bands for quasi-allowed excitations (Platt, 1956). The most complete symmetry among tetrapyrroles is present in the {d1-d9 ) metallo-porphyrins. Here (Fig. 45a) the Q-band absorptions are usually limited to two maxima referred to as a and B for peaks at longer and shorter wavelengths, respectively. As with Soret nomenclature, the present text uses numbering from longer to shorter wavelength to identify additional or 'non-typical' peaks. Thus, the aband(s) of vandyl-benzoetioporphyrin ([CXVIII]; Appendix A) will be found herein listed as a1 = 590.5 and a2 = 581.0 nm. The subtleties and theory of metalloporphyrin spectra are better left to the reviews of Buchler (1978) and Gouterman (1978).

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154 Tetrapyrroles which are not metallo-porphyrins, per se, are considered to exhibit spectra revealing a broken overall symmetry (Gouterman, 1978). Structual (Fig. 2) and spectral types of concern, for now, in tetrapyrrole geochemist r y include; free-base porphyrin (Fig. 45b), free-base dihydroporphyrin or 'chlorin' (Fig 45c) and free-base tetrahydroporphyrin or 'bacteriochlorin' (Fig. 45d) spectra. The visible absorption ('Q') bands of the free-base por phyrins and (di-, tetra-) hydroporphyrins are, for the most part, four in number. These are identified with Roman enumeration (I-IV) from lower to higher energies, as shown in Figure 45b-c. Classically, the description of free-base porphyrins has derived from the 'quality' of the visible absorption spectrum as related to chemical and electronic structure (Stern and Wenderlein, 1936b). These spectral-structural "types'' were reviewed by Smith (1975) and by Baker and Palmer (1978). Figure 46 contains the electronic absorption spectra of six freebase porphyrins from the present study and serve here as e xamples of the various spectral types. ETIOtype spectra (Fig. 46a) are characteristic of the porphyrin nucleus (Fig. 2) with si x or more B-pyrrole positions substituted with alkyl or essentially alkyl, as in the case of non-conjugated aliphatic acids (g.g. acetic, propionic, etc.), groups. ETIO-type spectra yield visible band orders of IV>III>II> I Differences between methyl (g g octamethylporphyrin [XLIX]), ethyl (g.g. octaethylporphyrin [L]) or mixtures of methyl and ethyl (g.g. etioporphyrins-I, -III [LI, LII]) are extremely subtle at best)

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10 (a (b (c -0 .8--0 6 --0.4-0 -W02J X u 0 1.0 (e (t (d <( 0 8 0 6 0.4 0 2 0.0 I I 350 I 550 750 750 350 550 750 3 WAVELENGTH( n m) Figure 46. Electronic absorption spectra of six main free-base porphyrin types.(a) ETIO-type, (b) RHODO-type, (c) OXO-RHODO-type, (d) PHYLLO-type, (e) DPEP-type, (f) BENZO-PORPHYRIN-type. Vl Vl

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156 RHODO-type spectra {Fig. 46b) result from the conjugation of a single strong electron withdrawing group, usually carbonyl (g g aldehyde, ketone, carboxylic acid), to the porphyrin periphery. This effect occurs whether conjugation is at a B-pyrrole or at a methine bridge ('meso') position and gives a III>IV>II>I band order. OXO-RHODO-type spectra (Fig. 46c), with a band order of III>II> IV>!, result when 2 'rhodofying' groups are opposite (g.g. ring A and ring C Fig. 2e) each other or a conjugated B-keto ester is present (cf. Smith, 1975). 2-0xo-phylloerythrin-ME [XXXV], containing an acetyl and cycloethanone moiety on opposite sides of the porphyrin ring, yields an oxo-rhodo spectrum (Fig. 46c). This is pointed out here since previous authors (cf. Smith, 1975) use examples in which one of the rhodofying moieties is always a carboxylic acid. This need not be the case, as shown (Fig. 46c). The PHYLLO-type spectrum (Fig. 46d), IV>II>III>I, results when four or more B-pyrrole positions are unsubstituted or a 'non rhodofying' (i.g. electron-donating or electronically neutral) moiety is present on a methine bridge ('meso') position. Classically, phylloporphyrin-XV [LVIII] serves as the example of this spectrum and is the compound after which the PHYLLO-type spectrum was named (Stern and Wenderlein, 1936b). Other standards in the present study yielding a true PHYLLO-type spectrum include porphyrin [XLVIII] and tetramethyl porphyrin [[. LVII]. Though similar in name, phylloerythrin [XXXII] does not yield a PHYLLO-type spectrum. Rather, a band order of III>II>IV>I results and was described by Smith (1975) as "oxorho dorhodo." Since all spectral 'types' infer a relationship between band order and chemical structure, the term 'oxorhodo-rhodo' suggests the

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157 presence of three rhodofying (i.g. strong electron withdrawing) groups. This is incorrect and either PHYLLOERYTHRIN-(alt. PE-) or ERYTHRO-type is suggested to describe spectra with a band order of III>II>IV>I We shall use PE-type herein, due to the importance of phylloerythrin [XXXII] in tetrapyrrole geochemistry A central compound in geochemistry is DPEP [XXVIII] and its carboxylic acid precursor, OPE [XXXVIIa]. These 'deoxophylloerythrins' yield visible spectra (Fig. 46e) with the unique band order of IV>I>II>III. The statement that OPE [XXXVIIa] and OPEP [XXXVIII] have PHYLLO-type electronic spectra (Dunning and Moore, 1957; Smith, 1975) is wrong and despite more recent attempts to correct this error (Baker et . 1968; Baker and Palmer, 1978) it often can still be found in today's literature. Since the finding, structural proof (Chicarelli et 1984) and in vitro synthesis (Clezy et 1988) of DPEP analogs possessing cyclopropano (6-membered) and cyclobutano (?-membered) 'isocyclicrings,' all yielding true PHYLLO-type (IV>II>III>I) absorption spectra, it is now known that the distortion of the porphyrin macrocycle via ring strain with 6,y-cycloethano (5-membered) substitution is responsible for the DPEP-type (IV>I>II>III) spectrum. That is, alkyl bridging from a B-pyrrole to adjacent methine ('meso') position alone is not sufficient. Resultant isocyclic, or exocyclic, rings without steric strain (6+ membered) yield spectra reminescent of meso-alkyl substituted species. Originally thought to be 'petro-rhodoporphyrins' (Fisher and Dunning, 1961; cf Appendix A), the monobenzo (b) porphyrins are now known (Baker et 1968; Barwise and Roberts, 1984) to be common

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158 constituents of petroleum and other bitumen. In the free-base form monobenzo-etioporphyrins [CXVI] exhibit a visible band order of III>I>IV>II (Fig. 46f). This spectrum is for the 'b' type benzopor phyrins with, essentially, conjugated butadiene substitution across B,B'-pyrrole positions. The presence of an exocyclic benzene-like substituent (6-membered, conjugated) between a B-pyrrole and mesoposition (benzo[a,t]porphyrin) yields a nearly CHLORIN-type (cf. Fig. 45c) spectrum with a band order of (Fig. A16). The benzo(a,t)porphyrins are unknown to date in bitumen and are mentioned here for both completeness and due to carbon-skeleton similarities to geologic pigments with cyclo-propano exocyclic rings. Phorbides, bacteriophorbides, chlorins and purpurins (Fig. 2 and Appendix A) are free-base porphyrins with one or two sites of ring conjugation reduced. Essentially, the CHLORIN-type electronic spectrum (Fig. 45c) is any in which band I, lowest energy or red-band, is of the greatest extinction in the visible range. Numerous variations within this theme do exist (Fig. 47) and, as with free-base porphyrins, relate directly to structure. The di-and tetra-hydroporphyrins ('chlorins') discussed below were prepared as given in Appendix A. A classical CHLORIN-type spectrum is exhibited by chlorin-e 6-TME [XXIII]. Here, the resultant band order is I>IV>III>II. This band order and even the position of the band maxima is similar for chlorine6-TME [XXIII] and that reported for chlorin (i.g. 7,8-dihydropor phyrin) and octamethylchlorin (Gouterman, 1978). This reveals that the chlorin nucleus, with ring reduction lowering "the degeneracy of the square ring," (Gouterman, 1978) is not only the dominant chromophore

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159 0. 350 550 750 350 550 750 WAVELE NGTH(n m) F i gure 47. Electronic absorption spectra of selected 'chlorins'. (a) Chl orin-e6 TME [XXIIIa], (b) purpurin-18 ME [XXVI], (c) pheophorbide-a ME [Vlb], (d) bacteriopheophytin-a[XXI].Solvent =ethy l ether.Band identifications are discussed in text.

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but that peripheral alkyl, or alkyl acting (i.g. -CH2CH2COOCH3 in chlorin-e6-TME [XXIII]) moieties, exert very little effect on the resultant spectrum. This contrasts with the fully ring-conjugated porphyrins, as discussed above. 160 Alteration of CHLORIN-type spectra does, however, occur when conjugated auxochromes, exocyclic rings and/or additional sites of reduction are present. This, then, encompasses the purpurins, phorb ides and bacteriophorbides, respectively (cf. Seely, 1966). An example of a PURPURIN-type electronic spectrum is that exhibited by purpurin-18 ME (Fig. 47b [XXXVI]). Here the band order is I>III>II>IV. However, the diagnostic feature of this spectrum, as belonging to a purpurin, is the red-shifted (bathochromic) position of band I (I= 696 nm) versus that of a typical chlorin (g.g. [ XXIII], I = 665 nm). The intense nature of band III (A= 538-542 nm) in the spectrum of purpurin-18 (Fig. 47b) appears to be related to the diketo cyclic lactone auxochrome rather than simple carbonyl conjugation alone. That is, purpurin-7-TME [XXIV], with a band order of lacks this feature. Chlorins in which a 6,y-cycloethano ('isocyclic') ring is present (Fig. 2e) are termed phorbides, or phorbins (cf. Seely, 1966). The spectra of phorbides with only vinyl and alkyl substituents (g.g. deoxomesopyropheophorbide-a ME [XII]) exhibit near classic CHLORIN-type spectra, though exact band position and relative ratios are diagnostic. However, chlorophyll derivatives retaining a 9-oxo-phorbide nucleus yield spectra with characteristics important to geochemical studies. Here (Fig. 47c), all of the 9-oxo-phorbides studied ([IIa], [IIb], [III], [VIa], [VIb], [VIII], [X]) reveal strong inflections

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161 ('shoulders') along the higher energy side of the Soret band. These near ultraviolet overlapping (n) bands are thought to be "separate electronically allowed transitions" (Weiss, 1978) but this has not been resolved. Still, the presence of these near ultraviolet overlapping bands in the Soret region, within the overall CHLORIN-type spectrum, stands and is used herein as a marker for a 9 -0XO-PHORBIDE chromophore. Further ring reduction of 9-ox o-phorbides yields the tetrahydro porphyrins called bacteriophorbides. The electronic spectrum of bacteriopheophytin-a [XXI] is given in Figure. 47d. The resultant band order is I>IV>II>III. Here, the 'bacteria' (tetrahydroporphyrin) nucleus is evidenced by both a large bathochromic shift in band I (l = 757 vs. 665 nm) and the hypsochromic nature of Soret absorption (l = 363 vs. 400 nm), relative to a 'typical' chlorin (g.g. [XXIIIa]). The change from free-base porphyrin to metallo-porphyrin, as given in the start of this section, results in a dramatic alteration of the electronic spectrum. That is, the 4-banded visible absorption is replaced by 2-Q bands. Gouterman (1978), and others (Buchler, 1978; Smith, 1975; Treibs, 1973), attribute this to enhanced ('square') symmetry within the system. In the most qualitative and near naive terms, it has often been stated that the change from free-base chlorin or phorbide to the metallo complex does little to change the electronic spectrum (Fischer and Stern, 1940; Hodgson and Baker, 1967; Scheer and Inhoffen, 1978). This is true only to the extent that a large band I absorption is retained and the resultant, more or less 4-banded (visible), spectrum is chlorin-like. Adding confusion to geochemical studies was the report of Hodgson and Baker (1967) that the metallo-pheophorbides yield

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162 "spectra similar to the free pigments". Had exacting electronic spectroscopy been recorded, or reported, it would have been more than clear to the organic geochemical community before now that it. is quite easy to discern metallo-from free-base dihydroporphyrins from such spectra alone, as detailed below. Metallo-dihydroporphyrins, most notably the copper complexes, were reported and visible spectra given in the tome of Fischer and Stern (1940). Little information, however, on the near ultraviolet (Soret band) absorption and its relationship to the visible can still be found for this class of pigments. This obviously excludes the chl orophylls, magnesium chelates of phorbides or in one case, a pheoporphyrin [XIX], for which a wealth of information exists (Appendix a: cf. Gouterman, 1978; Weiss, 1978). Jones et gj. (1968) reported the absorption spectra for the copper and zinc complexes of pheophy t i n-a [II], pheophorbide-a [VI], pheophytin-b [XV] and pheophorbide-b [XVII]. However, since the pheophytin and pheophorbide of either the '-a' or '-b' series have identical chromophores this gives, in reality, only 4 metallo-phorbide spectra, the Cu and Zn complex of each. Jones et gj. (1968) did report the wavelength maxima for these pigments and the ratios of Soret to band I absorption. About the same time, Hodgson and Baker (1967) reported spectra, in figure form alone, for the copper, nickel and vanadyl complexes of pheophytin-a [II] and pheophorbide-a [VI]. The lack of attention to detail, most notably exact positions of maxima, negates the import of that report beyond a most qualitative aspect. Thus, one focus of the present study was to synthesize a number of dihydroporphyrins and, pertinent here, their copper and nickel

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163 chelates. Given current thought on chlorophyll geochemistry in aquatic sedimentary environments (Baker and Louda, 1986a), Cu and Ni were chosen as the most probable species to be encountered. Two examples of metallo-dihydroporphyrins are given here (Fig. 48). As can be seen through comparison of the spectra for the freebase (solid traces) and metallo-chelates (dashed traces), each pigment exhibits a hypsochromic (blue) shift in band I position while that of the Soret has moved bathochromically (to the red). It is through of the e xact positions of Soret and band I absorption maxima, rather than band ratios or overall spectral 'appearance,' that the metallo-dihydroporphyrin nature of these and especially geochemical pigments can be told. This is more thoroughly d i scussed under 'Chromo-phore Manipulation and Identification.' Empirical Electronic Absorption Spectral Data Numerical data on the electronic spectra for the 119 known pigments (Appendix A) obtained, synthesized or derivatized for this study are given in Tables 10 and 11. Details of instrument calibration and wavelength accuracy are given within the footnotes of these tables. Aside from the obvious import of these data in base-line geochem-ical studies and fodder for previous discussion in this section, these data and their complete forms (full spectra) were used as the main comparators with the geochemical isolates given later. Further, these data form the basis of chromophore identification, not only through comparisons of knowns with geologic pigments but also by study of the

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1 0 -(a I I ( b 0 .8-l I w I I \ I
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Table 10. Electronic absorption spectra (phorbides,bacteriophorbides,chlorins and of free-base and metallo-dihydroporphyrins purpurins) and free-base porphyrins. CLASS (RNC)COMPOUND(n) FREE-BASE PHORBIOES: (lla)pheophytin-a (llb)dihydropheophytin-a (lll)pyropheophytin-a (V)9-00-pheophytin-a (VIa)pheophorbide-a (VIb)pheophorbide-a ME (VII)9-00-pheophorbide-a ME (VIII)pyropheophorbide-a HE (11)9-00-pyrophe ophorbide-a ME (l)esopyropheophorbide-a ME ME (11)9-00-mesopyrophcophorbide-a ME ME (1111)7-PDP-OOMPP-a (1111)7-POP-UOMPP-a (IV)pheophytin-11 (IVI)3-MOF-9-0D-pheophytln-b (XVII)pheophorhide-b ME (XV11 1)3-MOF-9-0U-pheophorbide-b (IXI)bacteriopheophytin-a (XX II) 2-aiiED/1-9-0D-bncter iophcophyt I n-o FREE-BASE CIILORINS AND PURI'URINS: (XXI(l)chlorln-e6 T H E (IIIV)purpurln-7 THE (XXV)OD-purpurin-7 THE (XXVI)purpurln-1 8 ME (XXVI1a)chlorln-p6 HIE (XXVIII)rhodin-g7 niE (XXIX)3-MDF-rhodin-g7 THE (XXX)7-oxo-octacthy l chlorin ME (b) ABSORPTION SOLV SORET IV Ill II EE EE EE ACE EE EE liCE EE J:E EE BENZ BENZ EE EE BENZ EE EE H EE EE EE EE Er: EF. EE EE EE EE CIIL EE 409.5 409. 5 409.5 396.5 409.5 409.5 396.5 409.5 396.5 405.2 4 I 0. 5 394.8 390.0 389.5 396. s 4 34 5 402.5 434,5 402.5 362.0 374.0 400.5 4 03.0 4 0 I. 0 4 07.0 399.9 426.0 4 03. s 408 395.5 sos.o 505. 0 505.0 500.0 505.0 505.0 500.0 sos.o soo.o SO I ; 0 504.0 497 497.0 49R.O 499.6 523 503.0 523 503.0 530 497.2 500, 0 502 SOl 505.0 498.6 521 SO I 0 S I 2 497.8 5 34.5 s 34 s 5 l4. s 534.5 534.5 534.5 530,5 533.5 (524) 523.8 534. 5 527.0 (552) (528) (552) (528) 6 3 5 (660) 529,5 540 530 54 I. 5 53 1 553 530.0 550 532 609 609 609 597 608 60B 597 608 597 600 603 586 5 85. 2 586 587 600 595 600 595 6 88 682 610. 5 635 611 639 615 600 605 615 588 666.5 666.5 666.5 65 I 5 666.5 666.5 65 I. 5 666.5 65 I. 5 656. 0 658.5 642.0 639.6 640.0 641.0 654.8 65 0 0 654.8 650.0 757. 0 715,8 665,0 678.0 6(>5. 0 695.5 670.8 654.0 661.5 (>4 2 64f>, 6 VISIBLE(d) BAND ORDER >I I >Ill >II >IV >Ill >II >IV >Ill >II >IV > II >IV >Ill >II > 1 V >Ill >II >IV > II >IV >Ill >II >IV > II >IV >Ill >II >IV >Ill >II >IV >I I >(Ill) > I V >II > Ill > I V > I I > I II >IV >II > I I I > I V >I l > (II l) >IV >II ) ( l I l ) > l V > l I > (II I) >IV >II ) ( l II) >IV >I I > II I >IV >I I > I II > I V >II I > II > l I I> I V > II > l V > I II > I l > l I I> I I > IV > l V > I II > II > I V > I I I > I I > I V >I I I > I I > I 1 > IV > II >IV > I I > I II S/1 ( e ) 1.9 1.9 1.9 3.4 1.8 1.8 3.4 I. 8 3,4 2.4 2. 5 4. 4 3.4 3.0 3.3 4. 7 3.6 4. 6 3.2 I 4 .f> 2. I 3.6 2. 7 1. 6 2. 8 7. I 3.0 s.o b O I 4 3 4 4 3 4 3 3 4 3 4 2 (cont. f-> (j\ V1

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Table 10 (cont'd). CLASS ABSORPTION HAXIMA(nm)(c) VISIBLE{d) (RNC)COMPOUND(a) SOLV(b)SORET IV Ill II I BAND ORDER S/1 (c) I FREE-BASE PORPHYRINS "DPEP"-NUCLEUS": (XX) 'pheophorbldeslcl/c2 EE 4l0 495 574 595 625 Ill >II> IV >I 25 0 (XXXII)phyl1oerythrin ME BENZ 4 20.0 5 21 562.5 586 639. 0 Ill >II > IV >I 200 0. (XXXIII)9-0D-phylooerythrin HE BENZ 406. 0 504.0 537.5 567.8 621. 2 IV > I II > Ill 90 0 (XXXIV)7,8-dloxy-OPE HE EE 402.6 506 544 585 641.0 I >IV >Ill> II 6,9 2 BENZ 422.8 528.5 572.5 598.0 650.5 Ill > I V >II > I 63 0 (XXXVI)2-aiiEUc9-0D-rE ME EE 400.8 500.5 536 567.5 620.8 IV >II > I >Ill 48 0 {XXXV lib) OPE BENZ 402,0 499.8 534 566,5 619.5 I V > I >II >Ill 38 0 (XXXV II I) llPEr BENZ 402.0 499.8 534 566.5 619.5 IV > I >II >Ill 38 0 BeNZ 402.0 500,0 534 566 619. 5 IV > I >I I > I I I 37 11 (XL) 2 011 E I' BENZ 402.0 500.0 534 566 619.5 I V >I >II >Ill 32 0 "RIIOOIN-NUCLEUS": (XLI)mesorhodln-I X ME BENZ 411.0 511.5 548.5 583 637.0 IV >I > I I I > II IS 0 (XLI)mesorhodin-IX HE EE 405.5 508.5 545.0 583.5 635.5 IV > I >II I > II IS 11 [XLI I)OD-csorhodln-IX ME EE 400.0 500.5 532.0 5 71. s 626. 0 IV> II I> II > I 25 0 (XLIII)mesovcrdln-IX M E BENZ 419.5 (510 ) (560) 6JJ 695.0 I > [IV)> I I> (Ill) 6 I (XLIV)OD-mesovcrdin-IX HE EE 408 508 5 38 582 640 IV>III > I 40 0 (XLV)didehydro-dcoxoesoverdin-IX HE BENZ 4 I 2. 5 535 568 612.5 667.5 I >II "Ill> IV 3. 3 (XLVI)pyrrorhodin-XV BENZ 4 I I. 0 513.0 55 I. s 587. 0 64 I. 5 I >IV >I II> II II I (XLVI)pyrrorhodin-XV EE 406.0 511.0 547.0 587.0 640.5 I >IV >Ill> II 10 I {XLVI I) 00-pyrrorhodi-XV EE 400.0 499.0 5 31.0 5 71. s 626.0 IV> Ill> I 34 (I "ETIO-NUCLEUS": (XLVIII)porphyrin(porphin) BENZ 396.0 489.5 519 563.2 6 16.5 I V >I I >Ill > I 300 0. (XLIX)octamethylporphyrin Clll. 398. B 498. 2 s 32.8 5 67.5 620. 8 IV >Ill> II > I 31 0 (L)octaethylporphyrln BENZ 4 0 I. 0 498. 5 53 I. 0 568.2 622.0 IV >Ill> II > I 30 (I (LI)etioporphyrin-1 BENZ 399.5 498.0 530.5 568.5 6 22.5 I V>III> I I > I JJ 0 {LII)etioporphyrln-111 BENZ 399.5 498.0 530.5 568.5 622. 5 I V >I I I> II > I 32 0 (LIII)pro toporphyrin-IX DHE BENZ 4 I 0. 0 5 06.0 54 0. B S7B.2 6JJ.O IV >Ill> II >I 28 II (LIV)hentoporphyrin free-base llHE BENZ 408.2 505. 0 539. 2 577.5 632.0 IV >Ill> II > I 39 (I (LV)csoporphyrin-IX DME BENZ 4 0 I. B 498.8 531. s 569.0 623.8 I V >Ill> II > I 3 II (LVI)dcuteroporphyrin-IX ONE BENZ 4 0 I. 2 49 7 0 529 568.0 621.8 IV>III> II > I 53 n [LVII)dcuteroetioporphyrin {f) BENZ 4 0 I. 0 49 7. I 529 s 6 8. 2 621.6 IV >I II> II > I 52 0 --.. .. 1,3,5,7-tetramethylporphyrin BENZ 4 01.5 495 522 566 618 II >I 40 II (LVIIIb)phyli o porphyrin ME BENZ 4 OB. 2 504.5 537.0 576.5 630.5 IV >II > I I I > I 140 0 (LIX)rhodoporphyrin M E BENZ 408.5 508 54 7. 8 576.2 6lS. O Ill> IV> II >I 140 (I (LX)pyrroporphyrln HE BENZ 4114.8 502.0 531.4 575.8 62 2. 2 I V >Ill> II >I 70 (I [CXVI) 'benzoetloporph yrin' BENZ 403. s 504.6 540.0 573. 5 (130.B I l l > I > I V >II 18 I ...... 0' (cont. 0'

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Table 10 (cont 'd). CLASS SOLV(b)SORP.T ABSORPT ION HAXIMA(nm) (c) VISIBLE (RNC)COMPOUND(a) IV Ill II I BAND O RDER S/ I (e) 1 / D IIIYDROPORPIIYR INS METALLO-PIIORBID ES: (!)chlorophyll-a EE 429 :sH 578 615 661.5 I >11 >III >IV (XIV)ch1orophyll-b E[ 442 5 (550) (568) (595) 64 2 .o I >II >Ill >IV (LXI)Cu-pheophorbide-a ME BtNZ 4 21. 1 505 547 603,0 650.0 I >11 >Ill >IV (LX11)Cu-9-0D-pheophorbide-a ME B ENZ 405.2 510 (540) (580) 6 21.5 I > IV> ( 11 ) > ( II I ) (LXIIIa)Cu-esopyropheophorbide-a ME BENZ 421.8 SOB 550 596 64 2. 8 I>II>III>IV (LXIIIb)Ni-esopyropheophorbide-a HE BENZ 417.6 498 538 593 640.8 I >II >Ill >IV (LXIV)Cu-9-0D-esopyropheophorblde-a HE BENZ 402.0 SOD (525) 566 614 o I >IV>(III)>!I (LXV)Cu DONPP-a ME EE 395.2 494.0 (523) 564.0 605. 5 I >II >IV>(III) NE BENZ 399.9 497 (525) 567 609.2 I >II >IV>(III) (LXVIa)Cu-7PDP-DOHPP-a BENZ 402.8 499 536 568 610.5 I >II >IV >III (LXV!b)Ni-7-PDP-DOHPP-a BENZ 399.8 494 525 566 609.8 I >II >IV >III (LXVII)Cu-pheophytin-b EE 438.0 520 -582 628.5 I >II >IV (LXVIII)Cu-3-MDF-9-0D-pheophytln-b EE 409.2 sos (5 48 ) -635 I >IV >(Ill) COPPER-CIILORINS/PURPURINS: (L XIX)Cu-chlorln-e6 TME BENZ 414 0 505 (540) (595) 641 I > (II)> IV> (Ill) (LXIX)Cu-chlorin-e6 THE EE 409,0 502 ( 540) (595) 634.6 I > (II)> IV> ( II I) {LXX)Cu-purpurin-18 HE BI!NZ 419 0 504 546 623 67 2.0 I > I I >IV> Ill (LXXI)C u-chlorin-p6 THE BENZ 412.5 SOl (5 3 0 ) (600) 646.5 l>(II)> IV>(III) (LXXII)Cup urpurin-7 TME BENZ 419.8 507 54 2 ( 6 20) 669. 5 I > ( I I )> IV> Ill (LXXIII)Cu-OD-purpurin-7 THE BI!NZ 414.4 508 552 (610) 642.5 l>(li)>IV >Ill (LXXIV)Cu-rhodln-&7 THE BENZ 4 15,8 509 548 598 648.8 I> II >Ill> IV (LXXV)Cu-3-MDF-rhodln-g7 TME BENZ 409.5 503 (54 2) (590) 634.6 I> I I >IV >Ill (LXXVII)Cu-mesopyrrochlorin ME EE 401,6 502 528 56 4 614.2 I > II >Ill> IV (LXXVII)Cu-mesopyrrochlorln ME BI!NZ 406.2 so I. 2 s 31 566 617.2 I > II >Ill> I V FOOTNOTES: ii)Jiilc-Ro,.an nucral cod e for cross -reference to IIIPendlx 1\, Com on or workina'namc elven(sce 1\t>pcn.tlx II). ODoxy-deoxo,MEethyl cster,DHEdlmethyl ester,TMEtrlethyl cster,MDF mothanol-desforyl,oiiEDA ohydroxyethyl-desacetyl,oiiEPEohydroxycthyl-doscthyi,PDEpropyl-descthyl,PDPpropyl-despropio,PEphylloerythrin,OPEdeoxophylloerythrin,DPEPdeoxophylloerythroetioporphyrin,DOHPPdeoxollesopyropheo ph orhide. b) SOLVENTS: EEcthyl ether,OENZbentene,ACEacetonc,CIILchloroform. 1.3 2.7 1.4 3.4 1.8 1.4 s.o 2.7 3.4 3 5 2.3 2.0 4 1.9 2.5 1.8 2.3 2. 2 2.0 6 6 6,0 I 0 II c ) Absorption maxlma(n)given as real numbers are !. values arc .t. In a nd parcnthcsircd figurrs for the center of a shouldcr(i.e.inflectlon point) arc!. 2n.Calibration of nbsorbance wns pcrfored with dilution s of K2Cr04(4D-i/l ) in O .OSN KOII(Rno,l967 ) .Wavelenath callbrntion was versus hollu oxide. d) Visibl e bands labeled I -IV with inc reaslna encrgy(decreaslng wavelength) and are preseiiTCTTn order of decrenslnJ extinction. e) Band ratlos(SSoret) given arc overages and Integer values are roundcd-off.Exact band ratio values(wlth the IICthod:.!.!!.!_cxtlnction)fluctuate with solvcnt,conccntratio n and spcctrophotorocter scal c(AUFS) used(see Text). 23 8.3 1 4 6. I I S 12 2.8 6 2 5.8 9. I II 6.5 5 4.8 5. I II 9.8 7.11 Ill s. z 4 5 6,S 6.R (j\ '-.1

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Table 11. Electronic absorption spectra of metalloporphyrins. CLASS ABSORPTION HAXIHA(nm)(c) UA !Ill t :AT I OS (RNC) COMPOUNO(a) SOLVENT(b)SORET(y,S) BETA(!!) ALP IIA (a) S/a(d) a / a (d) 'OPEP'-NUCLEUS (LXXV!li)Ni-PE ME BENZ 413.0 537 582.2 4. 5 3. 2 (LXXIX)Ni-9-00-PE ME BENZ 397.5 516 553.0 8.9 2.0 (LXXX)Cu-PE ME BENZ 416.2 548.0 594.8 6.4 2.6 (LXXXI)Cu-9-00-PE ME BENZ 4 04 2 529.0 563. 8 20 1.2 (LXXXII)Ni-2-0XO-PE ME BENZ 4 21.0 581 601.8 6.6 3.3 (LXXXI!I)Ni-2-ai!EDE-9-00-PE ME BENZ 399.8 516.0 554.0 9. 4 1.5 (LXXXIV)Cu-2-0XO-PE ME BENZ 420.8 561 611. 2 7 1 3.8 (LXXXV)Cu-2-aiiEDE-9-00-PE ME BENZ 405. 0 529.8 564. 0 16 I. 02 (LXXXVI)Ni-DPE ME BENZ 396.0 517.0 554.5 8 3 2. 1 (LXXXVI)Ni-OPE ME EE 392. () 513.5 550. 5 8.2 2 2 (LXXXVII)Cu-OPE ME BENZ 402.0 526.0 563.5 12 1. 4 (LXXXV!l)Cu-OPE ME EE 3n8.5 5 24. 0 561.0 I 2 1.4 (LXXXVIII)VO-OPEP BENZ 410.8 5 34.0 57 4. 0 I 2 1.3 (LXXXIX)Cu-7-PDE-OPEP BENZ 4 0 2. 5 526. 0 563. 5 1 2 1.4 (XC)Ni-OPEP BENZ 396.0 51 7. 0 55 4. 5 8.8 2. I (XC)Ni-OPEP EE 3!!2. 0 514.0 55 1. 5 8.0 2. I (XCI)Ni-7-POE-OPEP BENZ 396.0 5 I 5. 8 553.9 8. I 2. 1 (XCII)Ni-2-HOE-DPEP BENZ 396.0 517.0 5 54. 5 8. 7 2. 2 "Abelsonite" 'RIIOOIN-NUCLEUS' (XCIIJ)Cu-mesorhodin ME BENZ 4 09. 5 543.0 5 84.8 13 1.9 (XCIV)Cu-00-mesorhodin ME BENZ 4 0 I 2 526 565 19 1. 5 (XCV)Cu-pyrrorhodin ME BENZ 410.0 551 592 10 2 2 (XCVI)Cu-00-pyrrorhodin ME BENZ 399.5 526. 0 5 64. 5 20 1.6 f-" (]'\ 00

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Table 11 (cont'd). Class (RNC)CONPOUNO(a) 'ETJO'-NUCLEUS [XCVII)Ng-octamethylporphyrin (XCVIII)Cu-porphyrin (XCIX)Cu-octamethylporphyrin [C)Cu-octacthylporphyrin (CI)Cu-ctioporphyrin-1 (CI)Cu-etloporphyrin-1 (Cil)Cu-mesoporphyrin-IX ONE (Clii)Cu-protoporphyrin-I X DME (CIV)Cu-deuteroporphyrin-IX OME ONE (CVI)VO-mcsoporphyrin-IX ONE (CVII)VO-etioporphyrin-1 (CVII)VO-ctioporphyrin-1 (CVIII)VO-etioporph yrin111 (CVIII)VO-et1oporphyrin-111 (CIX)Ni-mesoporphyrin-IX ONE ( CIX)Ni-mcsoporphyrin-IX ONE (CX)N i -octacthylporphyrin [CX)Ni-octaethylporphyrin (CXI)Cu-pyrroporphyrin ME (CXII)Ni-pyrroporphyrin Nf: (CXIII)Cu-phylloporphyrin ME ( C X I V) N i -ph y I I o p o r ph)' r i n (CXV)Cu-rhodoporphyrin ME 'BENZO-ET10'-NUCLEUS (CXVII)Ni-'benzoetioporphyrin' (CXVIII)VO-'henzoctioporphyrin' (C:XIX)CII-'bcnzoetioporphyrin' ABSORPTION Mi\XHii\{nm) (c) SOLVENT(b)SOitET[y,S) Bf:Ti\ (Ill fiLl' IIi\ (a) BENZ 4 0!1 R 544.11 581.2 BENZ 39 3. 8 516. 0 550.0 BENZ 398.2 526.0 562.0 BENZ 398.8 525.5 562.0 BENZ 399.5 526.0 562.5 EE 394. 5 523. 5 560.0 BENZ 399.8 526.0 562.5 BENZ 408.8 5 34.4 57 2. 2 BENZ 398. 4 5 24. 5 560.0 BENZ 360. 0 4 71. 2 560 BF.NZ 407.8 5 34.4 57 2. 2 EE 40,,0 532 569 BENZ 407.0 534 5 71.0 EE 403.0 532 569 BENZ 407.0 534 5 71.0 Ef: 389.5 515.0 55 I. 0 BENZ 394.5 519.0 5 54.5 EE 389.5 5 I 5. 5 550.5 BENZ 393.5 517.5 552.5 BENZ 402. 8 521!,0 561.9 BENZ 400.5 523.5 552.5 BENZ 406.0 5 31.5 564.5 BENZ 403.8 55 2. 2 558. 5 BENZ 407. 5 537 580.5 Bf:NZ 40l1 5 528.5 56R,5 BENZ 415.5 546.5 590.5 Bf:NZ 4 0 5. 5 536.0 574. 5 81\NO lti\TIOS S/a(d) a/ll[d) 2 I 1.1 25 0.8 1 2 2.0 12 2. 2 II 2. 2 10 2 2 II 2. I 1 0 2 0 14 I. 9 9. I 0. 2 II 2.5 II 2. 5 II 2.3 II 2. 5 II 2. 3 s. 5 3.0 5. 4 2. !l 4. 2 2. 9 4 l 3. 2 I 5 I 4 8 4 I. 4 26 0.97 13 I 4 II 2. 3 4.0 4. 4 7. 6 3. I 7.7 3. I ...... 0\ 1..0

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Table 11 (cont'd). FOOTNOTES: a) RNC =Roman numeral code (see Appendix A). Names given are common names abbreviated as follows;PE = phylloerythrin, OPE= deoxophylloerythrin, OPEP = deoxophylloerythroetioporphyrin, 00 = oxydeoxo, HEOA = alpha-hydroxyethyl-desacetyl, PDE = propyldesethyl, MOE = methyldesethyl, ME = methyl ester, DME = dimethyl ester. Metals (Ni,Cu, Mg) are divalent except vanadium (V(IV) as V02--) and manganese(Mn(III)). b) Solvents: BENZ = benzene EE = ethyl ether. c) Absorption maxima given as real numbers are 0.2 nm, whereas integer values are 1 nm. Calibration of absorbance was versus serial dilutions of K2Cr0 4 (40mg/L) in O.OSN KOH (Rao, 1967), while wavelength calibration was versus holmium oxide. d) Band ratios are averaged values and integer values (10+) are rounded-off figures. Exact values fluctuate slightly with solvent, concentration and spectrophotometric scale( AUFS). ...... 0

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spectral changes imparted by simple in vitro derivatizations (metallation, demetallation, carbonyl reduction). 171 During the isolation of geologic tetrapyrrole pigments, often either the amount of final 'purified' isolate or the purity of same, or both, precudes any attempts at detailed structural analyses NMR). In cases such as these, the sensitivity of electronic absorption spectroscopy can be of considerable importance in allowing at least tentative identifications to be made. This sensitivity der i ves from the fact that the important dia gnostic features are the absorption maxima of the tetrapyrrole pigments and these present themselves above the non-pigment light absorbing background. Ultra-violet/visible spectroscopy (UV/VIS) not only allows the partial identification of tetrapyrrole pigments but is often the only method amenable to their quantitation. It is the opinion of the author that the basics in tetrapyrrole pigment analyses, namely UV/VIS, are becoming more and more neglected in recent literature reports of organic geochemistry. In fact, only about 10% of geoporphyrin publications even give absorption maxima and even less report full spectra for p igments reported as unequivocable structures. Chromophore Manipulation and Identification Two simple in vitro derivatizations were developed in order to chemically and spectroscopically 'probe' the chromophore of geologic tetrapyrrole pigments These tests are mainly aimed at the dihyropor phyrins (phorbides, chlorins) characteristic of early diagenesis, since

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they are not as yet amenable to 'routine' mass spectral analyses, as detailed later. 172 These 'probes' are the borohydride reduction of carbonyl moieties and analytical chelation by free-base pigments with monoisotopic ( 29Cu63) copper. Together, if the unknown pigment is a free-base tetrapyrrole with a cojugated carbonyl auxochrome, these two tests result in 4 UV/VIS data. These being the spectra for (1) the native pigment, (2) the oxy-deoxo-(00-) derivative, (3) the Cu-chelate, and (4) the 00-Cu-derivative. Pigments lacking conjugated carbonyl functionalities yield three data. These are the two spectra, native and Cu-derivative, and the negative chemical test. That is, negative results with borohydride reduction proves the absence of a conjugated carbonyl and thereby aids in chromophore identification. Sodium borohydride (NaBH4 ) is a mild reductant which acts only on aldehydes, ketones and acid chlorides (Brown, 1979; Chakin and Brown, 1949). Holt (1959) first employed NaBH4 in order to selectively reduce the carbonyl moieties of several chlorophyll derivatives. Based on the report of Holt (1959) and success with the method when applied to carotenoid studies {Louda, 1978), we initial tests with geologic tetrapyrroles (Baker and Louda, 1981). Results herein represent an expanded in-depth study on the utility of borohydride reduction as applied to both known (Appendix A) and geologic pigments. In the present study 22 oxy-deo x o ('00') tetrapyrrole pigments {Table 10-11; Appendix A) were formed through in vitro treatment with NaBH4 Borohydride treatment was performed by the addition of a few crystals of NaBH4 to the pigment (<100 in ethyl ether containing about 10% (v/v) anhydrous ethyl alcohol with mixing. The reaction

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173 mixture was flushed with dry N2 and stored at room temperature (ca. 26C) for 4-6 hours, with mixing every one-half hour Alternately, methanolic NaBH4 can be added to pyridine solutions (Holt, 1959). Reaction was stopped by the addition of water and the product driven into ethyl ether. Chromatographic purification, as given earlier, afforded the oxy-deoxo products in near quantitative yields. Resultant spectral data can be found in Tables 10 and 11 and Appendix A. Formation of the monoisotopic (29Cu63) copper chelates was as given in Chapter 2. It must be pointed out here, however, that the order of metallation with copper and treatment with borohydride is very important. That is, attempts to add Cu2+, as Cu63S04 to oxy-deoxo tetrapyrroles often lead to the partial re-oxidation of the product, reforming the oxo-pigment, as well as undefinable side products. Thus, in all cases with authentic or geologic pigments two aliquots were required for complete and verifiable chromophore manipulation. Aliquot "A' provided the spectra of the native pigment and the oxo-deoxo derivative. Aliquot 'B' was sequentially metallated with copper and then reduced with borohydride. This procedure is shown diagrammatically as Figure 49. Within this scheme, especially with geochemical pigments, the majority of pigment is retained in the native or copper derivatized form and is held for other analyses. Analytical treatment with NaBH4 was often at the 1-5 level in order to preserve unaltered isolates for additional study (g.g. MS, NMR). As stated in Chapter 2, was employed as resultant mass spectra, given below, are greatly simplified when compared to those obtained with copper of natural isotopic abundance. In addition, artifact formation through unwanted in vitro

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STANDARD TETRAPYRROLE -or-GEOCHEMICAL ISOLATE ('NATIVE' PIGMENT) (A) 1-5,ug majority saved (UV/VISJ *) (B) 1-5 )JQ Na 8 H4 C u63(2+) f t 00-DERIV Cu-1DERIV Na8H4 UV/ VIS 174 Figur e 49. A n a lytical scheme for the in vitro manipulation of tetrapyrrol e chromophores using sodium borohydride and mono -isotopic copper-63 sulfate.Abbreviations:" A and B refer to aliqu o ts,OD = oxy-deoxo, UV/VIS = determination of electronic absorption spectrum

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chelation (g.g. Zn, cf. Louda and Baker, 1981) is precluded via the above technique. 175 Two examples of the manipulation of dihydroporphyrin chromophores are given here. These, and other, data are also in tabular from within Table 10. Figure 50a contains the electronic spectra of pheophorbide-a ME [VIb] and its 9-oxy-deoxo derivative [VII]. Large hypsochromic shifts in both Soret (409.5 to 396. 5 nm: a = -13 nm) and band I (666.5 to 651.5 nm: a = -15 nm) absorption are found to occur upon reduction of the 9-keto auxochrome to the hydroxy (oxy) functional. Comparing hydroxy tetrapyrroles to the corresponding pigments with hydrogen substitution (g.g. [XI] with [XIII], [XXXIII] with [XXXVIIb] or [XXXVIII], one finds that the bathochromic shift imparted to the tetrapyrrole via substitution with an hydroxyl group amounts to only (+) 1 nm. Additionally, it is noted that the overlapping bands (n) to the near ultraviolet of the Soret band in the 9-oxo-phorbide (pheophor bide-a [VIb]; Fig. 50a, solid trace) spectrum are absent upon removal of carbonyl conjugation (9-0D-pheophorbide-a [VII]; Fig. 50a, dashed trace). This and similar observations lead to the suggestion of a 9-oxo-PHORBIDE spectral type earlier in text. The spectrum of pheophorbide-a ME ([VIb]; Fig. 50a, solid trace) is dramatically altered upon the chelation of copper (Fig. 50b, solid trace), in opposition to statements to the contrary (cf. Baker and Hodgson, 1967). The spectrum of Cu-pheophorbide-a ME ([LXI]; Fig. 50b, solid trace) reveals band I absorption at 650.0 nm and Soret maxima at 421. 1 and 401 nm. The lessening of the energy difference between the two principle absorptions, hyp$ochromic band I and bathochromic Soret

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1 0 I ( a I I ( b wO.B 1\1 u I z l I n: 0 6 n 0 (./) ro
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177 shifts, upon the chelation of copper by dihydroporphyrins was routinely found (Table 10) and the resultant spectra (Figs. 48a, 49b, SOb) are quite diagnostic. This type of spectral change in the dihydropor phyrins upon metallation extends as well to nickel (Fig. 48b, Table 10), magnesium (i.g. chlorophylls: Table 10) and zinc (Jones et 1968). Akin to the free-base form (Fig. 50a), Cu-pheophorbide-a ME [LXI] exhibits hypsochromic shifts in both band I (650.0 to 621.5 nm: = -28.5 nm) and Soret (421.1 to 405.2 nm: = -15.9 nm) upon reduction [LXII] of the 9-oxo auxochrome. Likewise, the fine structures of bands II and III is depleated as their extinction has decreased (Figs. 50a-b, dashed traces). Purpurin-7-TME [XXIV], a chlorin (Fig. 2c) containing a y glyoxylic acid auxochrome, serves here as an example of the 'chromo phore manipulation' (Fig. 51) of a non-phorbide dihydroporphyrin. Purpurin-7-TME [XXIV], as with the other purpurins (g.g. XXVI) is so named (1. purpura) from the purple hue attained by the bathochromic shift in band I absorption ([XXIV] A1 = 696.0 nm), relative to other dihydroporphyrins (g.g. 660-665 nm). The spectrum of purpurin-7-TME [XXIV] is shown as Figure 51a (solid trace) and is typical of the purpurin modification (cf. Fig. 47b) of a CHLORIN-type (cf. Figs. 45c, 47a) electronic absorption spectrum. Removal of carbonyl conjugation (y-glyoxylic to y-glycolic acid) through reduction with borohydride alters the spectrum (Fig. 5la, dashed trace) to that of a typical vinyl chlorin (cf. Figs. 45c, 47c, 48a). In line with the spectral behavior of other dihydroporphyrins (Table 10), the chelation of copper leads to concurrent hypsochromic band I and bathochromic Soret shifts in the product ([LXXII] Fig 5lb,

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1 0 11 (a I I ( b tl I 1\ w 0 8 u z <( c1) 0 6 0: 0 lf) CD <( 0 4 II l \ 0 0 I 400 6 0 0 800 4 0 0 600 800 WAVELENGTH(nm) Figure 51 Comparison of the electronic absorption spectra of (a) purpurin7 TME [XXIV] (b) copper purpurin7 TME [LXXII ] and their 'oxy-deoxo' derivatives(dashed) as obtained by reduction with sodium borohydride.Solvent = ethyl ether. 1--" -...J 00

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179 solid trace), relative to the free-base precursor ([XXIV] Fig. 51a). Treatment of Cu-purpurin-7-TME [LXXII] with NaBH4 generating the 'oxy deoxo' or '00' derivative [LXXIII], leads to the spectrum given as Figure 5lb (dashed trace). While the spectrum of Cu-00-purpurin-7-TME ([LXXIII] Fig. 5lb, dashed trace) is exceedingly similar to other Cu vinyl-chlorins [LXIX], [LXXI], [LXXV]: Table 10) it is dissimilar to the Cu-(non-oxo)-phorbides (cf. Fig. SOb, dashed trace [LXII]). However, it is the spectra and the spectral changes upon analytical treatment which allow one to identify the purpurin-? [LXXIII] chromophore. No other, known, pigment will give an exact data match for all four spectra. The above scheme (Fig. 49) and resultant data (Table 10; Figs. 48, 50-51) provided the author with an analytical methodology by which to identify the chromophores, and thereby the dihydroporphyrin nuclei, of numerous geochemical pigments This advance was especially important in tracing the defunctionalization of chlorophyll-a (I] during early diagenesis (Louda and Baker, 1986), and is detailed in the next chapter. Chromophore manipulation (Fig. 49) is obviously possible with porphyrins and metalloporphyrins as well. Phylloerythrin ME (XXXII] exhibits the OXO-RHOOO type spectrum (Fig. 46c) shown in Figure 52a (solid trace). The auxochrome effect of the conjugated 9-carbonyl (keto) function is easily removed, and thereby proven, through reduc .tion with borohydride. The resultant 'oxy-deoxo' derivative, 9-00phylloerhythrin-ME [XXXIII], was found to have the PHYLLO-type spectrum (cf. Fig. 46d) shown as Figure 52a (dashed trace).

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1 .0. ( a I I (b j I 0 8 i (I n w 1\ A II u I I\ II z II <{0. 6 d) I I \ f\ J v I n:: 0 \J \. I\ ) ,_.) \ (/) 'v \ m0. 4 II "-. <{ I "----......._ __ I o 2 I 1 \ I VtJ I \\ ,.. \ ---0 0 ' I 350 550 750 350 550 750 WAVELENGTH(nm) F i gure 52. Comparison of the electronic a bsorption spectra of (a) phylloerythrin ME [XXXII], (b) copper phylloerythrin M E [LXXX] and their oxy -deoxo' derivatives(dashed) obtained by r e du ction with sodium borohydride.Base lines are shifted upwards for the scal e expnaded spectra. Solvent = benzene. ...... 00 0

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181 Aside from yielding analytical test data, this experiment provides a result directly applicable to possible native geologic tetrapyrroles. That is, an alternate terminology for 9-00-PE-ME [XXXIII] could include the name 9-hydroxy-OPE. Further, the chromophore is identical to possible 9-hydroxy-OPEP species. The fact that the spectrum of 9-00-PE ([XXXIII] Fig. 52a) is a PHYLLO rather than OPEP (IV> I >III>II) type reveals a greater influence of hydro x yl substitution upon spectral fine structure than found with the other o x y-deoxo tetrapyrroles. That is, hydroxyl substitution on the dihydroporphyrin chromophore, be it of a phorbide or chlorin nucleus, does little to alter band order and is expressed only in subtle (.5-1.0 nm) shifts in maxima. In the present case, hydrox yl substitution at the ca rbon (Fig. 2) of the isocyclic ring appears to enhance the alkyl nature of the position #10 methylene to the point where electron donation at the -meso locus of the porphyrin macrocycle mimics that of a conventional -alkyl-porphyrin (cf. phylloporphyrin-ME [LVIIIb] Fig 46d). These data and interpretations may aid in future understanding of certain geological 'polar porphyrins' (see Baker and Smith, 1975b; Baker et 1978a-b), as the hydro x yl moiety is much more chromatographically active, relative to carbonyl and alkyl moieties, on convet i onal adsorptive media. Continuing with the in vitro testing of free-base porphyr i n spectra, the next step (Fig 49) is metallation with Cu63 In this case [XXXII] the product is Cu-PE-ME [LXXX] and yields the spectrum given as Figure 52b (solid trace). Relat ive to the copper chelates of purely alkyl porphyrins (g.g. [LXXXVII], [CI] [CXI] etc.: Table 11) with Soret/B/a bands at about 400/526/562 nm in benzene solvent, it was

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182 found that all absorption maxima bathochromically shifted and noted the presence of additional or overtone B-bands. Discussion of these features could not be found in major reviews of metalloporphyrin electronic spectra (Buchler, 1978; Gouterman, 1978) but appear analogous to the effects caused by the 9-keto moiety in protochlorophyll-a (= 7,8-didehydro-chlorophyll-a: see [I]) which lead to significant differences in the "x" and "y" electronic axes and the appearance of "daughter" transitions (Weiss, 1978). These additional-overlapping, or 'daughter,' bands were also found within the spectra of Ni-PE-ME [LXXVIII], Ni-2-oxo-PE-ME [LXXXII] and Cu-2-oxo-PE-ME [LXXXIV] (Appendix A). These inflections to the blue of the B-band plus the hard spectral data (Table 11) now become markers for the Cu or Ni oxo pheoporphyrin chromophore. Not unex pectedly, reduction of the 9-keto group, yielding Cu-9-00-PE-ME [LXXXI], results in a spectrum (Fig. 52b, dashed trace) very similar to the more familiar Cu alkyl porphyrins (Table 11). Metalloporphyrins containing conjugated carbonyl functional groups are also amenable to chromophore testing via the present scheme (Fig. 49). Pigments such as Ni-PE-ME [LXXVIII] yield characteristic spectra (Fig 53a) and the reduced Ni-00-PEME [LXXIV], exhibits a typical Ni-porphyrin spectrum (Fig. 53b). Though not done as such with standard pigments, it follows that demtallation of Ni-porphyrins and analytical treatment as in Figure 49 (free-base, 00, Cu, Cu-00) are viable alternatives towards chromophore identification with unknown (geologic) pigments. Classically, the term 'rhodin,' as fully discussed in Appendix A, is applied to both chlorins of the 'b-series' (3-formyl-

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1 .0...--------------, 0 8 w U0.6 z
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184 dihydroporphyrins: g.g. rhodin-g7 [XXVII]) and to true porphyrins containing keto substituted 6-membered (cyclopropano) rings in conjugation with the porphyrin chromophore nucleus. Due to the recent identifications of geologic porphyrins with 6-membered isocyclic rings ('pseudo-DPEP' or 'DPEP-6' : Louda and Baker, 1983) in geologic samples (Chicarelli et . 1984; Wolff et . 1984) interest in the porphyrin-rhodins as potential precursors has arisen. Further, geologic pigments with spectra akin to 'rhodins,' porphyrin and dihy droporphyrin type, have been reported and termed as "dio x y-deoxophylloerythrins" (Baker and Smith, 1975b), "chlorin-635" (Baker et 1976), or "chlorin-636" (Baker and Louda, 1980a). Pyrrorhodin [XLVI] yields the electronic absorption spectrum shown as Figure 54a (solid). The band order of I>IV>III > II fits a CHLORINtype visible spectrum (Fig. 45c) more closely than it compares to the standard free-base porphyrin spectra (Fig. 46a-f) In the pure state, as with the present standards, the fact that pyro-rhodin [XLVI] is a porphyrin and not a dihydroporphyrin like rhodin-g7 [XXVII] can be discerned from the Soret to band I (S/I) ratio. That is, the porphyrin r hodins yield S/I > 10 and the chlorophyll-b-series (dihydroporphyrin) rhodins display values of (Table 10: cf. Jones et . 1968). However, it can easily be imagined that impure geologic isolates, usually with a non-tetrapyrrole UV/VIS background (cf. Baker and Smith, 1975b; Baker et . 1976), cannot be definitely stated as porphyrin or dihydroporphyrin on the bases of S/I-values or visible band orders alone The developed scheme of chromophore manipulation (Fig. 49) allows definitive identification of the macrocyclic chromophore quite easily and with only trace ( < 1-2 quantities required.

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1.0 . (al I I ( b ,. 0.8 II w I \ u z I fl <{ 0.6 m /v \ rjl 0::: 0 II ___ ) 0.4 <{ j \ -o .o 1 ... '"''-" r 1 350 550 750 350 550 750 WAVELENGTH(nm) Figure 54. Comparison of the electronic absorption spectra of (a) pyrrorhodin-XV [XLVI], (b)'oxy-deoxo' pyrrorhodin-XV[XLVII] and their copper derivatives(dashed). Visible portion for the copper derivative is scale expanded and the baseline shifted upwards for clarity.Solvent = ethy l ether. 00 V1

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186 Borohydride reduction of the conjugated carbonyl in pyrorhodin ([XLVI] Fig. 54a, solid trace) results in the DO-derivative [XLVII] which exhibits the phyllo-modified ETIO-type (Fig. 46) visible spectrum shown as Figure 54b (solid trace). Aside from revealing a true porphyrin visible spectrum, 00-pyrrorhodin [XLVII] yields an S/I value of 34 (Table 10). That is, with few yet easily discerned exceptions, porphyrins and dihydroporphyrins exhibit S/I values of >>10 and <5, respectively (Table 10). At this point, the results of NaBH4 reduction and UV/VIS alone are adequate to classify the prime (macrocycle) chromophore as either porphyrin or dihydroporphyrin (see [XXVIII] and [XXI] in Table 10). Confirmation of the above conclusions is attainable through conversion of the rhodin to its copper chelate ([XCV] Fig. 54a-dashed) and subsequent reductive conversion to the 00 derivative ([XCVI] Fig. 54b-dashed). Both compounds yield electronic absorption spectra quite typical of metallo-porphyrins (Fig. 45b) and dissimilar to those of metallo-dihydroporphyrins (Figs. 48, 50-51: Tables 10-11). That is, the Cu-pyrrorhodin [XCV] and Cu-00-pyrrorhodin [XCVI] derivatives display a/B bands and yield large S/a values (Table 11). The results and discussion above reveal that with electronic absorption spectroscopy and facile in vitro chemistry one is easily able to 'probe' and identify the chromophore of tetrapyrrole pigments, be they synthetic or geochemical isolates. The resultant analytical methodology (Fig. 49) was found especially useful in the identification of geologic dihyrdoporphyrins, as these chl?rins and phorbides usually yield only uniterpretable pyrolytic (DIP-EI) mass spectra.

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Second-Derivative Absorption Spectroscopy 187 Frequently during the present studies, isolates or chromatographic fractions were encountered in which prime absorption bands, most notably the highly diagnostic band-! or a-band of free-base dihydroporphyrins or metalloporphyrins, respectively, were accompanied by over lapping side bands ('shoulder,' 'inflection') of uncertain origin. In order to aid in the interpretation of these more minor absorbing species, prior to selecting additional separation technique and/or attempting quantitation, the use of derivative spectroscopy was investigated. Derivative spectra were collected with a Perkin-Elmer model 575 spectrophotometer (Coleman Instruments) coupled to a PerkinElmer model C570-0729 (Hitachi Ltd.) derivative function accessory. Early on during this phase of study it was found that the first derivative lacked the sensitivity to adequately locate the positions of maxima appearing as overlapping or 'side-bands' in tetrapyrrole spectra. Thus, the more highly sensitive technique of second derivative spectroscopy was used exclusively herein. The manners in which overlapping bands affect the recorded or 'apparent' position and extinction values of absorption max1ma is well covered by others (Rao, 1967). Graphically, these effects are shown in Figure 55. Pertinent to the present study, one can well note, that for bands of similar widths and Gausian energy distributions, the formation of two distinct peaks relies mainly upon the wavelength separation of the contributing maxima and the quantitative contributions ('relative extinction') of each.

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5:1 0 r
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189 Based on the above, a variety of binary pigment mixtures were made using the standards (Appendix A} available. Choice of these mixtures were made with attention to the actuality and potentiality of similar melanges in geochemical samples. Unless otherwise given, the ratios cited for test mixtures are on a molar molar/molar)' basis rather than by extinction. Table 12 contains the seven sets of test mixtures thought to be most pertinent to geochemical analyses. Maxima given here and calcu lated differences derive from Tables 10 and 11 as well as from the in depth data given in Appendix A. Interest in the (-7 [XXIV], -18 [XXVI]) as a minor yet diagenetically important series stemmed from earlier studies on DSDP/IPOD cores (Baker and Louda, 1980a; Louda et 1980}. Herein, test mixtures of pheophorbide-a ME [VIb], as a spectra representative of non-purpurin type dihydroporphyrins ('chlorins'}, and either purpurin-7-TME [XXIV] or purpurin-18-ME [XXVIb] were made in mixtures of 6:1, 4:1, 2:1, 1:1, 1:2, and 1:4, on the basis of absorbance. Selected spectral overlays from this series is given in Figure 56. As can be noted from these spectra and Table 12, separation of band I maxima by more than about 25-30 nm yields separate peaks except at very low concentrations of one species Fig. 56b; 6:1, pheophorbide -a: purpurin-18}. Second derivative techniques allowed easy location of the 'true' maxima in these cases. That is, the recorded maxima for pheophorbide-a [VIb] and purpurin-18 [XXVIb] drift towards each other by about 2 nm when mixtures yielding similar absorbance (1:1, 1:2: Fig. 56b} are measured (cf. Rao, 1967). In the case of purpurin-? [XXIV], separation of band I maxima, relative to pheophorbide-a [VIb],

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Table 12. Binary mixtures of tetrapyrrole pigments and their band separation values used in the study of second derivative electronic absorption spectroscopy. PIGMENTS MIXED1 WAVELENG1H(nm)2 MAJOR I MINOR ALPHA OR BAND I POSITION (run) CDMPONENf MAJOR MINOR SEPARATION Pbide -aiPur7 666 678 12 Pbide-aiPur-18 666 696 30 Ni-P H I Ni-PE 555 582 27 Ni-PH I Ni-Benz 555 568 13 Ni-PH/VO-PH1 555 572 17 VO-PH21 VO-Benz 572 590 18 FOOTNOTES: 190 1 )P igment code: Pbide a = pheophorbide-a ME[VIb];Pur-7 = purpurin 7 -TME[XXIV]; Pur-18 = purpurin-18-ME(XXVI];NiPH = N i deoxophylloerythrin ME (LXXXVI] ;Ni -PE = Ni phylloerythrin ME (LXXVII]; Ni-Benz = Ni-'monobenzoetioporphyrin '[CXVII];VO-PHl= VO etioporphyrin-III [CVIII]; VO-PH2 = VO deoxophylloer ythroetioporphyrin[LXXXVIII]; Vo-Benz = VO 'monobenzoetioporphyrin[CXVIII] 2) Spectroscopic data reported in Tables X and XI,ro unded to integer values here.

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1.0 (a I I (bl z -0:: ::J 0.... 0:: ::J 0.... w l I\ /{\ \ I l I\ 1\ I u I t4 A-l/ if;\ 1 : 2 0 I 1 : 1 w I 2 : 1 0.... l.L 4 : 1 0 0 0 0 I -....;-'-... 1 -I -, 1 6 : 1 f= I I I I I I I I I I I I I I I I 1..0 f-->

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192 is only 12 nm and the true positions of both maxima are hidden in all cases (Fig. 56a). Again, second derivative recordings allowed l ocation of these two peaks. As an example of second derivative data (Figure 57), a geochemical fraction was chosen in order to point out a complication when comparing mixtures of neat standard and natural mixtures. In this case, as subsequent chromatographic separation proved, this fraction consisted of pheophqrbide-a [VIa] and purpurin-18 [XXVIa] as the free-acids, plus certain carotenoids absorbing at about 420-480 nm. Here, the apparent ratio of pheophorbide-a [VIa] to purpurin-18 [XXVIa] mimics that of the 6:1 to 4:1 synthetic mixtures (Fig. 56b), on the basis of relative absorbances. However, the band I contribution of purpurin-18 [XXVIa] appears only as a shoulder in the geochemical mix (Fig. 57a), while it is a true peak in the standard preparation (Fig. 56b). This alteration of spectral behavior when comparing geochemical and synthetic mixtures is most likely due to at least two, and possibly more, factors. First, a non-tetrapyrrole UV/VIS background, decreasing asymptotically from higher to lower energies, is often present. Second, the effect of isomers (alkylated, dealkylated, etc.) is unknown and most likely variable between geologic samples However, that the minor pigment in this mixture had the electronic chromophore of purpurin-18 [XXVIa] was easily shown through second derivative spec troscopy. Though the main interest was centered upon location of band I (696 nm: Fig. 57a), the additional datum of the presence of strong absorption at 538 nm (compound [XXVI], band III: Table 10, Fig 56b) afforded substantiation of the minor pigment as being purpurin-18 [XXVI] at least in chromophoric structure.

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1.0 'a) 406 0 8 w <( en a::: 0 (/) 0.2 +4 +2 l/) 1-z ::J >-0 II II I I 0::: !::: D 0::: <( -2 -4 350 450 550 650 (1-.,nm) 750 Figure 57. The zero order (a) and second derivative (b) electronic absorption spectrum of a geochemical chlorin' fraction containing pheophorbide-a and purpurin-18 pigments. Asterisks point out characteristic absorptions due to purpurin-18.Solvent = ethyl ether. f--" \..0 w

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194 During the development of a separation scheme {Fig. 27) with which to investigate the metallo-{Ni, VO-) porphyrins of bitumens, it soon became apparent that a rapid method with which to discern the probable identity of co-chromatographic species with alternate chromophores was needed. Second derivative spectroscopy provided the needed identifications after the following tests with standard pigments. Four test mixtures (Table 12) were investigated, again based on known geochemical co-occurrence of the various tetrapyrrole chelates. In each case, it was found that the minor pigment of interest in each binary mixture also exhibited a-band absorption at longer wavelengths than did the major pigment. This was especially fortuitous in that this absorption neighbored a spectrally clear region (ca. 1 >600 nm) and allowed heightened sensitivity in the region of the bands of interest (1 570-590 nm). The main goal of the use of second derivative technique with the metalloporphyrins was to extract the position of a-band absorption due the minor species. Therefore, for 3 of the 4 binary sets {Ni-PH/VO-PH, Ni-PH/Ni-PE, Ni-PH/Ni-BENZ: Table 12) arbitrary mixtures were made by doping a solution of the major with the minor pigment until the absorbance of the long wave a-band was of a relative height similar to that found with natural samples. Back calculations revealed that these three mixtures included about 4-20% (molar) of the minor pigment. Nickel alkyl porphyrins are now known to be accompanied by their 9-keto precursors the Ni phylloerythrins, in certain marine environments (Baker and Louda, 1982, 1986a). Figure 58a is the absorption spectrum and second derivative of same for a mix of Ni-DPE ME [LXXXVI] and Ni-PE-ME [LXXVIII]. The data in Table 12 reveals separation of the

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wlfl >I-z + >=> a:r wa:o 0<( a: o .... z-u <( -2 w If) -3 0 6 ,a) w u z <( 0 4 (() a: 0 If) (() <( 0 2 I 0 0 I I I ==J I I b) I lc) 500 600 400 600 400 500 600 WAVELENGTH(nml Figure 58 Electronic absorption (lower) and second derivative (upper) spectra of test mixtures containing nickel deoxophylloerythrin ME [LXXXVI] and (a) nickel phylloerythrin ME [LXXVIII], (b) nickel 'monobenzoetioporphyrin [CXVII] or (c) vanadyl etioporphyrin-III [CVIII].Solvent = benzene.Minor component added at about 10 molar percent. Absorption spectra(lower) and second derivatives(upper) recorded from 450-650 nm, with the identified alpha band of the minor component shown in the upper figures.

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196 contributing a-band maxima by 27 nm. In the present case, that being with standards, this amount of band separation is sufficient enough that the individual bands retain integrity as maxima ('peaks') and location by slow/stop scanning for maximal absorbance is adequate to locate their positions. However, the second derivative substantiates identification and allows easier interpretation with less pure geochemical mixtures. The co-occurrence of Ni-benzoporphyrins (g.g. [CXVIII]) with the more common Ni-alkyl porphyrins of the DPEP-and ETIO-series has only recently been shown (Baker and Louda, 1986b). The present electronic spectral data contributed to that report and others herein. Mixture of Ni-DPE-ME [LXXXVI] and Ni-benzoetioporphyrin ('Ni-BENZ' [CXVII]), with an a-band separation of only 13 nm (Table 12), resulted in the spectral data given in Figure 58b. Test mixtures included Ni-BENZ [CXVII] as 5-35% (molar) of the total. When Ni-BENZ [CXVII] was present at levels below about 3% it was undetectable. In those cases where the a-band of Ni-BENZ [CXVII] created a discernable shoulder or side band to the red of the nickel alkyl porphyrin absorbance Ni-BENZ), the second derivative (Fig. 58b) allowed positioning of this band (Aa = 568) and afforded extension of this technique to studies on Ni-geoporphyrin mixtures. Re-examination of the analytical scheme (Fig. 27), used herein for the study of geologic Ni-/VO-porphyrin mixtures, shows that during the first crude separation the presence of 'non-polar' (high-mw) vanadyl porphyrins in fraction 1 is possible. Examination of second derivative spectra now allows us to discern the identity of the chromophore responsible for how energy side bands ('shoulders') in the electronic

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197 spectra of otherwise Ni-alkyl-porphyrin fractions. In order to test the ability of the second derivative to discriminate between Ni-and VO-porphyrin a-bands, when the VO-species is minor, a combination of Ni-DPE-ME [LXXXVI] and VO-etioporphyrin-III [CVIII] was chosen. This mixture afforded the minimum separation of Ni-and VO-porphyrin a-bands (17 nm: Table 12), when considering only the OPEP-and ETIO-series (Table 11), and allowed a more stringent test. The spectra resulting from such Ni-/VO-mixtures are typified by the sample given in Figure 58c. The second derivative again afforded easy location of the maximum due to the minor (VO-PH: Aa = 571 nm) species. Due to a detailed investigation of vanadyl alkyl-plus benzo porphyrins, comparing electronic and mass spectral methods of quantitation, an exacting series of mixtures were prepared This series consisted of VO-benzoetioporphyrin (VO-BENZ [CXVIII]) forming 0.0, 1.0, 2.0, 4.0, 8.0, 16.0 or 32.0% (molar) of the total, with VO-etiopor phyrin-III [CVIII] making up the remaining majority In this study (Fig. 59), only at the 1.0% level could the presence of VO-BENZ [CXVIII] not be shown nor its a-band(s) positioned via second derivative spectroscopy. However, in 'routine' analyses VO-BENZ [CXVIII] might be overlooked at levels under 4-5% of the total. That is, until the benzoporphyrins constitute about 4-5 % of the total a noticable shoulder to the red of the dominant VO-alkyl-porphyrin a-band is not present. Thus, in cases where the minor presence of VO-benzoporphyrins could be geochemical significance, it is suggested that second derivative stud y of electronic spectra be employed. As revealed above, the application of second derivative technique with electronic absorption spectroscop y affords enhanced initial

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1 1 a) b) c) uL_._ U) 1-* z :J >-1 0: 1--2 w > -ld) e) f) 0 0 z 0 0 u w U) * 1 -2 I I I 500 600 500 600 500 600 WAVELENGTH(nm) Figure 59. Second derivative electronic absorption spectra of various test mixtures made with authentic vanadyl etioporphyrin-III[CVIII] and vanadyl 'monobenzoetioporphyrin' [CXVIII].Mixtures contain (a) 1.0, (b) 2 .0, (c) 4 .0, (d) 8 .0, (e) 16.0 and (f) 32.0 molar percent of the second component(see Table 14). Solvent = benzene.Asterisk indicates the alpha band of the VO-benzoporphyrin( 59 0.5 nm). ...... \.0 CX>

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identifications for a variety of the minor tetrapyrrole pigments present in geochemical arrays. Tetrapyrrole Quantitation Using Electronic Absorption Spectroscopy 199 Throughout the history of organic geochemistry, a variety of UV/VIS techniques for the quantitation or 'quantitative estimation' of geologic tetrapyrroles have been used. The earliest methods, aimed at the metalloporphyrins, included harsh demetallation techniques (HBr in acetic acid), acidic (HCl)/ organic partition and spectrophotometric determination (band I e xtinction) of the in vitro free-base porphyrins (Groennings, 1953; Treibs, 1936). Similar demetallation technique, but with quantitation by integration of the Soret band absorbance above background for porphyrin present as dications in the acidic reaction mixture, was also tried (Costantinides et gl. 1959). Quantitation of free-base porphyrins, derived by neutralization of demetallated (HBr-formic acid) products, with direct Soret band absorbancy has been reported (Sugihara and Garvey, 1964). Again, based upon demetallation and the generation of porphyrin dications, direct absorbance of the dication (A = 546 nm, = 17 x 103 ) visible maximum has also been used (Baker et gl. 1967; Erdman, 1965). All of the above methodologies include harsh acid demetallation and purification steps prior to quantitation. In all of these reports the severe drawbacks not only of pigment destruction but of non-reproducibility were admitted. In attempts to avoid the deleterious effects of demetallation, Sugihara and Bean (1962) reported metalloporphyrin quantitation in oils by integration of the native Soret absorption above background This

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200 technique, however, relied upon the colorimetric determination of total Ni and V and the supposition that the elemental V/Ni value was identical to that of the porphyrinic subset and extinction coefficients were approximated by interpolation between end members (Ni-PH, VO-PH). Further, the direct integration method was noted (Sugihara and Bean, 1962) to be inapplicable to bitumen with "low" (< 600 f..lg/g-oil: calculated from the value of 1.35 f..lmole/g-oil given in Sugihara and Bean, 1962) concentrations of total metalloporphyrins. A switch to the use of a-band absorbance and extinction coefficients for the quantitation of intact ('native') geologic metallopor phyrins came in the 1960-1970 years (g.g. Baker et 1976; Millson et 1966) and persists to date. As detailed below, all quantitation of geologic tetrapyrrole pigments in the present studies utilizes Beer-Lambert relationships and employ published millimolar (emM) or specific (a) extinction coefficients for the major low energy (a-band, band-!) absorption. When dealing with geologic tetrapyrrole mixtures or isolates, the use of these longer wavelength absorption bands is especially important in that the effect of non-pigment UV/VIS absorbing species is minimized. That is, the spectral 'signal-to-noise' ratio is enhanced. Aside from the metalloporphyrins, there are essentially two other classes of tetrapyrrole pigments which require attention as to their quantitation. These are free-base porphyrins and dihydroporphyrins, both free-base and metallo. Free-base porphyrins have classically been (Blumer and Omenn, 1961; Thomas and Blumer, 1964; Treibs, 1934a, 1935b) and are today (Baker and Louda, 1982, 1984, 1986b; Baker and Palmer, 1978; Louda and

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201 Baker, 1981) quantified using their characteristic band-! (see Fig. 46) absorption maxima (1 = 615-640 nm). Dihydroporphyrins are usually free-base species, excepting chlorophylls (Mg chelates: see [I], [XIV], [XIX]) per se, in aphotic sediments (Baker and Louda, 1986a; Keely and Brereton, 1986; Louda and Baker, 1986). The quantitation of chlorophyll degradation products, whether in water column detritus or sediments, has to date, for the most part, involved gross estimates using a single extinction value. It must be noted that the studies mentioned here had productivity, standing crop, mixed layer dynamics or similar goals and are often only marginally related to chlorophyll diagenesis in the strictest sense. Some of the first attempts to quantify chlorophyll and its breakdown products in natural waters involved the use of compl icated equations applied to UV/VIS spectra of crude extracts ( L orenzen, 1967ab; Parsons and Strickland, 1963; Rai, 1973; Richards and Thompson, 1952). Similarly, early examinations of fresh water (Val lentyne, 1955, 1960) or marine (Orr and Grady, 1957; Orr et gl., 1958) sediments relied on the use of the extinction coefficient for only 1 pigment, pheophytin-a [IIa], during 'quantitation' of total crude extracts. Oceanographic and limnologic interest in productivity and water column dynamics has lead to more intense research and greater advances in chlorophyll degradation, relative to organic geochemistry per se. The estimation of chlorophyll and total 'pheopigments,' having lost the chelated Mg atom, is often performed using the fluorescence intensity of crude extracts before (fo) and after (fa) acidification, which elicits total demetallation (Yentsch, 1965; Yentsch and

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202 Menzel, 1963). These data, CHL/PHEO via Fa/Fa, together with similar results from the studies using direct absorption spectrometric equa tions given above, do provide geochemical insight. That is, the documented rapid and essentially total conversion of chlorophylls to pheopigments in the water column directs the geochemist to consideration of the latter as the immediate geopigment precursors. Distinction between 'chlorophylls' and 'pheopigments' does not, however, aid in the quantitative-structural study of tetrapyrrole diagenesis. Certain water column studies are reported in which individual pigments or pigment-types are separated prior to quantitation A first and important advance in these studies was the formation of a distinction between and quantifying pheophytins and pheophorbides (Currie, 1962; Patterson and Parsons, 1963). That is, chlorophyll derivatives without Mg and either retaining or having lost the phytyl (C20H39) moiety. Chromatographic advances, including TLC (Garside and Riley, 1969; Jeffrey, 1974, 1976, 1980) and HPLC (Abaychi and Riley, 1979; Bidigare et gJ., 1985; Gieskes and Kraay, 1983; Mantoura and Llewellyn 1983), are now employed for the 'routine' separation and subsequent (TLC) or concurrent (HPLC) quantification of chlorophyll breakdown products in the water column. To date, the application of single separation step prior to the quantification of early diagenetic chlorophyll degradation products remains an elusive quarry. In the present study emphasis was placed upon separation techniques (Table 4; Figs. 21, 27) which would first split geologic tetrapyrrole arrays by gross polarity differences and thus allow the application of higher resolution techniques to discrimi nate among the more subtle structural differences.

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203 In the present study, all quantitative estimates were performed by measuring the long wave tetrapyrrole absorption above the non-pigment UV/VIS background. In most cases, the delineation of 'background' was obtained by a visual best French curve fit from the 'red' 700-900 nm) asymptote to the intercept of absorption at 450 or 475 nm for freebase or metallo species, respectively. In certain cases where the isolate exhibited 'dirty' UV/VIS (i.g. high non-pigment to pigment ratio) the same procedure was followed except the lower wavelength intercept was moved bathochromically to the region of lowest energy still yielding tetrapyrrole absorption, either as inflections or true maxima. Examples of background delineation, as utilized herein, are provided in Figure 60a-b. In cases involving the estimation of co chromatographic species with differing band-! or a-band absorption (g.g. pheophorbide-a [Vlb]/purpurin-18 [XXVI], Ni-alkyl porphyrins/Niphylloerythrins, VO-alkyl-/VO-benzo-porphyrins) each band was assumed to be perfectly Gaussian and separately extended to the non-pigment background. This is shown in Figure 60d and the absorbance recorded in each case is that from the maximum down to the nearest interference from background, either non-pigment or overlapping pigment extinction. The first test cases used to evaluate the quantification of tetrapyrrole mixtures containing different chromophoric species involved the mixture of known amounts of pheophorbide-a-ME [Vlb] with either purpurin-7-TME [XXIV] or purpurin-18-ME [XXVI]. These test mixtures were previously discussed in conjunction with the second derivative study and sample spectra were given in Figure 56. The exact known molar ratios and those calculated from resultant mixture spectra are given within Table 13. Calculation of each species contribution

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204 1 0 a) 0 8 0 6 A 0.4 0.2 w u 0.0 z
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205 Table 13 .. Quantitaion of dihydroporphyrins: Estimation of or purpurin-18 in the presence of pheophorb1de-a, as calculated from synthetic known A I A Pheophorbide-a[Vlb] 6:1 4:1 2:1 1:1 1:2 1:4 Pheophorbide-a(Vlb] 6:1 4:1 2:1 1:1 1:2 1:4 FOCJINai'ES : ACTUAL I Purpurin-7[XXIV] 3.53:1 2.35:1 1.18:1 0.59:1 0.29:1 0.15:1 I Purpurin-18(XXVI] 6.91:1 4.60:1 2.30:1 1.15:1 0.58:1 0.52:1 CALCUlATED (corrected) 6.86:1 (lnc) 3.82:1 (3.58:1) 1. 99: 1 ( 1. 66 : 1) 1.54:1 (0.99:1) 0.98:1 (0.46:1) 0.72:1 (0.25:1) 8.00:1 (lnc) 5.42:1 (lnc) 2.73 :1 (lnc) 1.54:1 (0 86:1) 0.82:1 (0 7 1 :1) 0.52:1 (0.36:1) Visible spectra of these mixtures are given as Figure 56. Mixtures made on the basis of absorption(A) and converted to molar ratios('ACTUAL') using the following extinction coefficients: Pheophorbide-a(Vlb], tmM = 52.76 at 666nm(Stern and Wenderlein,1935a; Purpurin-7[XXIV], tmM = 30.25 at 678nm(Stern and Wenderlein,1936b), Purpurin-18[XXVI], tmM= 58.25 at 696nm(Stern and Wenderlein,1936b). 'CALCULATED' molar ratios were made by direct measure of the resultant absorption for each species. '(corrected)' estimates were made following correction for overlappi n g absorption,as shown in Figure 60 and assumed Gaussian band profiles. 'lnc' = little or no change

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206 (individual and relative quantities), to the mixture involved drawing best-fit backgrounds and band overlaps, assuming true leptokurtic Gaussian band shapes (energy distributions), as given above. The present calculated values (Table 13) were obtained in two manners. First, when only the low energy ('red') side of pheophorbide-a ME [VIb] band I was extended in this manner always overest imated the presence of pheophorbide-a ME [VIb] was always overestimated due to an imparted hyperchromic effect on its band-! extinction in concert with the uncorrected additive effects of underlying purpurin based absorption Somewhat better estimations were possible when both contributing bands were drawn as Gaussian curves to their high and low energy sides. The recorded absorption of pheophorbide-a ME [VIb] or the coincident purpurin was then taken only between the maximum and the appropriate overlapping extinction. Even with such correction for overlaps in band-! absorptions, it was found that the species with the higher energy maximum, pheophorbide-a ME [VIb] in these cases, was slightly overestimated. Presumably, this represents the hyperchromic addition of less intense bands (g.g. band-II) from the species with the l onger wavelength band-! maximum (purpurins) to the shorter wavelength species. Though the total effects, hyperchromic and hypochromic, upon the recorded apparent absorption of component pigments cannot be completely corrected for, these studies provide a basis for judging the validity of similar estimations when applied to geologic pigment fractions. Lack of significant band-! maxima separation for the mixture of pheophorbide-a ME ([VIb], A1 = 666nm) and purpurin-7-TME ([XXIV], A1 = 678 nm), plus the broad band-! energy distribution ('band width') of the later, lead to near intractable spectra. In this case,

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207 errors ranged from 66 to 380%. However, when purpurin-7-TME [XXIV] was the minor species as in the 4 : 1 and 2:1 mixtures (A/A in Table 13), estimates of the correct order of magnitude ( <102 ) were possible In the case of mixtures of the phorbide with purpurin-18 ME [XXVI] the separation of band-! maxima was 30 nm (Table 13), allowing the individual peaks to be e x pressed. Here, the errors i n estimation ranged from 16 to 24% in concert with decreased amounts qf pheophorbide-a ME [VIb] and the resultant overestimation of same due to the uncorrected overlaps of absorption discussed above. Fortuitously, it is the purpurin-18 and not the purpurin-? chromophore which is encountered in early diagenesis (Louda and Baker, 1986) and the present study provides a measure of validity to the quantitative study of purpur in-18 in sedimenta r y bitumen. The geologic vanadyl porphyrins have long been known to be mixtures of VO-al kyl-(g.g. DPEP-plus ETIO-series) and benzo porphyrins (Baker and Louda, 1986a; Baker and Palmer, 1978; Baker et 1967; Barwise and Whitehead, 1980). To date, not one study involving the quantitation of vanadyl geobenzoporphyrins e xists. The abundance of VO-benzoporphyrins, relative to the quantified coincident VO-alkyl-porphyrins, via mass spectral intensities is possible (Baker and Louda, 1986b), but even this is usually not reported in the e xisting literature and mass spectral response efficiencies are also unknown. In an attempt to provide a quantitative method for the estimation of vanadyl alkyl-plus benzo-porphyrins in geochemical isolates, a series of mixtures with precisely (. 1 % ) known abundances of each were prepared The visible absorption spectra of mixtures containing from

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208 1.0 to 32.0 mole percent of VO-'benzoetioporphyrin' ([CXVIII], 'VOBENZ') in solution with VO-etioporphyrin-III ([CVIII], 'VO-ETIO' or 'VO-alkylporphyrin') are given as Figure 61. Using these spectra and the background/band overlap correction techniques given earlier, the apparent concentration (nmole/ml) of each component and the mole percentage as VO-BENZ were calculated (Table 14). Excellent agreement between the calculated and known values was found, though the VO-BENZ portion tended to be underestimated by about 15%. Considering those mixtures with 2-16% (molar) VO-BENZ, the range typical of geologic VOporphyrin arrays (cf. Baker and Louda, 1986a-b; Baker et g}., 1967; Barwise and Whitehead, 1980; Louda and Baker, 1987), we find means of 101.4% and 84.9% for the estimation of VO-alkylporphyrins and VObenzoporphyrins, respectively, in such samples. This implies that the quantitation of VO-alkylporphyrins is extremely good by direct absorption calculations using a-band extinction. Further, the estimation of VO-benzoporphyrins in mixed isolates appears to require a correction of 1.18 x (= 100.0/84.9: Table 14) to approach reality. In the present studies, the above data on the electronic spectral quantitation of vanadyl porphyrins was incorporated into mass spectral experimentation and the interpretation of analytical results with geologic pigments. The exacting quantitation of nickel and vanadyl geoporphyrins is extremely important to the study of tetrapyrrole geochemistry as well as in the assessment of organic maturational and epigenetic (g.g. biodegradation) changes. Aside from certain studies between 1935 and 1965, discussed earlier in this sectibn, little if any attention has been given to assessing the validity of geologic metalloporphyrin

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210 Tabl e 14. Estimatimation of absolute and relative amounts of vanadyl benzoporphyrins in mixtures with vanadyl alkylporphyrins(!). TEST MIX2 3 MOLE% cula t ed) % OF ACTUAL CONCENTRATION4 VO-BENZ voB Z 'o'actuai VO-ETIO VO-BENZ 1.0 1.1 (110 .0) 99. 8 110.1 2 0 1.8 ( 90.0) 98. 2 88. 9 4 0 3 2 ( 80.0) 98. 4 79.1 8.0 7 1 ( 88. 8) 100 6 88.1 16.0 12. 8 ( 80. 0) 108.5 83. 5 32. 0 25.7 ( 80. 3) 116.3 85. 3 x (2 -16%) ( 84.7) 101.4 84. 9 FOOTNOTES: 1)In vitro synthetic mixtur es made using VO-etioporphyrin-III[CVIII] and VO-'mon obenzoetioporphyrin [CXVIII] .Quantitation was via Beer-Lambert relationship using EmM= 31.6(VOOEP,see[L] : Fuhrhop and Smith ,1975 ) and -DTIM= 33. 88(Clezy and Mirza,1982) at 571 and 590. 5 nm for VO-ETIO and VOBENZ,respectively .Estimation of absorbance followed correctio n for background using a 'best-fit' French curve technique(see Figure 60) 2)Given as mol e percent of the mixture as VO-BENZ. 3)Values represent calculated o r apparent percent of the actual percentage of VO-BENZ. 4)Values given a r e percents of the actual concentrations (ng/mL) found after estimation of the amounts o f each component in the various mixtures

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211 quantitation with absorption spectroscopy. The present investigation was therefore a return to basics and the quantitation of nickel and vanadyl porphyrins, as doped into porphyrin-free petroleum fractions serving as a contaminating spectral background, was tested. Nickel-and vanadyl-porphyrin quantitation in bitumen fractions was assessed as follows: Hi-porphyrins; All of the aromatic/hetero aromatic containing fractions from a crude oil, chromatographically freed of nickel porphyrins (see Fig. 27) and their absorption, were pooled and served as an aromatic background: To aliquots of this background were added known quantities of Ni-deoxophylloerthyrin ME ([LXXXVI], Ni-DPE-ME) in benzene, a pigment with the exact chromophore of Ni-DPEP [XC]. The component and resultant electronic spectra are given as Figures 62a and 62b, respectively. Subsequently, background correction, as given earlier, and measurement of the absorption at about 552-555 nm allowed calculation of the apparent total Hiporphyrins (Table 15). Examination of these data reveals that, on the whole, Hi-porphyrins in petrogenic aromatic fractions are underestimated by about 8%. The mean found was that the calculated 92.0% of reality. Thus, a correction factor of 1.09 x (= 100.0/92.0: Table 15) was added to the quantitation of Hi-porphyrins isolated during the separation of petroleum like bitumen (Fig. 27). Nickel porphyrins exhibiting electronic spectra close to the 'purity' free of spectral background) of standards (Fig. 62a) were estimated directly and without this correction factor. VO-porphyrins; In like manner, test mixtures of a vanadyl alkylporphyrin (VO-OEP: see compound [L]) were added to an essentially petroleum resin-like back ground. In this case, the background consisted of Golden Eagle crude

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1.0 -r---------------, 0.8 w 0.6 <( 0) 0: 0 V) 04 0) <( (a 0.0 I I =;:--.... I i 350 550 750 350 WAVELENGTH, nm . 550 (b 3 X rr c .,
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Table 15. Estimation of the accuracy of calculating nickel and vanadyl porphyrins in the presence of nonpigment background. NICKEL PORPHYRINS! MIXIURE (See Fig.62) 1 2 3 4 5 VANADYL PORPHYRINS! MIX'IURE (See Fig.63) 1 2 3 4 5 6 7 8 9 10 FCXJTNOTES: 12 m1crograms,tota ADDED CALCUlATED PERcrnr-3 F.STIMATED 6.32 5.74 12.65 10.94 25.30 24.84 50.60 47.22 101.20 92.95 12 m1crograms,tota 90.8 u 86.2 98.2 93.3 91.8 MFAN = 92.0% ADDED CAlCULATED F.STIMATED 0.84 2.10 3.35 4.19 8 .39 20.96 33.54 41.93 83.86 125.79 1.15 2 .08 4.31 5.02 8.58 21.80 33.23 42.76 83.41 123.74 137.1 99.2 128. 5 119.7 102.3 u 104.0 99.1 102.0 99.5 98.4 MFAN(#l-10)108.9% MFAN(#510)100.9% 1) Mixtures made by adding Ni deoxophylloerythrin-ME[LXXXVI] or VO octaethylporphyrin(see [L]) to pigment-free aromatic or resin fraction o f petroleum,respectively.Background was checked for the absence of metalloporphyrin-like absorption with both zero-order and second derivative electronic absorption spectroscopy. 2) 'Ni porphyrins'calculated using [ mM = 34,82 at 554nm(Fuhrhop and Smith, 1975). 'VO porphyrins'calculated usingcmM= 26.14 at 571.5nm(Falk,1964). 3)"PERCENT F.STIMATED" refers to the amount calculated following the admixture of a known amount of metallo-porphyrin to the background. U At this stage the alpha band had become a true peak. At this stage the beta band had become a peak. 213

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214 oil from the Big Horn basin. Golden Eagle, on the bases of analyses of over 200 g of the crude, has been shown to be essentially void (<< 2 of VO-porphyrins (Baker et gJ. 1987) and the electronic spectra of the whole crude (Fig. 63a) lacks metalloporphyrin absorption bands. A slight indication of the vanadyl porphyrin Soret (A = 407 nm) could be found with second derivative technique but the visible region (A 450-900 nm), the region of interest, was found to consist only of the asymptotic background required herein. The component and resultant spectra for mixtures of VO-alkylporphyrin and petroleum background are given in Figures 63a and 63b, respectively. Application of the usual best-fit French curve background to these spectra (Fig. 63b) then allowed measurement of the VO-OEP a-band absorbance and calculation of the apparent total pigment in each mixture (Table 15). Considering first all ten mixtures, a mean of 108.9%, comparing calculated to known, results and implies an overestimation of 8-10% for the quantification of VO-geoporphyrins. However, if one examines the deviation above 100%, it is found that essentially all of this overestimation occurred in the first four test mixtures (Table 15). E x amination of the 'quality' of VO-porphyrin absorbance above background in the original spectra (Fig. 63b) one can note that in mixture 5, and continuing in all subsequent mixtures, the a-band absorption is present as a true maximum ('peak'). This contrasts with mix es #1-4 in which the aband maxima are in evidence as shoulders only Thus, if we consider only those mixtures yielding a-band as peaks (#510; Table 15) then a mean of 100.9%, with little fluctuation (-1.6 to +4.0%), results. These results militate direct calculation of VO-porphyrin concentra-

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I I I I PORPHY O .BJ\ /FREE RIN-I o1usicl'R"6lE 0 6 w u z <( lD a: 0 <( 0.2 0. 0 I '-I / ...._, \:::::::=-350 I I I I 650 WAVELENGTH(nm) 1 0 1 \ b) 0 8 0.6 w u \\ '\\\ I \\\VI\\ A 1 Crrr-1=2i14 z <( lD a: 0 <( 0.2 0.0 I I I I ---I I 350 450 550 650 750 WAVELENGTH(nm) Figure 63 Quantitation of vanadyl porphyrins using electronic absorption spectra contaminated by non-porphyrin background. (a) Spectra of VO-OEP[L](solid) and a crude petroleum determined to be porphyrin free(as indicated). (b) Test mixtures as given in Table 15. N 1-'Vl

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216 concentrations in fractions or isolates (Fig. 27) from bitumen without the addition of a correction factor, as found necessary for Niporphyrins. Throughout these studies, quantitation of tetrapyrrole pigments was via application of Beer-Lambert formulae to recorded absorbances and volumes and using the most appropriate extinction coefficients as gleaned from literature Table 16 is a compilation of extinction coefficients and the geologic pigments or fractions to which they were applied. All of the data given in this section was obtained through the investigation of the electronic spectral characteristics and behavior of authentic/standard tetrapyrrole pigments and quantitative techniques employed published extinction coefficients. These points are reiterated here as a partial disclaimer. That is, during the study of tetrapyrrole geochemistry, even though the principle chromophores are identical in many cases, the overall spectral effects of isomeric forms and mixtures cannot be adequately addressed. This drawback, especially in obtaining accurate extinction coefficients, is likely to persist, perhaps indefinitely, since each new sample will more often than not yield mixtures different from those before. However, the data base and conclusions herein now show that estimates well below order-of magnitude levels and usually with 100 5-10% confidence can indeed be obtained through attention to basics. Further, the qualitative aspects of electronic absorption spectroscopy, when coupled to simple derivatization techniques (NaBH4 63Cu-metallation), were shown to be effective tools in the identification of geologic tetrapyrrole chromophores, a first step towards structural elucidation. This latter point is

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217 Table 16. Selected standard pigments(1) their extinction coefficients, molecular weights and carbon numbers as used herein for the quantiation of geological tetrapyrroles. STANDARD COMPOUND2 mw 2(C#) 3 A.,run Ent1 ( a ) 4 Pheophytin-a[IIa]a 870 ( 55) 666.5e 43.03 ( 63.7 ) A (B) Pheophorbide a[VI]b 592 (35) 666.5e 52.76 c Chlorin e6[XXIII] c 596 (3 4) 665.0 e 53.25 c Bacteriopheophytin-a[XXI] 888 (55) 757.0e (7 6 0) (B) Pyropheophorbide -a[VIII] 534 (33) 666.5e 51.78 c MesopyrofheoJhorbide-a[X] 536 (33) 656.0e 47.51* D OOMPP-a XII 522 (33) 639.6e 43. 0 E Chlorin -p6[XXVII] 582 (3 3) 670.8e 46.66 F Purpurin-18 [XXVI] 550 (3 2) 695. 5e 58.25 D PE [XXXII] 534 (3 3) 639.0b 2.3 c OPE tXXXVII] 520 (33) 619.5b 6.54 c DPEP XXXVIII]d 476 (32) 619.5b 7.3 G Ni-PE tLXXVIII] 590 ( 33) 582.2b 17.0 H Ni-DPE LXXXVI) 576 (33) 554.5b 19.0 H Ni-DPEP .[XC] 532 (32) 554.5b 20.0 H Ni-Mesoporphyrin-IX[CIX]ef '532' (32) 551.5b 34.82 I Ni-'Benzoetio -PH' [CXVII) 540 (33) 568.5b J VO-Mesoporphyrin-IX[CVI] g '543' (32) 572.2b 26.14 I VO-DPEP [LXXXVIII] 541 (32) 574.0b 21. 0 H VO-'Benzoetio-PH' 549 (33) 590.5b 33.88 K Cu-Mesoporphyrin-IX[CII] (28) 562.5b 25.70 I Chlorophyll(s)c[XIX] 608 (35) 628 e 22.0 B FOOTNOTES: 1)See APPENDIX-A for structures,complete nomenclature and additional spectral information (cf. Tables X,XI). 2)Molecular weights(mw)and carbon numbers(C# )are given for free acid forms of carboxylic acid pigments or for the 'most common'gelogic pigment in certain cases. Abbreviations:OOMPP-a = deoxomesopyropheophorbide-a;PE= phylloerythrin;DPE -DPEP= porphyrin;'Benzoetio = 'monobenzoetioporphyrin (see[CXVI]in App.A). Geopigment guantitation:Aside from quantitation o f specific pigments, as given,certain values were used for geological tetrapyrrole arrays, these being(by superscript): a -c= fractions 'CELL-1' -'CELL-3'(see Table IV),repectively;d = freebase porphyrins;e = Ni-porphyrins; and i = Cu-highly dealkylated etioporphyrins(Cu-HDEs:Baker and Louda,1984). 3)Visible maximum recorded in this study(Tables X,XI.App.A).Spectra recorded in or indicated. 4) I: mM in lmM em and (a) in lg em (*)Value cited is for mesopheophorbide-a. (":-:':)Calculated as anti-log of reported log I: 5)REFERENCES: A=Stern and Wenderlein,1936a; B=Smith and Benitez,1955; C-Stern and Wenderlein,1935a; D= Stern and Wenderlein,1935b; E= Zelmer and Man,1983;F= Stern and Wenderlein,1936d;G= Baker et al. 1968;H= Hodgson and Baker,1967;I= Stern and Dezelie,1937d;J=Clezy et al.,1977; K=Clezy and Mirza,1982.

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218 especially important to the study of the dihydroporphyrins (phorbides, purpurins, chlorins) characteristic of early diagenesis. Mass Spectrometric Analyses Interest in the mass spectrometry of geologic metalloporphyrins first surfaced in the early 1960's. These initial studies included determination of the mass spectra for nickel etioporphyrin-III (Ni[LII]; Hood et gl., 1960) and vanadyl etioporphyrin-! ([CVII]; Mead and Wilde, 1961). In the realm of geochemical isolates, Dean and Whitehead (1963) reported the high energy MS of the vanadyl porphyrins in a petroleum distillate and indicated the presence of pseudohomologs from at least C28 to C33. However, they" ... accepted with reserve ... those data and cited the high probe temperatures required to distill the sample as an agent for artifact formation. Substantiation for the concept of 'homologous series' of metalloporphyrins in geologic samples came with r eports on the analyses of oil shale (Morandi and Jensen, 1964, 1966; Thomas and Blumer, 1964). Systematic investigations by Baker and co-workers at the Mellon Institute (Baker 1966; Baker et gl., 1967; Yen et gl. 1969) laid the base for the MS of geologic tetrapyrroles as known today Those studies proved the e x istence not only of pseudohomologous series but also of alternate structural types. Figure 64 is a compilation of known, suspected and suggested geoporphyrin nuclei. The history of the isolation and identification of these various structures can be found in a recent review by the author (Baker and Louda, 1986a) and will not be repeated here. The salient features of these structures (Fig. 64) rest with the concept of mass spectral series. That is, for any given

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a) RWRN...NR R N R b R R R R R e) R R g) R R R R N, N *, R b R R d) R R R h) R R Figure 64. Generalized structures of geoporphyrin mass spectral series.(a) ETIO series,(b)DPEP series,(c)DiDPEP series,(d) tetrahydro-benzo-DPEP series,(e)benzo-DPEP series,(f) benzo-ETIO series, (g) cyclopropano series, (h) cyclobutano series.R = H or alkyl.(Baker and Louda, 1986a). 219

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220 carbon number, these structures will yield molecular weight classes based on di-hydrogen deficiencies, when compared to the most simple porphyrin type (vi z. ETIO, Fig. 64a). Thus, considering the ETIOseries, with a theoretical minimal carbon number of C20 porphyrin [XLVIII] per se), one easily calculates a mass spectral series of 310 + 14n Daltons, where n is an integer, for this type of structure (Fig. 64a). In like manner, any porphyrin containing a cycloalkano ring struc t ure yields a pseudohomologous series of 308 + 14n m/z, where n is an integer above 2. This series is classically referred to as the DPEP-series, after true DPEP [XXXVIII]-like pigments (Fig 64b) containing a cycloethano moiety (Baker et al. 1967; Yen et al., 1969). Recently, due to the identification of geoporphyrins containing cyclopropane (Fig 64g: Chicarelli et al. 1984; Wolff et al., 1984) or cyclobutano (Fig. 64h: Fookes, 1983a; Ocampo et al., 1984; Wolff et al., 1983) ring structures, the term CAP-series, for phyrin, has been suggested (Eckardt et al., 1989). Relative to the ETIO-series ("M": Yen et al., 1969), all of the DPEP-or CAP-series porphyrins are considered M-2 in nominal mass. Not shown in Figure 64 is a possible tetrahydrobenzoetioporphyrin structure (cf. Fig. 64d) which would also fall into the M-2 grouping. Nominal masses at M-4 474 14n m/z) were first thought to represent di-DPEP type (Fig. 64c) structures (Yen et al., 1969). Recently, the M-4 geoporphyrins have been suggested (Barwise and Roberts, 1984) and proven (Verne-Mismer et al. 1987) to be tetrahydrobenzo-DPEP (THBD) structures (Fig. 64d). Porphyrins originally called 'rhoda' or 'petrorhodo,' due to electronic spectral characteristics (Howe, 1961; Millson et al., 1966),

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221 are now known (Baker et gj., 1967; Barwise and Whitehead, 1980; Kaur et gj., 1986) to be benzo-substituted DPEP-and ETIO-structures. These benzo-DPEP (BD: Fig. 64e} and benzo-ETIO (BE: Fig. 64f) structures fall into M-8 and M-6 series, respectively The rapid placement of geoporphyrin molecular ions into the correct type series is made possible only with the use of extensive tabulations. To ease this task, a simple FORTRAN program was developed (Table 17) to handle not only free-base pigments but also any chelated metal with or without axial ligands. Tables 18 through 22 are the generated nominal masses for the M-8, M-6, M-4, M-2 and M series in the forms of free-base, nickel, copper, zinc and vanadyl pigments, respectively. It must be noted that lines 12-13 (viz. THBE/DPEP) of the program (Table 17} are redundant, in that both are for the M-2 series, and one (THBE) should be deleted for future work. Prior to discussion of the mass spectral experimentation and data of the present study, a brief coverage of the parameters extracted from and discriptive of geoporphyrin MS is required. Baker and et gj. (1967} not only provided the benchmark for qualitative mass spectral analysis of geoporphyrin mixtures, but went on to develop quantitative measures. In all, 4 measures or 'parameters' were employed. These were the weighted average mass, band width, skewness and the DPEP-toETIO ratio. Of these, band width (= standard deviation) and skewness (= assymetry) have either found little use in geoporphyrin analyses or were replaced by geochemically more useful indices alkylation index: Baker et gj., 1977). Definition of these indices, by way of formulae, is presented here as Table 23. During the present study, it was found useful to generate certain numerical parameters for easier

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2 2 2 Table 1 7 F ORTRAN prog r a m forthe gen eratio n mas s tables for m o n obenzo-, tetra h ydro b e nzo-, ana logs o f D PEP and E TI O series po rphyrins o f n o m i n a l a nd a l kyl-t10LWT MOUlT rn1 tlRl : t?EOF t. 1 ,j. fJ, ? u:. 11. 1 1 J 1. 15. l b 1 7 10. 1 ? :!0. .:3. :"! ..,.,. _.,, ::!7 ::;o. 31. 3'' 33. -. .._.-y, 3 :,. c c c to 40 I M LOU!o( pr,our.;.M T O CALCUUo f [ fll[ J, WILL .., ' .,. IIC" '01 rr o I rt : M OLEC:ULroR lIEIGIIT SEf.:(:':S Oi ' : . . : ... Ll .JUt::r r Ci' o fi(:o)tl iiiJiif:Cfi ur I < r -1 <-' F'f [10 1 : 1 6 1 COHTINUE l DO 40 I=lr6rl MU TO W PR 1.1 r 1 7 < ' r =.!. 70 > ? r r: rm :1:, [10 YOU U I S H ANO THEf, I\ U N F'li UIT-:<. EtiTU: YCS OR t!O I r l SINGLE QUOTES ,,,...A ll .. A N S ' ; F oit!S EO, '.'ES' l GO TO l O U[IC R f IIIH 17.

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223 Table 1 8 N ominal m ass table for C 2 6 to C80 free-base porphyrins. U S E R rRGGRAh CNtEP D0 SO USING SINGLE O UOTCS. ; 'FREEBAS E ENTER M W O F CENTRAL MOLECULAR WEIGHTS O F FRE E-BA S E SERIES PORPHYRINS. CM\I:< NtJn [l[i"IZE rHf< [ f H(c[ [: ; cr L r f U 2 7 28 ... :.;.) 36 3'7 38 39 4 0 .t\:2 1 3 4 1 : l 1 6 1 7 IF) 4 ? 50 51 60 61 67 6U 69 70 71 7 }.'\ .:'5 336 400 4 1 4:8 442 45.!1 470 484 4?8 5:! 6 540 554 563 ss:: 591. 61') 652 6?4 70G 722 736 750 7.!,1 778 306 820 8 4 0 8 6 2 876 890 0 4 ? 1 ?46 960 974 998 1002 1016 1030 1044 1 0 5C 1')7:! 383 4 0 : 416 430 444 472 48U 500 528 542 .J.JO 570 59f! 7 5 :! 7SO 7<;'4 808 8:::!::! 850 8 6 1 878 '.:.:0 ':'4 0 962 976 990 1004 1018 10:>: 1046 1060 10:' 3<;'0 40,! 418 -l3:! 446 460 4 7 4 483 5 1 6 530 5 4 4 5 5 8 5 7:? 586 600 6 1 4 6 2::J 6 65. ) 670 68 o9:J 71::! 7::?6 71 0 754 796 8 1 0 824 830 G S:! 880 8'74 903 936 75C' 964 '77 3 992. 1006 1020 103 10'18 ll'6::! 1076 420 434 443 462 476 490 504 513 53:! 54t ; 574 60.: 6l.S 630 658 672 700 711 : "'42 756 770 79: 1 G12 826 840 3 5--t 868 ?l'J ri2 ?""1"'1 96.!.; ','' i 4 1022 t036 1 0 5 0 1 1078 4o: 49.) 504 5 1 C 602 6 3 0 64 700 71-l -.. ,,,.. ,,), o"\1;" \ ;,_j,_ t .., '.'8.) .;''il 1008 to::::: 1036 !050 10.:, 1 1 t '7'J I ,.J ...... 870 ll34 : : ,c.;;. > G > 11.)86 10\38 1 0'7 0 ::i :'! , 11.10 1102 1 1 0 4 1100:. 1l j,, c ; o 1114 11!.> 111G tl20 11::.1 tt.::::.: 7 ? 1120 1130 11.3:::! 1134 1 13 : u:. so 1 142 1148 114:3 .. rOL : '..JISII ;\lfiJTIIEr\ :iiHI I : : r r ::r.: r:.:S :JF : 1/0 [,< : r:IGLE ClUOT[::;.

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Table 19. Nominal mass t able for C26 t o C80 nickel p orphyrins. -1 '. '. .. toO :o USHI G SI!IGLC llUCTt:S. ; N ENTCR MW OF ,H(J/1 <:.; > MOLECULo)f; WEIGIITS OF N I C KEL SERIES PORPHYRINS. 224 C;)R E < iIUtl E < !::llZ i o lCN::E TIIDD THE< E DPf ETIO :::s ::!9 30 31 34 3 5 37 J 3 ? '10 4:! -l3 48 4? 50 5 1 ... 53 54 55 5 6 60 61 .5::! 63 6-1 66 !:.7 68 6? 70 7 1 73 456 470 484 4'/8 51: 568 sa:: 6::? 1 6.38 66.S bOO 694 708 _, ... ,., '--I .l\. 750 761 / 7 U 7S"2 BC.:. s:o 83 <14:3 86: s;, :1'.'0 904 91C }3:.: ?46 960 ?7 983 too:: 1016 1030 1044 1058 107::: 1086 1100 111<\ 1 t.::.:: 1 l I 2 ; 1 :,., 11 :"0 1 1:; I ll'J:J 1!0 YOU .-,;l UTHEr; r.uro, 61: 62.; 6 4 0 65-1 663 682 6?6 '710 72q ';' 3 3 J!j2 i66 780 794 803 8", 836 850 864 873 '1(:6 9.!0 ?34 948 96:::! 976 990 1001 1018 1032 104 6 1060 1074 1088 1 10::! ll l: 1 J 7 ll8..J 1::!0 0 Erll ER fCS or; !!(; !fl :; I N0LC liUO !'t;S . ;['j 530 ..,,_ 586 614 628 642 656 670 684 69t; 7 1 : 726 740 754 768 782 796 810 8::!4 838 866 ceo 094 90C 936 950 964 978 992 1006 1020 1 034 104 8 1062 1076 1090 1 104 111:J )1' 1"'1 11.'> 1 1"l0 11/l 1 I 440 462 476 4$ 504 519 546 560 574 602 630 6 4 4 658 67::! 686 700 7 1 4 729 770 7 8 4 798 81: 840 854 868 882 096 910 938 952 966 980 994 1008 1022 1036 1050 1064 1078 1092 1 106 1120 113 1 11 11.:.::! 11 :' .s 11 ')0 1201 e-.. .., ... 5 1..!. 57 1 .=,0..': 630 644 658 67::: 68<. 70\i 7t; ..., .. ,,, I -.. i /5:0 770 701 79a 82b 340 854 868 ::JU: ou ? 1 0 924 938 9M 980 994 1008 1022 1036 1 050 106 4 1078 1092 1106 1120 1134 1 1 1 3 11.:..:: 11 7!. 11?'.) 736 noo 814 8 4::! 8So 070 898 91:! 9:::.S '? 4 0 968 982 996 1010 10::!4 1033 105::! 1066 1080 1094 11013 11 ::!2 tl::b 1 t 1! 70 11':':; l:')t

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Table 20. Nominal mass table for C26 to C80 copper porphyrins. ( EIHU\ POiil'll '(l'\111 l"II-'C\FRECt: .. ,:;[,rll::I'.LLETC. ; DO :.;a US I1!G S HIULC (I Ul] rc:: ) 'COF'f'Er\' MW OF ATOMtS> > .S3 W f. Ll, ;lll S !IF SERH: S F'Or\F'HYI'diiS, HiDE (tFEr E:TIO 225 ---------------------------------------------------( .:.., '29 .30 31 3'2 .33 33 3 6 37 38 39 40 41 'l2 43 58 6 3 ... 6<-67 c. 'I ;'0 ..,I 1":'. -, .. J4,1 531 :55? 587 6() 1 6 1 5 6:?? .S43 c57 .S71 685 '".J'') 713 741 753 769 783 7?7 811 33'1 :35-:: f.!:J 1 951 91..5 oJ7':' 9?-:. 10'.)7 10'21 1035 104? 10.SJ 1077 10"'1 1 l'J5 t t 1'.' t 13J I,'. (1.'.: l 11 1:.).:) 4:;) 463 477 4'?1 505 519 533 547 Si.l 589 603 617 631 645 073 687 701 715 7:::ry 7-1: ':'0:--' '}Hl '19':: 100'/ 10'2 : l 1 0.57 1107 11:? l t 1.!!: 1 !S' II.'. ; I') l t : :'9 4"' 3 507 521 535 5 0:,.) 577 1 605 619 633 647 6.St .',8? 703 717 7J1 7 4 5 75? 77;'; 787 BOt 81::i 8'l. j 3!:i7 sa::; 81 1 9 'It:! '127 941 1011 to::s 10S3 tO.S:' tOOl 110'1 t 1 :!:! 1137 11:;)1 11.:;,, llr:' tl' .. i 53:' 57'? 60;' 6:1 635 6'1'7 66.5 677 691 70S 7 t ? 733 747 76[ 775 78? 803 8 1 7 El31 84:::; 8 7 3 807 901 r;'15 9'l:l 943 957 971 99Y 1013 10 105S 1083 10'.'7 t 111 11."':5 1 13'7 l : f..;' 1101 11? 5 1:.:v? / 481 495 509 537 6\r' 621 635 6 4 ? 663 .S77 .S'?t 71 (') 7 3 3 717 761 775 70? 803 817 031 85? 073 St.,., 94.3 957 9:'1 ?99 1 013 1 0 '27 1041 1055 10'?7 11: 1 t! 1 J..:? . t J 1 I.:;: t 1 ''5 1.::0? 4S: qn 5 I 1 5Ct -. . ... 763 777 791 cos S 1 '.' :!75 :]11') 9 (:.1 ? 1 7 t 945 :001 1 015 11):.!9 0 4 3 1057 !\1/1 t l113 1 l J [ .......... .,..,J._I t t .,: 1

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Table 21. Nominal mass table for C26 to C 8 0 zinc porphyrins ._I : !.. -. :... :.. ._:;: il' . : ... "'I! ..... EIHCr.: f OF:f -'H"i fdr: f"IF"l' oHCt.EL, [ rc. i DU S O QUUfCS. > 'ZINC' ENfER M W O F CENfRn L .: 6 4 MOLECULAR WCIGHT3 OF :6 27 30 31 33 34 .... 6 ;: 143 -162 4 '76 490 5 1!) s:s: 5 7 1 588 60: 6 1 6 630 644 653 672 / )f) 71-1 7:{ 8 750 7 7) 7 8 4 798 81: 8 4 0 4H.-, v .... 9 1 0 92-1 9 3 8 c;::;:: 960 ')71 llh): l 1030 1 C.'. l I 0/('l 10?..:: 1100:, 11 ::.> J 1,;: J 1 o:; 1 ..: 11 /.", l l ';'<' t .. :0'! r:co::E 150 478 4 9 2 506 520 53-l 548 7 C'.! 716 730 744 ..,, .. .... ... v ' -tO t O 1 0:.! 1 1 039 1 0 5 2 t OOO 1 10:: : I ,;., : 1::. 0 . . ... : t l')..! 1..!"..'6 THf 734 74:: 762 776 790 0<14 81:3 8,_) 1000 101'1 10:-!U 0 1:: 105.-. 1 l llt..: t 2 .:i tJ :0 I 11.'.:! 1 1 ? t 1210 t : r sr I."!"(.J v0..1 5 3 0 5 ? l 6C:; 1000 1 0 l 1 I J2C t () l.' l,>S.:: 1 (. :: ll l :"1: It I : 11.' t: :: lJ .. : ; 11 l t: .. 1 .... .: r ru ... ..... ., ; 4C: 51: ,J ........ '5: h ) 55l 5{'); ] 6 8.) 6'} 4 7C'(I 76 1 3 0 6 020 8J.i 8''' IC':>O I t t 1 1 0.1 11!' : :":": I : i l :v I ::: 1 1 1 ':' : "'11 -l. 226

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Table 22. Nominal mass table f o r C26 to C80 vanad y l porphyrins . .. ; -Mtl 1:1 C::iJ I r;. 703 71 7 7::1 .., 801 81 043 857 871 913 ?f.<' 9 -1011 lt;? 10:':3 1 0.!.;' 1081 1! f'? 11:3 t.l :.. t 1 1.:.:; 1 1 tt:: t:11 .-ou w : .. -'' Uf:nJ\ 551 579 5?:5 607 b:!.L 635 ,!,4'.'> b63 67"! [,U 1 705 7 1'7 .l()IJ 10:7 10-ll 1'-1:"6 1 1111 I 1.5? 1 1 :;3 11!.,'_7 I J ;[ tlf< ltll fi! :.l r!uLE Ull:JrES : r!O HWFf' llf: t'F; roLL 1 r ' 11 1.3 1127 t 1 1 II::;:; Jlb( 119.' t :Jt 471 485 49? 5!.., 541 ,.J..J..J 56 ? 503 59:' 611 639 653 667 68t b95 709 723 7J7 751 77? 7?!. un: e:1 835 8 } 977 891 919 9:!3 947 ?6t 0'7"-.. J 9C'Y 1003 1017 10:11 1045 107:.1 10C7 1101 1115 1 1::!9 1 tU 11:.7 1171 11?9 1 l."! 513 :041 0\l/ 3:1 835 849 !M3 077 891 905 '?1? 933 ':"47 C? ... ;L 975 ?t:'? JOOJ lOt:' 1031 10 1 0!:;? 1073 1087 t 101 1115 11::'1 lHJ 1157 1 ;> J 11CS 119"' 1:t.: _,. -. '' .., l3 ,:,41 '"')'H .. ; -' ... /'; 91'i 963 977 1005 101.;> J ()J3 1047 1 ')!, l 10'':'0 t J 0.5 111: 1.1 t I ; -; l tt:::.' If .... I 1 .. t ., ... 1 I :; : 227

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Table 23 Indices derivable from tetrapyrrole mass spectra. SYMBOL NAME CALCUIATION11 D I E DPEP-to-ETIO RATIO nD I nE % D PERCENT DPEP ( EID I ( EID + EIE)]100 A I. ALKYLATION INDEX ( (CN-32)I]I EI ;for CN > 32 x WEIGHTED AVERAGE MASS l:IM I r.r XD,XE X of DPEP or ETIO SERIES AS FOR X USING ONLY ID or IE XBD X of BenzoDPEP SERIES AS FOR X USING ONLY IBD CD,CE CBD AVERAGE CARBON NUMBER OF Cs= CN' + ((X -MCN') I 14.00 ] DPEP,ETIO or BenzoDPEP SERIES s s FOOTNOTES: REFERENCE Baker et al. 196 7 Barwise and Park,1983 Baker et al., 1977 Baker et al. 196 7 Baker et al. 196 7 11-HS STUDY THIS STUDY 11 I = normalized intensities from averaged and isotopically corrected mass spectrum; D = deoxophylloerythroetioporphyrin series(alt.CAP= et al. ,1989; mass of D = E -2,see Baker et al. ,1967);E =etioporphyrin series;CN =carbon number; Mi= nominal mass of porphyrin series members(see Tables 17 -22) ;BD = 'benzo-DPEP'series(see e .g.cmpd. [CXVI]); C =average carbon number of the series(s) in question; CN' = the carbon number with a nominal ffiass less than or equal to the weighted average mass of series in question and leaving less than one methylene equivalency(i .e. 14.00 amu)as a remainder; MCN' = the nominal mass of CN'(see text). N N 00

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229 comparisons between samples and amongst the tetrapyrrole types (g.g. free-base, Ni, VO) from the same sample. These additional indices (Table 23) include; percent benzoporphyrins (% BPH),. weighted average mass of the benzoporphyrin series (X80 X8e), and the average carbonnumber of each series (C0 CTHBO' C80 C8e) Also included, is the recalculation of the DPEP-to-ETIO ratio into the form of percent DPEP (%0), considering only the DPEP-plus ETIO-series total, as suggested by Barwise and Park {1983). All of the indices in Table 23 are, in essence, self-exp l anatory by their formulae. However, the average carbon number, a totally new concept in geoporphyrin MS, both merits and requires certain examination. Essentially, the average carbon number (C) of any geoporphyrin series evolves from the weighted average mass (X) for that series as follows: C equates to the carbon number of the next lowest integer (nominal) molecular weight (Tables 18-22) plus the decimal obtained by dividing 14, a methylene equivalency, into the remainder of the real number weighted average mass minus the integer value above. As an example, a Ni-DPEP assay might be found to yield X0 = 509.3. Checking the appropriate nominal mass table (Table 19) one finds that 509.3 amu falls between C30 (504 m/z) and C31 (518 m/z) Ni-DPEP. Thus, the C0 here becomes 30.0 plus (509.3-504)/14 or 30.38 (=30.4). The utility of the average carbon number derives from the ability of the researcher to perform direct comparisons between porphyrin series. Herein, good correlations between geologic free-base and Ni-DPEP series were found when C values were compared for the same sample. Figure 65 is the averaged mass spectrum of a 'typical' vanadyl geoporphyrin array and serves as an example for MS nomenclature and

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24 32 40 carbon number Figure 65. Averaged, isotopically corrected and normalized low voltage (6-8eV, 45-60uA) mass spectrum of a vanadyl geoporphyrin array. DPEP(solid) and ETIO(dashed) series are delineated,but the benzo-porphyrin series are omitted for clarity. N w 0

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231 indices, as used herein. The main features of all such geoporphyrin MS are the envelopes of the DPEP-(=CAP:solid trace) and ETIO-(Broken trace) series. Summation of mass spectral intensities (I) gives the basic descriptive parameter, the DPEP-to-ETIO (D/E) ratio. This data set also yields the percent-DPEP ( %D). Both D/E and %D, as used herein, ignore alternate series (BD, BE, THBD) and are based on D + E = 100 (Table 23). When referring to 'alkylated' attention is directed at members with carbon numbers of C-33 and above. Thus, the alkylation inde x (A.I.; Table 23) is a weighted measure of the presence or absence of these species As with D/E, A.I. is calcualted only for the DPEP-and ETIO-series. Studies are underway to re-examine the calculation of A .I. based on a C-34+ redefinition. That is, since a c-33 DPEP structure, namely 7-PDE-DPEP [XXXIX], is both theoretically possible by the Treibs' scheme (Baker and Louda, 1986a) and known to e xist in natural samples (Verne-Mismer et gj. 1986) it may be more geochemically correct to consider 'alkylated' species as only those at C34 and above. Further, the absolute relationship of C33 DPEP to C31 plus C32 DPEP may, theoretically, yield paleoenvironmental information (g.g. Eh} when applied to immature (pre-catagenic) samples. In essence, obtaining a mass spectrum requires that a sample molecule(s) be introduced in the gas phase, ionized, accelerated, separated by mass and detected. During direct insertion probe electron impact mass spectrometry (DIP-El-MS}, as used herein, routine operator control extends only to the volatization and ionization functions. Throughout the course of these studies, the effects of probe/source temperatures and ionization voltages on resultant porphyrin mass spectra were scrutinized.

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232 Originally, it was known that the mass spectrum of geoporphyrins changed in concert with probe temperature (Baker, 1966), but seemed to stabilize at some point (Baker et gj., 1967; Yen et gj., 1969). Further, the problem of fragmentation of these alkylporphyrins, leading to confusing patterns of overlapping ion intensities, was found to be reduced at low (6-12 eV) ionization voltages (Baker, 1966; Baker et gj., 1967; Yen et gj., 1969). From these reports the practice of low voltage EI-MS for geoporphyrin analyses evolved. However, the erroneous use of a single scan (S. E. Palmer; pers comm. 1986) for the description of geoporphyrin arrays (Baker et g}., 1967, 1978b; Palmer et gj. 1979; Smith and Baker, 1974) also became routine. Approxi mately 10 years ago, it became evident that geoporphyrins were sufficiently heterogeneous enough to yield selective volatization and recording MS over the entire range of volatility was required (Louda and Baker, 1981; Quirke et gj., 1982; Shaw et gj., 1978). The effect of this 'fractional distillation' is easily seen within Figure 66 and is true for free-base nickel and vanadyl geoporphyrins. Examination of these (Fig. 66) and other data reveals that the progress of volatilization is governed by both carbon number and structural type (i.g. series). Vaporization in accord to carbon number occurs as expected and proceeds from low to high. Volatility by series was found to be: ETIO > DPEP > THBD > BENZ. Thus, mass spectral scans obtained during geoporphyrin analyses progress from the appearance of low carbon number ETIO species, through a maximum of mid-range (C28 to C33) ETIOplus DPEP-series, to the higher weighted DPEP-plus BENZO-members. It must be stressed that at no time during the EI-MS analysis of a geopor phyrin mixture does a spectral scan occur that is even close to being

PAGE 261

233 al FREE -SASE b ) NICKEL c) VANADYL co I I ; I\,' I Jll 24 28 32 36 24 28 32 36 26 30 34 38 42 CARSON NUMBER Figue 66. in the mass spectra of (a) free-base, (b) nickel and (c) vanadyl geoporphyrins during the volatilization step of DIP-EI-MS.Vertical axis reflects course of volatilization from top down(scan#/total scans). Samples:(a)Monterey shale,(b)DSDP/IPOD 64-479-34-S,(c) North Oregon Basin crude oil.

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representative of the whole (Fig. 66). Throughout this study all reported mass spectra constitutes overall, averaged, normalized, and isotopically-corrected data. 234 Aside from examination of the volatility patterns for natural geoporphyrin mixtures, several binary mixtures of synthetic standards were examined. The data on these mixtures not only gave insight into 'fractional distillation' during MS analysis, but also provided both a comparison of MS and UV/VIS quantitation and a check on isotopic (Ni58/Ni60 C13 H2 ) corrections. One question which the author has never seen addressed in the literature, aside from assumption, is the state of the porphyrin ion as it leaves the source. That is, even though it is known that porphyrin M+ ions originate and are detected as intact species lacking a single electron, are they still fully aromatic pigments upon regaining an electron or has some unknown rearrangement occurred? This question was simply and definitively answered through the collection and re-analysis of metalloporphyrins which had undergone volatization, ionization, acceleration and left the source. In this case, the residue from several MS analyses on nickel and vanadyl geoporphyrin mixtures was extracted from the electric sector entrance s lit, approximately 1 em from the point of ionization/expulsion in the source block, and reexamined by UV/VIS and MS. Figure 67 is the electronic absorption spectrum of the slit residue and indicates the presence of intact Niand VO-porphyrins. The mass spectrum of this melange revealed the presence of these chelates and the carbon number range and series complement was in-line for a random mixture of the sample history of

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0.6 w u z <( 0 (j) dJ <( 0.2 0.0-+----r--r------"T--r---r----.-------l 350 450 550 650 WAVELENGTH, nm 235 Figure 67. Electronic absorption spectrum of the tetrahydrofuran soluble extract of the residue on the ion source exit slit following the MS analyses of nickel and vanadyl geoporphyrin isolates.Solvent = tetrahydrofuran.

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236 the instrument Thus, it can now be stated that geoporphyrins distill and undergo EI-MS as chromophoricall y intact spec ies. Concei vably, the most important instrument parameter controlling the quality and reproducibility of geoporphyrin EI-MS is the ion ization voltage. Characteristically, the ionization potential for porphyrins is considered to be about 8.0 eV (Baker, 1966; Baker and Palmer, 1978; Baker et gJ., 1967). However, the value of 8. 0 0.2 eV stems fro m an EI-MS study of tetraphenylporphyrin (TPP: Meot-Ner et gJ., 1973) and may or may not be directly applicable to all porphyrin MS. In general, it is both given and accepted that 'low-voltage' (g.g. 8 -14 eV, 40-60 EI conditions yield 'mainly parent ions' (cf. Baker et gJ., 1967; Baker and Palmer, 1978). During the development and refinement of ElMS technique for the present studies, the effect of ionization potential was i nvestigated using both synthetic standards and natural geoporphyrin mixtures. Typically, high-energy (70 eV, 260 EI mass spectra for alkyl porphyrins resemble the e xample given here as Figure 68, VO-DPEP [LXXXVIII]. Though e x acting (i.g. high resolution) in terpretation and peak assignments have been made for similar metallo-alkyl-porphyr ins (Budzikiewicz, 1978; Jackson et gl., 1965), mention of the sa lient features will be made here for the sa k e of comparison to l ow-voltage MS and application to geoporphyrin mixtures. The obvious features of the high energy MS of VO-DPEP ([LXXXVIII], Fig. 68) inc lude a greatly dominant parent or molecular ion (541 m/z), prominant losses of methyl groups (M-15, M-15-15, etc.) via benzylic cleavage (Jackson et gJ., 1965), and a well-developed doubly charged region {224-271 m/z) i n which the M2+ ion is nearly one-fourth as abundant as the molecular

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>-1--V> z + .......... L[) ..-1 L[) + .......... L[) ..-1 2 ......__, + 2 2t 300 Figure 68 Uncorrected real-time hig h voltage (70 eV,265 uA) mass spectrum of vanadyl d eoxophylloerythroetioporphyrin(VO-DPEP [LXXXVIII]).M = molecula r ion (541 m/z). N w -....J

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238 (base) peak. These data are presented as normalized values within Table 24. A conspicuous ion at 539 m/z, due to the formation of a M-2 species, appears in the mass spectra of VO-DPEP [LXXXVIII] and numerous other metalloporphyrins when determined with the present instrument This peak (M-2) can be minimized by using low sample sizes and decreas ing the time of e x posure in the probe at high temperatures (g. g > 250C). Seemingly, the use of larger sample sizes (5-10+ and elevated source-probe temperatures, in attempts to mimic geoporphyin runs, leads to conditions (t-T) conducive to the formation of supera r o matic (M-2, M-4: Table 24) ions This effect was minimal or nil at lower sample loadings of neat pigments or during geoporphyrin analyses. The main losses in the high energy MS of alkyl-porphyrins involve the loss of methyl from ethyl (R from R-CH2 -via benzylic (B) cleavage: Budzikiewicz, 1978; Jackson et gl., 1965; Quirke et gl., 1989). However, as seen from the data in Figure 68 and Table 24, following the loss of two methyls via B cleavage further loss occurs via competition between B-(-15 from ethyl) and a-cleavage in which the entire alkyl moiety (-29, -CH2CH3 ) is removed. This also appears augmented with the superaromatic species (M-2, M-4: Table 24). In order for 'routine' mass spectral analyses be applied to the typing or qualitative analysis of geoporphyrin mixtures, one must aspire to technique which generates only the molecular ion Given that geoporphyrins are mixtures of porphyrin structural types ('series': Fig. 64) differing by dihydrogen equivalents ( 2 m/z) and each series consists of a pseudohemology ( 14 m/z), the determination of distributions via high energy MS technique is clearly inadequate and leads to not only uninterpretable but erroneous data. That is, overlaps of ion

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239 Table 24. High energy (70eV, 260 uA) electron impact mass spectrum o f vanadyl deoxophylloerythroetioporphyrin [LXXXVIII] recorded at probe and source temperatures of 290C. SINGLY CHARGED SPECIESU OOUBLY CHARGED SPECIES ION LOSSES m/z I norm ION SPECIES m/z I norm M+ 541 100 M2+ 270.5 22 (M-2/ M 2 539 5-10 (M2) 2 + 269.5 3 (M4)+ M -2 2 537 2 4 (M4) 2 + 268.5 3 (M15)+ 2 + M -15 526 17 (M15) 263 5 ( M -30/ M -15-15 511 4 (M-15-15) 2 + 255.5 8 (M33)+ 2+ 254 2 M 2 2 -29 508 3 (M-2-2 29) (M45)+ 496 2+ 248 5 M -15-15-15 6 (M-15-1515) (M47)+ M-2-1515-15 494 5 (M-2-15 151 5) 2+ 247 5 (M59)+ M -151 5 -29 482 5 (M-1 5 -15-29) 2+ 241 6 (M61)+ M 2 -15-1 5 -29 480 <4 (M2 -15-1529) 2+ 240 6 (M-75)+ 466 )2+ 7 M-215-29-29 <4 (M-2-15-29-29 233 (M77)+ 2+ M-2-215-29-29 464 <2 (m-2-2-15-2929) 232 7 FOOTNOTES: 11 Losses given as integers refer to the following species: 2 = H,H ; 15 = 013 ; 29 = CH2013. I is the normalized intensity after equating the most abundant pea k norm (ion ) to 100.0.

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240 species due to the presence of various parents, each with its own fragmentation, and the losses from other parental speices only complicates the picture. Added to the problem of direct peak overlaps is the fact that the fragmentation of porphyrins during El-MS generates distinct metastable ions. A high gain representation of the singly charged region from the 70 eV spectrum of VO-DPEP [LXXXVIII] is given as Figure 69. The elevation of the baseline in the regions where fragmentation losses, or lower alkyl homologs would appear if present, becomes yet another complicating factor in high energy MS when applied to geoporphyrin analyses. Similar patterns were found during the high energy MS survey of VO-etioporphyrin-I [CVII], VO-etioporphyrin-III [CVIII], Ni-DPEP [XC], Ni-octaethylporphyrin [CX] and other metallopor phyrin standards (Appendix A) used herein. Various techniques have been and are still being examined for the 'routine' mass spectral assa y of porphyrins. Chlorophyll-a [I] and certain analogs have been examined by field desorpt io n (FD-MS: Dougherty et gl., 1980) and 253Cf-plasma desorption (Hunt et gJ., 1981) techniques but similar studies with geoporphyrins are wanting. Contrasting these 'soft' ionization modes are studies using collision activated dissociation (N2-CAD-EI/MS/MS: Quirke et gl., 1989) and chemical ionization daughter mass spectrometry (H2-CI-MS/MS: Sundararaman et gl., 1984). Both of these tech niques are yielding valuable structural insight on single geoporphyrin species but do little in the way of t y ping the entire mixture. Thus, to date, the technique of low voltage El-MS remains the most facile and, seemingly, accurate method for the qualitative analysis of geoporphyrins.

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a) 2 XT1 I I I I I I I I I I I I I x10 500 m/z b) I I I I I I I I I I I I I 500 Figure 69. High gain expansion of the high voltage ( 70eV, 265uA) mass spectrum of VO-DPEP[LXXXVIII]. (a)Ten-fold expansion of the region 440-560 m/z in Figure 68. (b) Ten-fold expansion of "a".Solid line tracing delineates baseline elevation due to metastable ion phenomena. N 1-'-

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242 In order to obtain reliable and reproducible MS of geoporphyrin mixtures the investigator must assess the behavior of standard and geologic pigments in his/her individual instrument During the present studies much effort was expended in setting instrument parameters such that only molecular ion and high signal-to-noise spectra resulted. Low voltage experiments were performed with the V.I.V. (variable ionization voltage) control on the DuPont 21-491 BRand filament voltages were recorded and calibrated for each new filament by connecting a DVM (digital volt meter) across the input leads to the source. It was found that, when the V.I.V. control (10 turn, 50 KQ potentiometer) was zeroed, the lowest ionization voltage attainable was 4.5 eV. All low voltage settings yielded a current of 45-60 depending upon the filament and its usage history. Figures 70 and 71 are selected low voltage El-MS for VO etioporphyrin-III [CVIII] and VO-DPEP [LXXXVIII], respectively. As can be seen from these data and for only this instrument, the presence of a M-15 peak and the accompanying metastable baseline perturbation is significant at about 9 eV and increases in concert with voltage to a maximum at 7 0 eV (cf. Fig. 68: high voltage). Diminution of M-2, M -15 and m* (M to M-15) was found at 4.5 to 6.0 eV for both species, as well as other standards (Appendix A) and natural geoporphyrins. Curiously, these directly recorded ionization voltages are below the 'accepted' appearance potential of ca. 8 eV for porphyrins (cf. Baker et g}., 1967; Meot-Ner et g}., 1973). This phenomenon is l ikely the result of running MS herein with a 'hot' source (290-350C) in order to minimize fouling and instrument down-time. That is, at elevated temperatures ionization is likely to occur via concert between energetic and ionic

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(a) 4.5-6. 0 eV (b) 8.0-10 .0eV (c) 12.0-14.0 eV J I I 500 mjz I 550 ( ) 243 Figure 70. Changes in the mass spectrum of vanadyl etioporphyrin-III[CVIII] as ionization voltage increases. ( a ) 4.56 0 eV (b) 8.0-10.0 eV, a n d (c) 12. 0 -1 4 0 e V

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4 5 -6.0eV 8.0-10.0eV 12.0-14.0 eV I I 550 f 244 Figure 71. Changes in the mass spectrum of vanadyl deoxophylloerythroetioporphyrin[LXXXVIII] as ionization voltage increases. (a) 4.5-6.0 eV, ( b ) 8 0 -10. 0 eV, and (c) 12. 0 -14.0 eV.

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245 {electron impact) processes. The practice of beginning geoporphyrin MS analyses with a 'hot' source stems from the heterogeneity of such mixtures and the need to maintain source temperatures above that of the probe in order to prevent the sublimation ('condensation') of pigment following vaporization. The determination of MS at m inimal effective probe/source temperatures was limited to the characterization of synthetic standards (Appendix A) and was determined to not be directly applicable to the development of technique for geoporphyrin analyses other than in affording broad guidelines. The presence of a metastable ion (m* = (daughter)2/parent, in m/z: Bieman, 1962) for the loss of the first methyl (M-15) from the two sample vanadyl pigments (Figs. 70-71) was found to occur even in the 4.5 eV spectra. Even though this baseline perturbation is minimal, its existence in the absence of the actual M-15 ion is remarkable and hitherto unreported. For now, the only explanation possible i s that at very low voltages (4.5-6.0 eV, 45-60 the benzylic cleavage of ethyl substituents still occurs but only so as to generate negative ions or unknown neutral species. Subsequent to the determination of instrument parameters which will yield only porphyrin molecular ions with standards, application to geoporphyrin mixtures was scrutinized That is, are the same patterns of mass spectral behavior actually exhibited by 'real-world' geologic samples. Figure 72 is the high energy (70 eV, 260 mass spectrum of the vanadyl porphyrins isolated from the asphaltene fraction of a Boscan (Cretaceous, W. Venezuela) crude oil. As can be discerned from this figure, quantitation of the individual series and carbon numbers is impossible. That is, due to sucessive overlaps of fragment and

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+ < D3o) (030) ++ T ---.,-1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I' I I I I I I I I I I 200 300 m 1 z 400 500 600 Figure 72 Uncorrected real-tim e high energy (70eV,260uA) mass spectrum of the vanadyl geoporphyrin array isol a ted from Boscan asphaltenes.Scan collected near the end of volatility (scan 39 of 47) for the sample( fraction "Main/Al-lO".Figure 35,Table 7)used. N .p(J"\

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247 molecular ions, plus baseline elevation from several metastable peaks correction to reality would constitute a mathematical nightmare, at best. 'Routine' mass spectra during the present studies therefore involved the careful distillation of sample while monitoring 70 eV (260 spectra and, as soon as porphyrin ions appear, the instrument was set to an ionization voltage as low as possible for actual data collection. That is, in practice, a compromise between lower ionization voltages, sensitivity and signal-to-noise ratios had to be arrived at early-on in the analysis. Routinely, 4.5-6.0 eV was desired but, in cases of low sample size or with 'dirty' final isolates, 8.0-12.0 eV spectra were also determined. The ability of very low ionization voltages to yield extremely clean mass spectra is well illustrated in Figure 73 in which sequential scans of the VO-porphyrins from a Boscan sample were alternated between 4.5 and 14.0 eV (45 It is easily noted that the amount of noise, and thus error, is enhanced in the 14 eV scans (Fig. 73a,d), relative to the 4.5 eV spectra (Fig. 73b-c), due to even this minor amount of fragmentation. As an example, one can note elevated peaks at 498 m/z (M-15 from C-30 DPEP, 513 m/z) and 483 m/z (M-15-15 from C-30 DPEP, 513 m/z) in the first14 eV scan (Fig. 73a). These peaks are entirely absent in the following 4.5 eV scan (Fig. 73b). Therefore, it is quite easily concluded that the most error in EI-MS, without due care, is imparted to the lower carbon numbers, since fragment ions from higher mass parents overlap consider ably. Certain problems do arise as one proceeds to successively lower ionization voltages during geoporphyrin mass spectral analyses, as with other compounds (cf. Biemann, 1962; Budzikiewicz et 1964). First,

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a) I I I I I I I I I I I I I I I I I I c) I I I I I I I I I I I I I I I I 500 600 b) 030 1 1 1 1 1 1 1 -r -T-T r 1 1 1 1 1 1 d ) mjz I I I 1 I I I I I I I I I I I I I 500 600 Figure 73. Alteration of the mass spectra of a vanadyl geoporphyrin array due to changes in the ionization voltage.Sequential scan s made at 10 second intervals at (a) 1 4 .0, (b) 4 .5, (c) 4.5 and (d) 14.0 eV(45 uA) N CX>

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249 sensitivity decreases. Examination of relative sensitivities for geoporphyrins revealed that, for this particular DuPont 21-491B, the average sensitivity at 14. 0 eV (45-60 pA) was about 15-35% that at 70 eV (240-260 pA). Below 14.0 eV sensitivity tended to decrease somewhat further (ca. 10-20% of 70 eV values). However, at very low ionization voltages (< 8-10 eV} the overall decrease in sensitivity for porphyrin molecular ions ceased to be a problem since the non-porphyrin back ground essentially disappeared. The net effect of decreased sensitivity at low energy was also offset by the use of larger sample size (1-5 which, theoretically, also decreased sub-sampling bias. The second problem with low energy mass spectrometry is that the instrument tends to de-tune. In the present case, this was found not to present a problem since the instrument was set via beta-focus (i.g., detector rather than TIC monitor) technique to a resolution between 1:1500 to 1:2500 at zero percent valley. However, as a check on the ion-optics associated with acceleration and the electrostatic sector functions during actual analyses, fine tuning of the repellers and the focus slits was performed (alpha-focus) at 70 eV every 5-10 scans following the start of data collection. Typically, 20 to 40 scans (100 sec dec-1 ; 40-70 m/z) were acquired during geoporphyrin mass spectral analysis. Following the acquisition of the raw mass spectral data, an average normalized and isotopically corrected spectrum was calculated, using formulae for C-H-N compounds as given in Biemann (1962}. In the cases of Ni-porphyrins and especially with mixed Cu-/Ni-highly dealkylated etioporphyrins (HOEs: Baker and Louda, 1984) the, primary,

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bi-isotopic nature of these transition metals required correction as we 11. 250 The requirement for mathematical manipulation of summed mass spectral data during isotopic correction prior to normalization militated that certain checks on observed versus calculated isotopic abundances be performed. Essentially, the isotopic corrections of importance in geoporphyrin MS are (1) those associated with the M+2 peak due to 6C13, 1H2 and 7N15 and (2) the second most abundant isotope of Ni (28Ni60 ) and Cu(29Cu65). According to Biemann (1962), the M+2 peak varies slightly with the degree of unsaturation and, thus, for porphyrins (g.g. DPEP [XXXVIII] C32H36N4 ; ETIO-III [LII] C32H38N4), being closer to an empirical 'CH' than either 'C' or 'CH2', the formula used was: (1.10 X /ICH)2 (M+2) = -----200 Employing the above formula, the calculated or expected values for the (M+2)+ peak abundance, as percent of M+, for C-20 to C -50 por phyrins were determined and are given here as Table 25. M+1 calculations (Biemann, 1962) were included for the sake of completeness. Performing a check on the validity of the above calculations consisted of examining the (M+2)/M relationships for both individual scans (i.g., variability) and overall mass spectra for several standard pigments (Appendix A) of various carbon numbers. The data in Table 26 reveals that the calculated and observed values for M+1/M+2 peaks are in extremely close agreement for the variety of tetrapyrroles selected here. This conclusion holds for both high (70 eV, 260 and low

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Table 25 (M+l) and (M+2) mass spectral peak enhancement due toisotop i c contributions, as calculated for C 20 through C 50 alkyl porphyrins. CARBON NUMBER PERCENI'AGE OF PARENf(M) PEAK NON-OXYGEN CONTAINING ALKYL (M+1) (M+2) vo-AIRYL PORPHYRINS (M+2) 20 23. 4 2.4 2 6 2 1 24. 5 2. 7 2 9 22 25. 6 2 9 3 .1 23 26.7 3.2 3.4 24 27.8 3.5 3 7 25 28. 9 3. 8 4. 0 26 30.0 4.1 4.3 27 31.1 4 4 4 6 28 32.2 4 7 4 9 29 33. 3 5 .1 5.3 30 34. 4 5 4 5 6 3 1 35.5 5 8 6 0 32-------------------36.6-------------6.2-------------6 4 33 37.7 6.6 6 8 34 38 8 7 0 7 2 35 39.9 7 4 7.6 36 41.0 7 8 8 0 37 42.1 8 3 8.5 38 43 2 8 7 8 9 39 44. 3 9 2 9.4 40 45. 4 9 7 9.9 41 46. 5 10.2 10.4 42 47.6 10. 7 10.9 43 48. 7 11.2 11.4 44 49.8 11.7 11.9 45 50.9 12.3 12. 5 46 52.0 1 2.8 13.0 47 53. 1 1 3 4 13. 6 48 54.2 1 3 .9 14. 1 49 55. 3 14.5 1 4 7 so 56. 4 15. 1 1 5 3 FOOTNCJTES: (M+1) and (M+2) values calculated for alkyl porphyrins using the equat i o n s of Biernann(1962)for compounds only C,H and N, as follo w s : (M+1) = [(1. 1 x #C) + (0 .36 x #N)] This was simplified for tetrapyrroles,containg only four nitrogen atoms,to the following; = [(1.1 X #C) + ( 1 .4)) (M+2) = ( 1 1 X #CH)1 / 200 For Vanadyl (VO) porphyri ns,or other porp hyri ns co ntaining only a sin g l e oxygen atom (M+2) values a r e t o be adjusted upwards by 0 2(Bi emann,1962). 251

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Table 26. Mass spectra survey of M I M+l I M+2 relationships for several free-base, vanadyl and mono-isotopic nickel porphyrins. (M + 1) (M + 2) 4 CDMPOUND 1 CODE COMPOUND 1 (C#/Ottf 3 Vi(eV) obs I calc obs I calc (L] (CX] [CX]run#Z (XXXVIIb) (X) (LVI] (LVI] (CVIII) (CVIII) (CVIII] (CVIII] [CVIII] [LXXXVIII] [LXXXVIII) OEP 58Ni-OEP 58Ni-OEP OPE-ME meso-PP&-ME Deutero-IX-DME Deutero-IX-DME VO-ETIO-II I VO-ETIO-III VO-ETIO-III VO-ETIO-I II VO-ETIO-III VO-DPEP VO-DPEP (36/0) (36/0) (36/0) (34/2) (34/3) (32/4) (32/4) (32/0) (32/0) (32/0) (32/0) (32/0) (32/0) (32/0) 70 70 70 70 70 70 14 4.5 8.0 12.0 14.0 18.0 14.0 70 45.0/41.0 40.5/41.0 40.5/41.0 38.8/38.8 37.9/38 .8 34.7/36.6 35.1/36.6 36.6/36.6 35.8/36.6 37.5/36.6 37.5/36.6 37.4/36.6 36.6/36. 6 37.2/36.6 FOOTNOTES: 1) Compound c o de and abbreviations can be found in Appendix A. 2) C# = CARBON NUt-IBER, 0# = OXYGEN NUMBER. 3) Vi = ionization voltage in electron volts(eV) 10.9/7.8 8.4/7.8 7.7/7.8 7.4/7.4 7.3/7.2 7.6/6.6 6.8/6.6 6.2/6. 4 5.8/6.4 6.5/6. 4 7.3/6.4 8.2/6.4 6.2/6.4 6.4/6.4 N5 4 43 55 10 6 3 3 3 3 3 3 3 5 4 4) and (M+2) peaks ca lculated as given in Table XXV (calc).Observed(obs) relationship measured fran mass spectra after equating the height of the molecular ion (M) to 100.0. 5) N = number of scans averaged into the r ecor d of observed (M+1) and (M+2) heights. N l.rl N

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253 (4.5-18.0 eV, 45-60 spectra. The above may appear moot but, as far as the author can tell, substantiation of the calculations given in Biemann (1962), as applied specifically to geoporphyrin analyses, has never been reported. An important point in the present case is that it is only the summed values (TIC) for each peak which afford the correct relationships of M/M+1/M+2. That is, random fluctuations, often at the level of -20%, do occur between scans. However, in all cases studied (Appendix A: 119+ pigments) the summed values yielded the correct relationships. This point further emphasizes the need for collecting and summing spectra over the entire range of volatility before applying isotopic corrections Given that the overall summed MS of free-base and VO-porphyrins (Table 26) yield near perfect fits between the calculated and observed M/M+1/M+2 relationships due to the non-major stable isotopes of H,C,N and 0, the estimation of porphyrin ions containing bi-('poly-') isotopic metals was next scrutinized. In the present case, the mass spectra of tetrapyrroles complexed with the pentaisotopic transition metal nickel were examined. Excepting cases in which nickel geopor phyrins are analyzed after harse demetallation, the relationships of nickel isotopes and geoporphyrin series (Fig. 64) required classification and substantiation. According to published figures (g.g. Heath, 1966) natural nickel consists of 5 stable isotopes as follows; Ni58 (67.8%), Ni60 (26.2%), Ni61 {1.2%), Ni62 (3.5%) and Ni64 (1.2%). In line with mass spectral usage these equate to the following normalized values: Ni58 {100.0), Ni60 (38.6), Ni61 (1.8), Ni62 {5.3) and Ni64 {1.8). As can be seen from

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this distribution, the relationship of (2.59:1) i s the most important. 254 First, a variety of 'expected' mass spectral distributions for nickel porphyrins of various carbon numbers were calculated. In these cases, it is important to note that the actual distribution of any Ni porphyrin ion, molecular or fragment, is made up not only of the various nickel isotopes but (M+1) and (M+2) peaks for each isotope due to the tetrapyrrole ligand. Thus, the calculation of 'expected' patterns and the deconvolution of observed (geoporphyrin) mass spectra is rather complicated, relative to the free-base or vanadyl pigments. Table 27 contains the calculations and normalized values for the 'expected' mass spectral patterning of 5 samples of Ni-po rphyrins with carbon numbers between C22 and C44. These calculated patterns are also shown in histogram form as Figure 74. As can be told from these data, the primari ly bi-isotopic nature of nickel porphyrins becomes diluted at higher carbon numbers and back-correction with geoporphyrin distributions is of paramount importan ce Further, though it has become 'routine' to consider only relationships (g.g. L ouda and Baker, 1981) the Ni62(M) plus contribution to Ni58(M+4) can reach levels as high as 10% or higher, in the higher carbon number geoporphyrins (cf. C44: Table 27), and should not be dismissed. This becomes i mportant onl y w i th samples high in E-4 to E-8 series porphyrins (Fig 64) of high carbon number. To date, the vast majority of geologic Ni-porphyr ins have been found to consist of mainly DPEP-(E-2) and ETIO-(E) series porphyrins and to extend from C22 to o nl y about C36. T hus, any errors connected with the consideration (Louda and

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255 Table 27. Changes in t h e over t isotopi c pattern of nickel porphyrins due t o carbon n u mbe r increases: Calculated iso topi c d i stributions for C22 CJO, C32, C36 a n d C44 nickel por p h y r i n s CARBON NUMBER m/z PEAK/Ni-ISOTOPE(1 ) C-22 395 396 397 398 399 400 401 402 C-30 507 508 509 510 511 512 513 514 C -32 --s-34 535 536 537 538 539 540 541 542 C -36 --s-90 591 592 593 594 595 596 597 598 C -44 --ri74 675 676 677 678 679 680 681 682 M MH 11+-2 M 11+-1 11+-2 M M+1 11+-2 M 11+-1 M+2 M 11+-1 11+-2 NORMALIZED INTENSITIES(2 ) 100.0 38.6 1.8 5.3 1.8 100.0 38.6 1.8 5.3 1.8 100.0 38.6 1.8 5 3 1.8 100.0 38. 6 1.8 5.3 1.8 100.0 38.6 1.8 5.3 1.8 25.6 9.9 0.5 1.4 0.5 34.4 13.3 0 6 1.8 0 6 36.6 14. 1 0 6 1.9 0 6 41.0 15. 8 0.7 2 2 0.7 47. 6 18. 4 0.9 2 5 0 9 2.9 1.1 0 1 0.2 0.1 5 4 2.1 0.1 0.3 0 1 0 1 6.2 2 4 0.1 0.3 0.1 7 8 3 0 0.1 0 4 0.1 10. 7 4.1 0.2 0.6 0.2 I 100.0 25.6 41.5 11.7 6.9 1.5 2.0 0.5 0.1 100.0 34.4 44.0 17. 2 6. 0 2 1 1.9 0.6 0 1 100.0 36. 6 44.8 15.2 8 3 2 0 2 1 0 6 0 1 100.0 41.0 46.4 17.6 9 0 2 3 2.2 0 7 0 1 100 0 47. 6 49. 3 20. 2 1 0.3 2.7 2.4 0.9 0 2 FOOillO'lt.S (1) PEAK designation given relative to the porphyrin molecular ion (2) Calculated according to t h e distribution of given in Table XXVIII,and the cont ributions of H and C to (11+-1) and (M+2) peaks,as described in Table XXV.

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>-1-(/) z w 1-z 0 w N _J <{ 2 0::: 0 z a)C-22 b) C-30 c) C-32 d)C-36 e)C-44 ------------------------------I II I I I I I ,, I I I 390 400 500 510 I I ., .r I "I 530 540 mjz I I ., I I I I I I "J I I 590 600 670 6 8 0 I I J Figure 74. Changes in the apparent relationships of M/M+1/M+2 peaks in nickel porphyrins due to increases in the carbon number of the ligand. (a) through (e) are the calculated mass spectra(Table XXVII) for C 22 through C-44 Ni porphyrins, as indicated, M+2 and M+l peaks are followed with solid and dashed lines,respectively. N VI 0\

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Baker, 1981) of nickel as being primarily bi-isotopic (Ni58/Ni60; 2.59:1) are likely to be e x ceedingly small (g.g., < 1-2 % ) 257 The calculation of 'ex pected' MS patterns for nickel porphyrins can easily be e x tended to mixtures as well, and these used to check deconvoluted geoporphyrins. One such e xample is given here for the theore tical, and often observed (Louda, this study), 1:1 admixture of C30 NiDPEP and C30 NiETIO (Table 28). As both the numerical and visual (offset: Table 28) data reveal, the amount of Ni-C30-ETIO (NiE30) would be overest imated by 44.0 % if appropriate back correction for isotopic contributions were not included and had these been geoporphyrin data. Second, patterns of nickel porphyr in distillation within the mass spectrometer (DIP-El-MS) were investigated the author e x pected to discern a slight fractional distillation of Ni porphyrin in which the lighter isotope (Ni58) would be above its e x pected values early-on during a run and decrease towards the end. Conversely, an increase in the relative abundance of Ni60 with the progress of Ni porphyrin distillation was e x pected. This was found not to occur Quite unex pectedly, it was found that the relative amounts of Ni58-/ Ni60-porphyrins, at any given carbon num ber (Figs 75-76), underwent wide and random fluctuations from the beginning of volatilization until the consumption of sample. These fluctuations yielded no discernable trend and remain unex plained However, when summed (TIC) by m/z and isotopically corrected for (M+2) due to CHN, the overall observed relationship of Ni58 to N i60 was found to be quite close (% ) to known values These data yield two valuable conclusions regard ing porphyrin and geoporphyrin MS analyses First, accuracy at the level

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Table 28. Cal culation of the 'expected' mass spectrometric pattern for a n hypothetical 1:1 mixture of nickel deoxophylloerythroetioporphyrin and nickel etioporphyrin at carbon number thirty. INTENSI1Y m/z MOLECULAR M(1) M+1(2) M+2(2) 1(3) Inorm' ( 3 ) I MS HISTOGRAM(4 ) 504 58N'D 1 30 100.0 --------100.0 69.4 505 --------34.4 ----34.4 23.9 506 58N. E + 60N' D 1 30 1 30 100.0 + 38.6 ---5.4 144.0 100.0 507 61N'D 1.8 47.7 49.5 34.4 1 30 ----508 60N' E + 62N' D 38.6 + 5.3 0.6 7.5 52.0 36.1 ?:: H 130 130 til 509 61N'E 1.8 14.4 0.1 16.3 11.3 1 30 62 64 H 510 N1E30 + N1D30 5.3 + 1. 8 0.6 2.3 10.0 6 9 tJ N 511 H --------2.4 0 .1 2.5 1.7 512 64N.E 1.8 0.4 2.2 1.4 1 30 ---513 --------0.6 ----0.6 0.4 I 500 5 1 0 514 -----------0.1 0.1 <0.1 I m/z FOOTNCJI'ES 1) Intensities of nicke l isotopes taken as 58Ni;60Ni/61Ni/62Ni/64Ni = 67.8/26.2/1.2/3.6/1.2, based o n percent natural abundance, or 100.0/38.6/1.8/5.3/1.8 ,following normalization. D= deoxophylloeryt h roetioporphyrin(=CAP= cycloalkanoporphyrin)type porphyrin, E=etioporphyrin. 2) (M+l) and (M+2) va l ues calculated as give n in Table XXV. 3) E I = sum of individual intensities; Inorm' = renormalized intensities for the overall distribution. 4) Graphic representation of !norm' profile. 520 N V1 CX>

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>-1-tf) 240/250 T(P/S,C) 280/285 z WI-'; 60 o-z 8 rv r i 1 \;J v '1-f--YVJ "\:ff'v'<'\\' ,\1 'V' v m I Z @: 40 V1 Ni60(M+) N Ni58(M+2r _J E 20 <(0 2m 0..._... 01 I I I I I I I I I I I I I I z 0 40 SCAN 80 NUMBER 120 Ni62(M+) Ni 6 1 (M+1 t Ni 6CM+2t Figure 75. +Scan to scan variability in the relationship of M+2 and M+4 to the molecular ion (M ) of nickel octaethylporphyrin[CX] during DIP-El-MS. Upper and lower traces record the relationship+of the M+2 (592 m/z) and M+4 (594 m/z) peaks, respectively, to the molecular ion(M, 590 m/z).High voltage(70 ev, 265uA) M S .. N Vl 1.0

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80 240/265 T(S/P.C) 280/285 20+ + ........... 2 ..__... en 1.[) z 70 N -E "'1" (Y) 1.[) 60 0 -0 50 0 20 40 SCAN *(534 + 536m/z = 100% 60 NUMBER 80 ......... u Q)Q) ...... roO -('1 :::l-._ u.--cua> -....... ('1 --(() L Q) lll .D 0 ........... (\j + 2 ..__... en 1.[) I 30z a:1 +.........._ 2: ..__... 0 co I 40z N -E co (Y) 1.[) ........ 100 Figure 76 Scan to scan variability in the relationship of the M + 2 peak of nickel etiporphyrin-I during EI-MS Solid and dashed lines show the calculated a nd found means for the Ni-58/ Ni-60 relation. Low voltage (8-10 eV,45uA) M.S .. N 0"1 0

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261 of % is indeed possible during so-called 'routine' analyses. Secondly, consideration of single scans as geoporphyrin data, at least for El-MS, should be avoided at all times. That is, the true relationships of isotopic and series, as detailed later, distributions rests in the entire sample and not in any single 'snapshot' (scan) taken during the distillation process (DIP-El-MS). Concerning the behavior of Ni-porphyrins in the mass spectrometer, the last set of experiments involved comparing the overall (summed TIC) observed and calculated isotopic distributions for several standard pigments. Sample data are given for sets of C-32 and C-36 Niporphyrins within Table 29. As stated for the study of Ni-porphyrin distillation patterns (Figs. 75-76), the overall (TIC) emergent relationships of nickel isotopic peaks, including CHN contributions, found during MS analysis match closely the calculated or 'ex pected' values using published isotopic values (Biemann, 1962; Heath, 1966). In the present case, considering Ni58 as M+, errors of % and % can be claimed for M+2 (Niro) and M+4 peaks, respectively. Several investigations on the mass spectral behavior and quantitation of vanadyl porphyrins in mixtures of standard pigments (Appendix A) were performed during the present study. Those included herein were 1:1 (molar) mixtures of (a) VO-DPEP (C32, [LXXXVIII] plus VO-ETIO-III (C32, [CVIII]) and (b) VO-ETIO-III (C32, [CVIII]) plus VO-OEP (C36, see [L], and (c) various molar percentages of VO-'benzoETIO' (C32, [CXVIII]) with VO-ETIO-III (C32, [CXVIII]. These experiments were aimed at discerning volatilization patterns and relative quantitation for vanadyl porphyrins by series and carbon number.

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Table 29. Observed versus calculated distribution isotopic peaks in the mass spectra of C 32 and C 36 nickel porphyrins. PEAK IDENTIFICATION(Z) ......... ......... ......... N ..... ..... f f f ....._, ..._, ..._, ::X: ::X: 5: p.. p.. ...... ...... ..... z z z co 0 ..... V") -.!) -.!) ......... N ......... + + + f ..... ......... f ......... ......... ......... ..._, L L L L :::c ....._, ....._, ....._, ....._, ....._, p.. ::X: ::X: ::X: ::X: ::X: ...... (1) p.. p.. p.. p.. p.. z ...... ..... ..... ...... .,...o z z z z Zo.!) v (3) N ( 4 ) CMPD[CODE] co co 0 ..... N V") V") -.!) -.!) -.!) + l. C# = 32 -CALCUlATED100.0 36.6 44.8 15.2 8.3 100.0 34. 6 44.2 14.9 8.6 14 6 NiDPEP XC 100.0 35.2 43.1 16.7 8.0 14 10 NiETIO[LII] 100.0 37.7 45.7 17.7 11. 0 70 93 C# = 36 -CALCUlATED 100.0 41.0 46. 4 17.6 9.0 NiOEP[CX] 1 00.0 42.6 46.6 18.0 9.1 70 140 NiOEP[CX] 100.0 41.3 47.8 1 9 .2 8 0 14 4 NiOEP[CX] 100.0 42.0 47. 9 20. 3 10. 7 70>': 13 FOOINOTES: (1) Compound name and Roman numeral code can be found in Appendix A. (2) Peak identification s refer to nickel isotopic spread and M+1 plus M+2 contributions due to the isotopes of carbon,hydrogen and nitrogen,as detailed in Tables XXV and XXVIII. (3) V. = ionization voltage.Spectra determined on a DuPont 21-4918 l. at F.A.U. except for "-.':" which was collected on a Varian MAT Ol5-DF at M.S.U.(East Lansing,Mich ) (4) N = number of scans averaged for reported data. 262 of

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263 Figure 77 (solid trace) is a plot of the distillation of VO-DPEP [LXXXVIII] and VO-ETIO-III [CVIII] during the mass spectral (8 eV, 45 analysis of a 1:1 molar mixture. Initially the MS derived ratio of the 2 is about 1 : 1 but, beginning with scan #7 (Fig. 77, solid trace), it was found that VO-ETIO-III [CVIII] was preferentially distilled earlier than VO-DPEP [LXXXVIII], even though the latter is 2 mass units less in weight. It is suggested that, as temperatures sufficient for the volatilization of these metalloporphyrins are approached and minimally achieved, a melt or 'eutectic' state occurs in which similar species self-associate and distill from the probe in accord with their true volatility behavior. A phenomenon akin to a molten state rearrangement must be occurring during the MS analyses of metallopor phyrins, be they synthetic or geochemical mixtures, since the initial scans of a run often contain a variety of ions (series, carbon numbers) which only become dominant near the end of the analysis. Whatever the explanation for the first quarter of this and other analysis is, it is apparent (Fig. 77) that VO-DPEP (541 m/z [LXXXVIII]) is less volatile than VO-ETIO-III (543 m/z, [CVIII]) and, thus, dominates spectral scans during the latter part of a MS analysis. The relative amounts of VODPEP [LXXXVIII] and VO-ETIO-III [CVIII] found via summing mass spectral intensities, including isotopic (i.g., M+2) correction (Table 25), were 51.7 and 48.3%, respectively. This is in e xcellent agreement (<%) with the 1:1 molar ratio of the mixture as made via quantitation with electronic spectroscopy At least for these two vanadyl alkyl-porphyrins, this suggests excellent similarity in mass spectral sensitivities, including ionization efficiencies.

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PROBE TEMPERATURE(indicated,C) El 250 300 El ...-... 30(70) 0 w + I I\ 9 I \ ,.."\ .......... 40 (60) I \ 0 Q_ I I \ w I I 0... I .... --. \ wl 0 \ I 50 (50) 0 > ,.' I I + r-I ,,I 91 z 60(40) -w '-"' u I --0::: w /::; 6 6 /::; 0... 70(30) 0 1 5 10 1 5 20 25 SCAN NUMBER Figure 77 Se lective volatilization of vanadyl porphyrins during DIP -EI-MS;changes in the VO-ETIO to VO-DPEP ratio at C 32 Test mixture was 1:1 molar in VO-DPEP[LXXX VIII] and VO-etioporphyrin-III[CVIII], as determined spectrophotometrically .Solid line shows V0-0/VO-E,with arrow delineating the trend.Dashed line records the total ion current for both species.Triangles indicate significant(ca.10C) increases in probe temperature. Low voltage (8.0 eV,45uA) mass spectra. N (j\ .j:"-

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265 The idea of a rearrangement or 'melt' period and the sputtering or 'burping' of metalloporphyrin sample prior to the system undergoing direct fractional distillation is reinforced through examination of the plot of 1otal (porphyrin) ion urrent (TIC: Fig. 77, dashed trace) for the MS analysis of VO-D32 plus VO-E32 As the instrument (DuPont 21-491-B) used lacked computer control, notably TIC/probe temperature feedback, thus, TIC was manually controlled During this, and all other MS analysis, the temperature of the probe was increased slightly after the first appearance of porphyrin ions in an effort to maintain a relatively constant TIC. In this manner the sensitivity settings of the mass spectrometer (amplifcation) and recorder (filtering) could be left at their initial settings and corrections for overall sensitivity changes were negated. Excepting an overall and relatively constant increase in probe temperature during this run (Fig. 77), events within the TIC profile bear mention. First, a sharp increase (or 'sputter') in TIC, during which VO-D32 and VO-E32 are approximately equally abundant, occurred during scans 2-5, without a drastic change in probe temperature. Second, three sharp or stepped increases in probe temperature were performed in an effort to maintain a stable (ca 200-250 mm, x100 galvanometer) RIC (relative ion current) for the most abundant ion on a scan by-scan basis. These three sharp increases ("A"; delta in Fig. 77) occurred at scans 13, 18 and 22 and elicited riC increases during scans 14-15, 20-21 and 23, respectively. The last increase (scan 23) was notably small and preceded the consumption of sample. It should be noted that the increases in TIC, following the initial sputtering (scans 3-5), occurred without drastically altering

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the (fractional) distillation of the individual vanadyl porphyrin series (D vs. E: Fig. 77, solid trace). 266 A visually graphic illustration of the selective volatilization ('fractional distillation') of VO-etioporphyrin-III ([CVIII], E32 543 m/z) over VO-DPEP ([LXXXVIII], 032 541 m/z) is provided in Figure 78. As shown, scans #9 and #22, out of 28 total, contain 66.9% (33.1%) and 36.6% (63.7%) VO-ETIO-ILI (VO-DPEP), respectively. The next MS experiment with vanadyl porphyrins involved comparison of the volatilization within a single series (viz. ETIO-) by differing carbon numbers. In this case, a 1:1 molar mixture of VO-etioporphyrinIII [CVIII] and VO-octaethylporphyrin (cf. OEP[L]) was prepared using UV/VIS techniques. A priori, the author full expected to find a MS scan series in which the E32 (543 m/z) compound dominated the early spectra and the E36 (599 m/z) member would prevail near the end of the run. However, such was not the case. As seen in Figure 79, the observed trend was for the relative percentage of VO-ETIO-III ([CVIII], E32 ) to increase slightly throughout the run. In fact, only in the first scan did the C-36 ETIO-compound (VO-OEP[L], 599 m/z) dominate. This observation is highly peculiar and is in direct opposition to both accepted thought on 'distillation' in concert with molecular weight and trends observed during the present studies with geoporphyrin mixtures. Further, after summing the individual ion currents for 541 and 599 m/z, it was found that the present MS analysis indicated molar percentages of 56.7 and 43.3, respectively. Thus, without reason to doubt the precision of UV/VIS quantitation, it appears that VO-OEP (VO-[L]) i s volatilized/ionized with less efficiency than VO-ETIO-III [CVIII] under DIP-El-MS conditions at 8 eV. Structurally, however, VO-OEP (VO-[L])

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SCAN# 9 D/E 30/70 >I-(/) z w 1--z 0 w N _j <{ 2: 0:: 0 z I 540 I ---. I I I 550 22 67/37 (0+2) m/z 540 550 Figure 78. Selective volatilization of ETIO over DPEP series porphyrins during DIPEl-MS. Scans number 9 and 22 from the run+shown in Figure 77.Dotted line indicates the abundance at 543 m/z due to the (M+2) contribution N 0" --.1

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PROBE TEMPERATURE (indicated (L 70(30) 230 275 w 200 0 \ \ 0 / -9 / / -\ / / .._... / 0 '.--. / 0... 1-60(40) w I 1oo 0 > 1-50(50) +I w z 61 w u :::1 0::: w 0... 40(60) 0 1 5 10 15 SCAN NUMBER Figure 79. Volatilization profile obtained during the DIP El-MS of a 1:1 (molar) test mixture of vanadyl etioporphyrin-III[CVIII] and vanadyl octaethylporphyrin (see [L]).Solid line is the percent ratio of VO-ETIO(C -32) to VO-OEP(C -36).Dashed line is the total ion current obtained b y summing 543 and 599 m/z.Thin solid line with arrow indicates overall trend. N (j\ 00

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269 is unlike any known, or likely to be found, geoporphyrin. At present, only the extreme symmetry and/or the ease of yielding multiple (M-15) ions can be offered as explanations for the above observations. Similar tests with alternate mixtures of VO-ETIO-series porphyrins are planned in order to sort this phenomenon. E x cepting the present problematic case of VO-OEP (VO-[L]), the observed volatilization pattern for vanadyl (geo-) porphyrins of the same mass spectral series (cf. Fig 64) is indeed from lower to higher carbon in concert with increasing probe temperature. The final experiments on the mass spectral behavior of standard pigments involved the analyses of the mixtures of VO-ETIO-III ([CVIII], E32, 543 m/z) and VO-'monobenzoetioporphyrin' ([CXVIII], BE32, 537 m/z) previously discussed under the section on electronic absorption spectroscopy Data obtained from the estimation of VO-BE in admixture with VO-ETIO-III by both UV/VIS and MS techniques are compared in Table 30. These mixtures ranged from 1 to 32% (molar) VO-BE and bracket the observed range of 4-12 % (molar) for benzoporphyrins in natural vanadyl geoporphyrin mixtures. Aside from the determinat ion of molecular weight during structural verification for this benzoporphyrin (Clezy and Mirza, 1982), the present report appears to be the first to deal with the mass spectral behavior of the benzoporphyrins and, more specifically, the quantitation of same. Examination of the data in Table 30 reveals that UV/VIS tends to underestimate the VO-benzoporphyrin(s) by about 16% while MS overestimates these pigments by about 10% These generalizations were arrived at after ignoring the data for the 1% VO-BE preparation, as it is near the limit of detection Since the VO-benzoporphyrin(s) appear to more

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270 Tabl e 30. Estimation of vanadyl benzoporphyrin s i n mixtures with vanadyl alkylporphyrin s : Comparison of mass spectr ometric a n d e lectronic absorption techniques. TEST MIXIURE ( 1) KNO\.JN mole% VO-BENZ-PH F.STIMATED P;l.AR PERCENTAGE W/VIS % l \ M.s.(J) % l (2) re re 1.0 1.1 110. 0 0 1 10.0 2 0 1. 8 90. 0 2 1 105.0 4.0 3 2 80. 0 3.4 85.0 8 0 7.1 88.8 8 6 107.5 16. 0 12. 8 80. 0 20. 5 128.0 32. 0 25. 7 80.3 40 4 126.0 MEAN(2-32%) 84.7 110.3 FOO'INOTES: 1) Formulation of known mixtures and determination of VObenzoporphyrins in these by W(VIS technique is descibed in Table XIV. 2) % 1 = the percentage estimated as related to the actual. re 3) Molar percentages estimated via mass spectrometric methods entailed the summation of VO-benzoporphyrin[CXVIII] and VO-etioporphyrin-I[CVII] peaks at 537 and 543 and covered the entire range of volatility for each(see Fig.80J

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271 efficiently volatilized/ionized during MS analysis and given that 'real world' geoporphyrins are exceedingly complex melanges, this author both uses and suggests the electronic spectral technique for the overall (total) quantitation of benzoporphyrins. Two complimentary facts lead to this conclusion. First, UV/VIS is highly specific for the VObenzoporphyrin chromophore and its precision is essentially i ndependent of analytical conditions Second, exacting quantitation of the VObenzoporphyrins during repetitive MS analyses, as will be shown, is more subject to errors associated with sample size, probe temperature ramping and other factors which alter volatility profiles. The vanadyl benzoporphyrins have been found here to be less volatile than their purely alkyl counterparts, both during the MS analysis of geoporphyrins and with the present synthetic mixtures. Figure 80 contains the 'distillation' profiles for the 8, 16 and 32% VO-BE [CXVIII] mixtures with VO-ETIO-III [CVIII]. Even though the VObenzoporphyrin (VO-BE32: 537 m/z) is 5 amu 'lighter' than the VO-ETIO compound (VO-E32: 543 m/z), the former exhibits significantly less volatility than the latter. This must be a reflection of increased aromaticity and stronger rr-rr overlap. The same, yet obviously more subtle, trend was found for the 1, 2 and 4% VO-BE [CXVIII] preparations. Graphically, the selective volatilization of these two vanadyl pigments can be seen in Figure 81. Here, the change in spectral dominance during the analysis of the 16% (molar) VO-'benzoetiopor phyrin' [CXVIII] is easily noted. Indeed, scan #4 contains only 7.3% VO-BE while in scan #22 VO-ETIO-III has disappeared and 100.0% VO-BE remains.

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PRO BE TEMPERATURE(indicated, o c ) -250 ( 320 ) -z 100 0 0 0 0 ._ - 0000 ..... 0 0:: 0 >...... I I (L z \ -20 0:: -80 0:: \..._ >-\ I \ 0 -. .... I-6 0 \ . \ QN (L(Y) '--'\ N -zE 1--L[) \ W I'-w 60 I \ 0 0 I > t-\ 0 >rn \ 0 20 (\J \ (Y) u \ ..._.,. ......__, 0 100 1 1 0 1 5 20 25 S C AN N U M B E R Figure 80. Comparison of the volatilization of vanadyl etioporphyrin-III(C-32,543 m/z[CVIII]) and vanadyl monob e nzoetioporphyrin (C -32,537 m/z[CXVIII]). Solin, dashed and dotted lines represent synthetic mixtures containing 8 16 and 32 molar percent vanadyl 'monobenzoetioporphyrin'(see Table 30). N -....J N

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SELECTED SCANS FROM THE BeV MS OF A MIXTURE OF 84/o VQ-ETIO-ill(C32E,543m/z)and 16/o VO -BENZETIO(C32BE,537m/z) SCAN NO. 4 (TIC,mm) (340) 100 >-r-80 lf) z w r-60 z EJ 40 N _j
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274 Following the mass spectral analyses of several synthetic mixtures and observations on numerous geoporphyrin arrays the following order of volatility is concluded: ETIO
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,.-... u 0 -6400+ Q) ........ cu u "'0 c 300 ...._, w 0:: ::J 0:: 200 w Q_ 2: w 1-w 0) 0 0:: Q_ 100 (33) B D --BE ---THBD ------0 ---------------E l ---2 2 3 2 4 2 carbon number PROGRESSION O F ANALYSIS (TIME) J ( S C A N NUM BER) ... ,... Figure 82. S uggested overall profile for the DIP -EI mass spectrometric analysis of geoporphyrins. Probe temperatures are for reference only and will vary with sample, instrument a n d operator.Distribution kites and carbon numbers indicate the progress of volatilization only and are also only for reference.Abbreviations: BD= benzo-DPEP, BE= benzoETIO, THBD = tetrahydro-BD, D = DPEP, E = ETIO. Phases I -V (see Text). N '-l Vl

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276 absent. Phase III consists of elevating the probe temperature (220-2600C) until, at 70 eV (260 porphyrin ions (450-500 m/z) appear. At this point the mass spectrometer is rapidly retuned to maximize the ion-optic focus and both the low voltage (4.5-12 eV) and sensitivity settings to be used throughout the run chosen. Often, a slight cooling of the probe, to avoid unwanted non-monitored porphyrin ion loss, is included to allow time for the requisite tuning. Continual but increases in probe temperature during phase IV affords a relatively constant porphyrin TIC and the collection of MS data. As decreasing TIC and scan profiles indicate that the sample i s nearly consummed, the probe temperature is sharply elevated (phase V) in order to volatilize the remaining high carbon number species and involatile benzopor phyrins. Typically, 15-50 scans are collected, summed, isotopicallycorrected and normali zed, in that order, to yield the 'average' geoporphyrin mass spectrum. In order to test the repeatability of the above techniques in obtaining geoporphyrin mass spectra one could make a statistical number of runs (g.g. 10+). In the present study, due to the lack of computer ized data acquisition, this was not possible due to time constraints and the amount of sample which would be consumed. Further, during repeated runs on the same sample the intrinsic familiarity with that sample would undoubtedly, even if unconsci ously, lead to unwanted bias and enhanced run-to-run similarities. Rather, the mass spectrum of the vanadyl porphyrins isolated from a Monterey sourced petroleum of 32 API gravity and 1.9 % sulfur was redetermined two and a half months following the original analysis. These spectra and the indices derived (Figure 83) reveal that, with certain exceptions, the reproducibility

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(a ) RUN No. 1 RUN No. 2 24 28 32 36 40 44 CARBON NUMBER (b) DPEPs ETIOs RUN1(r---' ,. C32 -RUN 2(---) Figure 83 Repeatability of geoporphyrin mass spectral analyses. Low voltage (4. 5 6 0 eV, 45uA) DIP-EI mass spectrum of a vanadyl porphyrin array from a Miocen e petroleum. "Run No. 2 (indicated) was performed months after the first run. (a) Average mass spectra.DPEP(solid) and ETIO(dashed) series are delineated,as indicated. Benzoporphyrins (ca. 8%) are deleted for clarity. (b) Overlays of the DPEP-and ETIO-series N distributions for Runs #1 (solid) and #2 (dashed).

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278 in obtaining precise geoporphyrin EI-mass spectra is quite good. As can be seen from the individual spectra (Fig. 83a) and overlays of the DPEP-or ETIO-series for these analyses (Fig. 83b), reproduction of the overall geoporphyrin distribution ('envelope') is excellent, as is the precision of percent-DPEP determination. The highest variability in geoporphyrin analyses, as suggested by tests with standard pigments, is evidenced in the determination of the benzoporphyrin series and the higher carbon numbers (C36+) of all series. Evolving from mass spectral experimentation on both standard and geoporphyrin mixtures certain error ranges in the determination of an average geoporphyrin mass spectrum and the indices (Table 23) extracted are concluded. These are as follows: percent-DPEP, where DPEP + ETIO = 100.0 (%); weighted average mass by series (C0 Ce) or overall (X) distribution (%); alkylation index (A.I., %); and percent benzopor phyrins (%). Error in the last index is, of course, decreased considerably if reported from electronic spectral data, as given in detail earlier in text. Concluding Remarks The investigations reported within this chapter were designed to provide the field with a single base-line study covering the analysis of tetrapyrrole geochemistry throughout the diagenetic-catagenetic metagenetic continuum. Except for the analysis of non-pigment oxidative and/or thermal breakdown products, we feel that this has been accomplished. Application of these analytical data and developed practices to the investigation of chlorophyll biogeochemistry is

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CHAPTER 4 RESULTS AND DISCUSSION II: GEOCHEMICAL INVESTIGATIONS 279 The study of tetrapyrrole geochemistry began in the 1930s with the works of Weigelt and Noach (1932), who reported chlorin-like compounds in the leaf residua of brown coals, and Treibs {1934a-b, 1935a-b), who isolated porphyrins and metalloporphyrins from various "fossil fuels." The landmark publication of Alfred Treibs'' {1936) drew the precursor product relationships (Fig. 1) between chlorophyll-a [I] and DPEP [XXXVIII], as well as between heme (cf. [LIV] and etioporphyrin-III [LII]. Thus, began both organic geochemistry and the study of tetrapyrrole pigments in that field. The study of the complete geochemical history of chlorophyll and related tetrapyrroles has been slow to develop. Aside from several pertinent studies in the 1930s (Conant and Hyde, 1930; Thayer, 1935; -Treibs 1934a-b, 1935a-b, 1936; Weigelt and Noack, 1932), ver y little in the way of novel insight emerged until the advent of modern mass spectrometric analyses (Baker, 1966; Baker et gl., 1967), which allowed a first look at the real complexity of the geological porphyrins. The intervening period (ca. 1936-1965) did yield certain insight but mainly from the petroleum chemist's interest in the Ni and V(VO) porphyrins as industrial catalyst poisons (Dunning and Moore, 1957; Erdman et gl.,

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280 1956, 1959) or as indicators of paleo-productivity (Daley, 1973; Kemp and Lewis, 1968; Vallentyne, 1954, 1955). The late 1980s, continuing into the 1990s, saw the emergence of truly molecular organic geochemistry and application to the geopor phyrins. To date, some 70-odd geoporphyrin structures have been reported (e.g., Callot et gl., 1990; Chicarelli and Maxwell, 1984, 1986; Chicarelli et gl., 1984, 1987; Eckardt et gl., 1991; Ekstrom et gl., 1983a; Fookes 1983a-b; Krane et gl., 1984; Ocampo et gl., 1984, 1985a-b, 1986, 1987; Prowse and Maxwell, 1991; Quirke and Maxwell, 1980; Quirke et gl, 1979, 1980b; Storm et gl., 1984; Wolff et gl., 1983) including diastereoisomeric forms of some (Bareham et gl., 1990). The determination of geoporphyrin structure is a near Herculean feat and one which usually requires 'big science,' that is--a group effort. In the cases mentioned above, three main groups are responsible for more than 90% of the known geoporphyrin structures. These are at Strasbourg, France (Universite Louis Pasteur); New South Wales, Australia (CSIRO) and Bristol, United Kingdom (University of Bristol). Further, two samples, Gilsonite and Messel oil shale, have been the most intensively studied. Until geoporphyrin identification attains a more 'routine' status, structural and diagenetic studies must rely on each other but not necessarily coexist. Therefore, the present investigations were undertaken on a wide variety of samples, using somewhat classical techniques, in order to describe the phenomenology of chlorophyll biogeochemistry and to provide a first approximation to the reactions involved and the forces behind each step. Then, in the near future, it shoul d be possible to

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meld structural studies with examination of sample suites to provide the best overall picture of tetrapyrrole diagenesis. 281 The present chapter will follow the divisions of tetrapyrrole geochemistry as set forth by Baker and Louda (g.g., Baker and Louda, 1983, 1986a; Louda and Baker, 1986, 1990). These divisions are diagenesis, catagenesis, and metagenesis and are taken directly from the precepts of modern organic (see Tissot and Welte, 1978). However, the application of these divisions and the subdivision by tetrapyrrole reaction sequencing is the focus of the present studies. The following sequence of topics will be discussed in order and serves here as an outline for the present chapter. The topics covered herein are: chlorophyll alteration prior to incorporation into sediments (senescence, herbivory); early diagenesis, oxidative alteration and reductive defunctionalization; mid-diagenesis, aromatization of dihydroporphyrins; late-diagenesis, chelation of nickel and initial release of bound porphyrins; catagenesis, release of vanadyl porphyrins from kerogen and changes in the 'quality' of metalloporphyrins (i.g., switch from DPEP-[alt. CAP, = to ETIO-series dominance and initial dealkylation); metagenesis, the continued dealkylation and eventual destruction of all tetrapyrrole structure. Following the above, selected special topics will be covered and include: aspects of petroleum metalloporphyrins; benzoporphyrins; the source of ETIO-series porphyrins, including in vitro thermal modeling; and, suggestions for certain standardization of analytical technique for better inter-study comparisons.

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Chlorophyll Alteration Prior to Incorporation into Sediment 282 A variety of changes in the quality and quantity of chlorophyll and other source {biologic) tetrapyrroles are possible prior to these pigments becoming a part of the non-living organic matter (OM) of sediment. Such changes include (1) "pheophytinization," the loss of magnesium from chlorophyll {Aronoff, 1953; Baker and Louda, 1982, 1986; Brown, 1963; Brown et gl., 1977; Conover et gl., 1986; Currie, 1962; Daley and Brown, 1973; Gillan and Johns, 1980; Hendry et gl., 1967; Hodgson et gl., 1960; Koyama et gl., 1968; Lamort, 1956; Nienhuis, 1981; Parsons et gl., 1977; Rothemund et gl., 1934; Schuman and Lorenzen, 1975; Shiobara and Taguchi, 1977; Strickland and Parsons, 1968; Vernet and Lorenzen, 1987; Yentsch, 1983; Yentsch and Menzel, 1963; {2) pheophorbide formation by the loss of both magnesium and phytol, primarily due to herbivory (g.g., Baker and Louda 1986; Blumer, 1965; Blumer et gl., 1963; Brown et gl., 1977; Carpenter et gl., 1986; Conover et gl., 1986; Currie, 1962; Daley, 1973; Daley and Brown, 1973; Eckardt et gl., 1992; Furlong and Carpenter, 1988; Gray and Kemp, 1970; Hallegraeff, 1981; Hendry et gl., 1987; Jeffrey, 1980; Keely and Brereton, 1986; King and Repeta, 1991; Lorenzen, 1967; Orr et gl. 1958; Prowse and Maxwell, 1991; Rothemund et gl. 1934; Schuman and Lorenzen, 1975; Vernet and Lorenzen, 1987; Wang and Conover, 1986); (3) "allomerization", the formation of hydroxy and hydroperoxide deriva-tives as precursor steps to further pigment alteration and/or destruction (Aronoff, 1953; Brown et gl., 1977; Daley, 1973; Daley and Brown, 1973; Gray and Kemp, 1970; Hendry et gl., 1987; Holt, 1958; Hynninen, 1979a-b; Louda and Baker, 1986, 1990); {4) chlorophyllase activity

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283 leading to chlorophyllides and pheophorbides (Barrett and Jeffrey, 1964; Hallegraeff, 1981; Hendry et gl., 1987; Holden, 1961, 1970; Jeffrey, 1974, 1980; Jones, 1979; Moreth and Yentsch, 1970; Strain, 1954; Vallentyne, 1960; (5) release of cellular acids catalyzing reaction with molecular oxygen (Aronoff, 1953; Daley and Brown, 1973; Hoyt, 1966a-b; Owens and Falkowski, 1982; Schuman and Lorenzen, 1975; and (6) rapid photooxidation involving single oxygen (Carpenter et gJ., 1986; Daley and Brown, 1973; Furlong and Carpenter, 1988; Gillan and Johns, 1980; Hendry et gl. 1987; Rontani et gJ., 1991, 1992; Vallentyne and Craston, 1957; Welschmeyer and Lorenzen, 1985; Yentsch, 1965). All of the changes given above lead to only two fates for water column chlorophyll, destruction or survival. In the first case, it is known that destruction, especially photooxidative, is the primary route and may involve allomerization and the formation of colorless compounds which still retain phytol and 4 nitrogen atoms (Henry et gl., 1987; Jen and MacKinney 1970a-b; Johns et gl., 1980; Park et gl., 1973; Simpson gl., 1976). As to the survival of chlorophyll tetrapyrrole skeletons, it is generally accepted that only about 0-5% (X -1%) of chlorophyll produced in the euphotic zone survives, primarily as pheophorbides (Baker and Louda, 1980a, 1982, 1986a; Carpenter et gJ., 1986; Currie, 1962; Daley, 1973; Louda and Baker, 1986) and pyro pheophorbides (Eckardt et gl., 1992; Keely and Brereton, 1986; King and Repeta, 1991; Prowse and Maxwell, 1991), for eventual incorporation into and possible 'fossilization' in sediments (Carpenter et gJ., 1986; Daley, 1973; Furlong and Carpenter, 1988; Kemp and Lewis, 1968; Orr gl., 1958; Yentsch, 1965).

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284 The above, albeit short coverage of c hlorophyll a lterati o n and destruction as pertaining to cell u l a r senescence, deat h and h e r b i vo rous recycling serves mainly to rei nforce a tremendous e nigma. T hat is, even given widespread interest i n chl orophyll, as rel ating to productivity/ standing crop studies and the C-c y cle in genera l (g.g. Broecke r 1974; Bunt, 1975; Deuser, 1971; Fen che l and Straaup, 1971 ; Gardiner, 1943; Goldberg, 1958; Gordon et at., 1 980; Hal l and Moll, 1975; Hargave, 1979; Harvey, 1934; Jeffrey, 1980; Parso n s et gl. 1977; Raymont, 1967; Richards and Thompson, 1952; Ryther and Yent sch, 1957; Sellner, 1981; Steeman-Nielsen, 1975; Stri c k land a n d Parson 1 968; UNESCO, 1966; Yentsch, 1965, 1983) the destru ction of chl orophy ll, estimated to be at the level of one-billion tons annually, is still unknown as to its mechanism(s) (Beevers, 1 976; Hendry 1 987; Hendry et gl. 1987; Holden, 197 0; S impson et gl., 1976). However i n most degradation schemes, the production of chlorophyl l derivative allomers (C-10 hydro x y: see F i g. 3, cf. compound [ IV]) is included as a key step (Hendry et gl., 1987). Aside from the participation of allomer ized derivatives in destruction, it i s known from i n vitro tetrapyrrole chemistry that al l omerization can lead to a variety of tetrapyrrole pigments lacking the isocyclic ring (i. g., pur purins, chlorins: see Fische r and Stern, 1970; Fuhrhop and Smith, 1975. cf. Appendix A). During the present studies, preliminary invest i gations which were meant to reveal certain of the pre-depositional alterations of the various chlorophylls were performed Further, these stud i es will also serve as a check on analytical technique and potential artifact formation (g.g., 'pheophytinization').

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Viable Algal Samples Certain observations made during the preparation of known or standard chlorophylls (viz., -a[I], -b[XIV] and -c1/-c2 [XIX]), described in detail in Appendix A, are directly applicable to the biogeochemical studies herein. 285 During the preparation of the chlorophylls-c1/-c2 [XIX] from the pelagic brown alga {Phaeophyta) Sargassum sp., it was found that when samples were extracted fresh, significant generation of polar chlorins {11 = 665 nm) occurred (see Fig. A6a: Appendix A). However, if the Sargassum sample was first stored frozen, these 'polar' chlorin artifacts failed to form {Fig. A6b). This result suggests that enzymes, with or without the assistance of cellular (viz. organic) acids, present in the cells of Sargassum lead to the alteration of chloro phyll-a [I] via pheophytinization, loss of phytol and oxidative changes of the isocyclic ring. That is, freezing, as well as other treatments (g g boiling, sonication), has long been used for the deactivation of chlorophyll altering enzymes (Holden, 1976; Jeffrey, 1962; Smith and Benitez, 1955). That these 'polar' (poly carboxylic acid) chlorins arise from the alteration of chlorophyll -a [I] is a conclusion by the author based on the fact that the chlorophyllsc1/-c2 [XIX] are porphyrins, as opposed to the di-and tetra-hydroporphyrin nature of the other chlorophylls, and, as such, are greatly less susceptible to oxidative alteration {cf Fischer and Stern, 1940; Fuhrhop and Smith, 1975). The above observation lends support to the idea that disruption of living algal cells in an oxic environment, as with mastication and ingestion during zooplankton feeding upon phytoplankton, can lead to

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286 enzymatic and o xi dative alteration of chlorophyll (viz -a [I] cf. Brown et sJ., 1977; Carpenter et sJ., 1986; Conover et sJ., 1986; Daley and Brown, 1973; Hendry et sJ., 1987; Louda and Baker, 1986; Prowse and Maxwell, 1991). Further, these results pointed to the absolute necessity to include an enzyme deactivation step (g.g., freezing) wherever one deals with living chlorophyll containing tissues. The green alga C losterium sp. Chlorophyceae), purchased from Carolina Biological Supply Inc. (U.S.A.), was utilized as a mimic for viable water column green phytoplankton. In this case, freezing the sample first allowed the extraction of the chlorophylls without the generation of oxidized artifacts. The total extract of this sample of Closterium sp. is given as Figure 84a. This e xtract also allowed a test of second derivative spectroscopy for the location of hidden or 'masked' maxima, due to overlapping absorption bands (cf. Rao, 1967). Following separation into the principal components, chlorophylls -a [I ] and b [XIV], it was shown that second derivative technique works well with mixed tetrapyrroles. The red alga Acrochaetium sp., purchased from Carolina Biologica l Supply Inc. (U.S.A.), was to be a natural source of chlorophyll-d (see Fig. 7). Chlorophyll-d was originally reported in the red algae Erythrophyllum delesseroides and Gigartina agardhii (Manning and Strain, 1943) and very few reports (cf. Holt, 1965; Smith and Benitez, 1955) exist since then. However, extraction of Acrochaetium sp., both fresh (Fig. 85) or frozen (not shown), failed to produce even a trace of absorption at ca. 420 and 690 nm (sec Holt, 1961; Holt and Morley, 1959; Smith and Benitez, 1955). Having failed to find any chlorophyll d in this alga, collections of various species of Rhodophyta were made

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1 0 1 0 430 b ) a ( 1 \ c) 429 ---b 454 r I I 0 8 I I I 661.5 II I I \\. \ Vl I I I-w z U0. 6 I => z I <{ 662 i:i: 0 ro I a:: I <{ 0 a:: Vl I I-iii I 644 a:: I <{ -2 I I I I I I I 02-l I -6St:f I I I Ol.Jt ,_, I 02-r"'i I I J I -41 I oo] I o o I \ I I I I I I I I 350 450 550 650 750 350 450 550 650 750 3 0 450 550 650 750 (i.,nml (;.,nm) Figure 84. Electronic absorption spectrum (a) and second derivative (b) of pigments extracted from the green alga (Chlorophyta) Closterium sp. and the electronic absorption spectrum of the isolated chlorophyl l a (solid) and -b (dashed). Solvents = (a/b) Acetone, (c) ethyl ether. N 00 ........,

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1 .0-, ------(/) w 1-U06 z z :::> <{ (!) 0:: 0 (/) 1-" (!) 0:: <{ 2 0.2 -4 0 0 350 350 450 550 650 ?50 Figure 85. Electronic absorption spectru m (a) and its second derivative (b) for the total pigment extract of the red a lga Acrochaetium sp . Band I absorption is reported at 690 nm for "chlorophyll-d"(Smith and Benitez, 1955) and the arrow indicates both its position and abscence. N 00 00

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289 along the coast of Southeast Florida (Delray, Pompano, Boca Raton, Deerfield), the Florida Keys (Elliot Key, Big Pine Key) and in the Tampa Bay area. Again extraction of these species (ca. 10 unidentified), either fresh or frozen, failed to yield detectable chlorophyll d. These results support Jeffrey (1980) who states: "I have never come across chlorophyll-d or -e in any of my pigment work. If they are 'real', they must occur only erratically and in small amounts." Chlorophyll-d may therefore be an artifact, possibly related to the known high lipoxygenase (peroxidase) activity of the Rhodophyta (Jacobi, 1962; Peterson, 1940). Coupling this known oxidative enzyme activity with the breakdown of cellular substructure during aging ('senescence'. cf. Beevers, 1976; Hendry et gl., 1987) it is possible that chlorophyll-d. as a chlorophyll-a degradation product, could form. Indeed, it is reported that treatment of chlorophyll-a with very dilute potassium permanganate led to the oxidation of the 2-vinyl moiety and, through a glycol intermediate, produced the 2-formyl (viz. 'chlorophyll-d') derivative (Holt, 1961). Therefore, chlorophyll-d appears not to exist in vivo in Rhodophytes. The diatom (Bacilliariophyccae) Synedra sp. was purchased from Carolina Biological Supply, Inc. (U.S.A.) and utilized for an ageing (senescence/death) experiment. In this case, half of the sample was frozen for several days and extracted. This portion ('FRESH') yielded primarily chlorophylls-a [I] and -c1/-c2 [XIX] upon analysis (Table 31). The remaining half was flushed with nitrogen, sealed and stored in the dark for two months d., 1404 h.). At this time the sample was frozen, thawed and extracted. Subsequent analysis (Table 31)

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290 Table 31. Tetrapyrrole pigment composition of the diatom Synedra sp. in fresh-viable and dark stored-dead states(1). t-DlAR PERCENTAGES ( 3 ) PIGMENT(2 ) FRESH (5) DARK-S1DRED ( S) ''VIABLE" "DEAD" Chlorophyll-a[ I] 91 <1 Chlorophyllide a <2 2 Pheophytin -a[IIa] 3 57 Pheophorbide-a[VIa] 1 28 Chlorophylls c1/c2[XiX] 3 12 ::!: a 7 :E: c (4) 33:1 7 : 1 FOO'INarES : (1)Samples purchased as "concentrated quarts"from Carolina Biological Supply,Inc .(U. S A ) (2)Cross referenced to known standards as given in Appendix A Chlorophyllide-a,described in Chapter 1,is chlorophyll-a without the esterified phytol moiety (3)Calculated following separtion over microcrystalline cellulose(Table 4)and using extinction coefficients as given in Table 16 (4)Rat i o of the sum of chlorophylla plus derivatives to sum of chlorophyllsc1/c2[XIX] (S)Extraction of the "FRESH" and "DEAD" aliquotes was following storage overnight at 40F. "DEAD" equates to an aliquote which,upon receipt,was flushed with nitrogen in the dark, stoppered and stored in the dark at room temperature(20 22C) for 2 d;1404h )

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291 revealed the total loss of magnesium from chlorophyll-a [I], resulting in the production of pheophytin-a [IIa] and pheophorbide-a [VIa]. This conversion of chlorophyll-a [I] to the 'pheopigments' is apparent even upon e x amination of the UV/VIS spectra (Fig. 86) for the 'FRESH' (solid) and 'DEAD' (dashed) samples In addition, a more rapid destruction of the chlorophyll-a [I] nucleus, relative to chlorophylls c [XIX], was noted. That is, the chlorophylls-a/-c ratio in the 'FRESH' diatoms was found to be about 33: 1 and this value decreased to about 7:1 following senescence and death (Table 31). E x amination of the chlorophylls-c [XIX] fraction from cellulose column chromatography (see Results I), certain clues as to the demise o f chlorophyll-a can be obtained. In this case, the UV/VIS spectrum (Fig 87) reveals the presence of the 'polar chlorin acids' described earlier during discus sion of Sargassum e xtracts. That is, at least with the diatom Synedra, it appears that the breakdown of cellular organization during aging and death elicits enzymatically mediated destruction of the chlorophyll-a structure. Specifically, splitting of the isocyclic ring and generation of poly carbo xylic acid chlorins appears to have occurred. Whether these compounds are truly i ntermediates during the opening of the tetrapyrrole macrocycle is unknown at present. In a closely related study, Yentsch (1965) reported that when the diatom Phaeodactylum sp. was kept in the dark for 270 hours (ca days) all chlorophyll-a disappeared with 1/3 being converted to pheophytin-a and 2/3 being destroyed No mention of the chlorophylls-c was made. In a hypothetical sea or lake devoid of predators one might surmise that only the chemistries of senescence (intracellular) and

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1.0-.---------------------------w U0. 6 z <{ d) 0::: 0 (/) m0. 4 <{ 0.2 )'-667 663"'t 0.0 292 350 450 550 (f\,nm) 650 750 Figure 86. Electronic absorption spectra of the total pigment extracts of fresh/viable(solid trace) and twomonth post-mortum/dead(dashed trace)diatoms(Bacilliariophyceae:Synedra sp.).Solvent=acetone.Asterisks draw attention to chlorophylls-c absorptions.

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0.8 w u 20 6 <( m a: 0 (./) C00.4 <( 665 0.0 --1----.----,----,-----===l 293 350 450 550 650 750 WAVELENGTHJnm Figure 87. Electronic absorption spectrum of the polar ( "CELL-3" ) fraction isolated from an extract of 2 -month dead diatoms(Synedra sp.: see Table 31).

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294 environment (extracellular) would be acting upon the chlorophylls as phytoplankton, algae and bacterial, sinks through the water column. This, of course, is not the case. Thus, grazing of phytoplankton and tripton (detritus) and the packaging of excreta into fast-sinking fecal pellets enter the overall scheme of chlorophyll biogeochemistry Sediment Trap Samples It has been estimated that nearly all of the primary organic particulate matter in the sea is consumed (Steele, 1974). Assimilation is, however, rarely 100% (Riley and Chesta, 1971). Herbivore and carnivore consumers typically package excreta into fecal pellets which often fall through the water column at a much faster rate that the individual food items. This so-called ''fecal express" (Gowing and Silver, 1983; Silver and Bruland, 1981) is now known to be one of the, if not the main, routes for the transport of organic matter from the photic zone to underlaying sediments (see g.g. Bruland and Silver, 1981; Gagaosian et gl., 1983; Honjo, 1980; Staresinic et gl., 1983). In order to examine certain of the pre-depositional changes in phytoplankton chlorophylls, sediment trap samples collected in the -upwelling region off Peru were studied. These samples were provided by Woods Hole Oceanographic Institution (Drs. R. B. Gagosian and J. W. Farrington) and were collected using a floating 1ediment 1rap (FST: see Staresinic et gl. 1982) by the R/V Knorr at 15 10's x 75 35'W (Gagosian et gl., 1980). A world site map, showing not only the location of these Peru sediment trap samples (PST) but including all geochemical sampling sites, for the present work, is given here as Figure 88. Detailed locations of the sediment trap samples FST-16/-17,

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!>"' j) 56/57 r *58 180' 9 BAK. 71 ._ 295 o' Figure 88 World map with samp l e locati o n s delineated. N umbers refer t o D SDP/IPOD Leg (NN) or Leg -Site(NN-NNN). Abbreviatio n s :BAK=Bakk e n Formation,W e s t ern Canadian Basin; MO=Monterey Formation,onshore/offsh ore California;BSL=Big Soda Lake,Farralon,Nevada;TB=Tampa Bay,Florida;PAP=Pond Appl e Peat bog, F l o rida;ML=Man g rove Lake,Bermuda

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296 as wel1 as the Peru sediments, are shown in Figure 89. Table 32 contains selected pertinent data for the sediment trap samples analyzed during the present study. The location of these sediment traps within the water column was such that FST-16 (Z = 11 m) was at the base of the euphotic z one (I 0.01 Io) and FST -17 (Z = 53m) coi ncided with the seasonal thermocline (Gagosian et 1980). It must be remembered that these traps collect sediment, that is, materials which are falling out of the water column. Therefore, 'living' phytop lank ton are likel y to be present in only trace amounts. The hydrographic data for this region (Gagosian et includes the now classic determination of chlorophyll and pheopigments by fluorescence before and after (fo/fa) acidification ('pheophytinization': see Yentsch and Menzell, 1963). In the region of 15S the chlorophyll-to-'pheopigment' rati o i s reported as being more than 10:1 i n the euphotic zone and less than 0 5:1 at the thermocline and below (Gagosian et 1 980). These data indicate a quite rapid and nearing completion loss of Mg from chloro phyll (viz -a). Going on the basis that chlorophyll-a, per se, i s reported from on-board on-site hydrographic studies and given that the samples studied herein were frozen for about one year prior to e xtraction, it appears likely that a portion of the pheophytin-a [IIa] reported here in Tabl e 32 was actually collected as chl orophy ll-a [I]. In order to even estimate the proportion of chlorophyll-a in the tetrapyrrole flux, one would need to e xtract and analyze these pigments immediately upon retri eval of the sediment trap. However, for geochemical source and precursor studies, the potential e xistence of chloro phyll-a [I] in this water column detritus is nearly moot as the loss of

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297 BC5- ICILOMETtRS Figure 89. Site map for sampling locations off the coast of Peru near l5S.(Redrawn from GagGsian et Samples described in Tables 32-33).

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Table 32. Floating sediment trap data(1). SAMPLE PARAMETER FST-16 LOCATION DATE TIME DEPI.DYED DEPTH,subsurface(Z) POC FLUX(2 ) PONFLUX 15.4'x75.1'W 3/12/78 0850-1555 CHLOROPHYLL DERIVATIVES(J) 'IDTAL YIELD ( 4 ) CONCENTRATION(S) DISTRIBUTION ( 6 ) PHEOPHYTIN-a PHEOPHORBIDE-a I 'PHORBIDE-686 '' POLAR CHLORINS Clllorophylls-c L -c FOOTNOTES: 11m 18.7mgC/m2/h 2.5mgN/m2/h 92.62 ug 22.2mg/gC 74.9% 11.7% nd 4.3% 4.3% 22.3:1 FST-17 15.4'Sx75.6'W 3/12/78 0904-1608 53 m 34.8mgC/m2/h 2.4mgN/m2/h 67.50 ug 8 .4mg/gC 45.8% 42.3% 2.1% 4.0% 5.8% 16.2:1 298 1-Sampling data from Gagosian et al.,1980,1982.POC=particulate organic carbon. 2-PON(particulate organic nitrogen)data from Lee and Cronin,1982. 3-This study. 4-Given per one-eigth sample split,as received. 5-Calculated using the following data:POC flux;sample splits received; and trap sampling area of 0.26 m2(cf.Gagosian et al.,1983b). 6 -Distribution determined following chromatography over microcrystalline cellulose(See Chapter 4, Table 4).

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299 magnesium is both facile and an accepted phenomenon of senescence and herbivory (refs. in Baker and Louda, 1986a; Hendry et 1987). E x amination of the distribution of chlorophyll derivatives in these sediment traps (Table 32) allows certain trends to be distinguished as discussed below. As forming sediment leaves the photic zone and passes through the thermocl i ne, on its way to the bottom, significant loss of phytol occurs. This of course, is signaled by the increase in pheophorbide-a [VIa]. Reprocessing of phytol from chlorophyll in these waters has been reported as leading to a significant production and flux of 6,10,14-trimethyl/pentadecan-2-one due to oxidative degradation aided by passage through herbivore guts (Volkman et . 1983). As covered during the introduction to this chapter, the removal of magnesium and phytol (from chlorophyll-a [I], generating pheophorbides-a [VIa], are known to occur during the heterotrophic activities of zooplankton It must be noted here that it is entirely possible (probable) that within the isolates labelled "pheophytin-a" (Table 32) e xist a certain percentage of non-phytyl pheophorbide-a esters. That is, it has only recently been discovered that pyro-pheophorbide-a steryl esters exist in water column detritus and surface sediments (Eckardt et 1991, 1992; King and Repeta, 1991; Prowse and Max well, 1991). Further the e xistence of pyro-pheophytin-a [III], known to occur in aquatic/marine depositional settings (Baker and Louda, 1982; Keely and Maxwell, 1991; Keely et . 1988), is likely as well. The alteration of pheophytin-a [IIa] leads, with settling, to the production not only of pheophorbide-a [VIa], and probably pyro pheophorbide-a [VIII] (cf. Keely et . 1988, 1990) but also to

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300 certain distinct alteration products. In the present case, the generation of the "poly" (viz. di-/tri-) carboxylic acid chlorins ("POLAR CHLORINS" in Table 32) is observed andis discussed more fully later, as they may relate to oxidative pigment destruction (cf. Louda and Baker, 1986). the pigment given as "PHORBIDE-686", so named after the type of absorption spectrum (see "Introduction", Fig. 47) and the (nm) position of band I, has previously been isolated from marine diatomaceous sediments (viz. DSDP/IPOD Leg 64, Sites 477 and 479: Baker and Louda, 1982). During that and a subsequent study and Baker, 1986) Phorbide-686 was tentatively identified, on the basis of the UV/VIS spectrum before and after treatment with sodium borohydride, as a pheophorbide-a derivative with additional carbonyl conjugation at the C-2 position (g.g. 2-acetyl-2-desvinyl-pheophorbide-a). However, study here reveals that the 'additional' carbonyl moiety cannot exist at the expense of the vinyl group. That is, following reduction with NaBH4 the resultant in vitro derivative (Soret = 396, I = 651.5 nm) is indistinguishable from 9-oxydeoxo-pyropheophorbide-a [IX] (Table 10) and does not mimic 9-oxydeoxo-mesopyropheophorbide-a [XI]. That is, treatment of the 2-acetyl-pheophorbide-a (cf. Louda and Baker, 1986) with NaBH4 would produce 2-a-hydroxy-ethylpheophorbide-a which would yield an electronic absorption spectrum (cf. Fischer and Stern, 1990; Gouterman, 1978; Weiss, 1978) more akin to 9-oxydeoxo-meso pyropheo phorbide-a ([XI], Table 10, Soret = 394.8 nm, I= 642.0 nm). Given the identification of a 132,172-cyclopheophorbide-enol from a sponge (Porifera: Darwinella oxeata) and its probable conversion from dietary chlorophyll-a (Karuso et . 1986), it now appears likely that our previous "Phorbide-686" isolates (Baker and Louda, 1982; Louda and

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301 Baker, 1986) and the one reported here (Table 32, FST-17) are similar, if not identical, to this compound with its condensed propionic acid moiety. The similarity between "Phorbide-686" and the 132,172-cyclo pheophorbide-enol (Fig 90), whether an in vitro known (Falk et . 1975) or the isolate from a sponge (Karuso et . 1986), is too striking to overlook Further studies are planned with "Phorbide-686 (hplc, NMR) and, for now, we can but tentatively identify it as a 132,172-cyclopheophorbide-a-enol and state that it probably forms via a dehydration/condensation reaction following the formation of pyro pheophorbide-a (see [VIII]) during residence in the alimentary canal of a consumer. This compound (Fig 90) will be returned to in subsequent sections as a probable precursor to geologic chlorins and porphyrins with dual fused isocyclic rings (g.g. Bareham et 1990; Callot et 1990; Keely and Max well, 1990). The last observations to be made here regards chlorophyll d ynamics in the Peru sediment traps revolve around pigment loss and the potential fate of chlorophyll-c. As organic matter falls through the water column much of it is recycled through various trophic levels These processes are so complete that average OM survival to underl y ing sediments is usually given as ca. 0 .1-1% (g.g. Menzel and Ryther, 1970; Tissot and Welte, 1978). These estimates are for the World Ocean as a whole and OM survival to under lying sediments is a known and definite function of the length (i.g. depth) of the water column from the euphotic zone to the sediment interface, as moderated by such factors as o xygen tension (i.g., o xic vs. anoxic) and sedimentation rate. Therefore, survival of primary production (OM) into aquatic/ marine sediments can range from 2-10+% as well (g g Deuser, 1971;

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-H20 Figure 90. The dehydration/cyclization of pheophorbides-a to 132 ,173-cyclopheophorbide-a enol(See Falk al.,1975;Karuso w 0 N

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303 Gagosian et gJ., 1982; Holm-Hansen, 1970; Honjo, 1980; Kemp and Lewis, 1968). In productive areas across the world ocean, typical OM survival rates with depth in the water column are 10% to the base of the euphotic zone (Gagosian et gl. 1982, 1983b; Honjo, 1980; Lee and Cronin, 1983; Lorenzen et gl., 1983; Wakeham et gl., 1980}, 4-6% to mesopelagic depths (Z 100-700 m, 10C isotherm: Denser, 1971; Gagosian et gJ., 1982, 1983b; Holm-Hansen, 1970; Honjo, 1980} and 0.1-1.5% to bathypelagic regions (Z 1,000 m+; 4 to 10 isotherms: Gagosian, 1982; Holm-Hansen, 1970; Hanjo, 1980). Hydrographic data collected on-site (Gagosian et gl., 1980) and subsequent studies on the OM flux at (Gagosian et gl., 1983b; Lee and Cronin, 1982; Wakeham et gl., 1983} provided the following insights as to primary productivity, OM flux and chlorophyll dynamics during the collection of the sediment trap samples (FST-16/-17) analyzed presently. During 'normal' (non-bloom) conditions, upwelling induced high productivity ranged about 4-6 g C/m2/day and, correspondingly, chlorophyll-a was present at about 20 'Normal' (non-bloom) productivity was mainly due to the photoautotrophic activities of the pennate diatom Thalassionema nitzchoides and the centric diatom Thalassiosira eccentrica, both of which could also be found in quantity in the fast sinking fecal pellets of the anchoveta Euqraulis rinqens. In the general area of 15, but not at the sites of FST-16/-17 (Fig. 89) during collection times (Table 32}, intense phytoplankton patch iness due to blooms of various dinoflagellates of the genus Gymnodinium were also noted. Within these blooms the concentration of chlorophyll a rose to nearly 75 At site, an OM flux of about 450 mg C/m2/d to the bottom of the photic zone (Gagosian et gl., 1983b}. They also

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304 calculate that flux to the base of the photic zone includes 11% of primary productivity but only 2% (5 mg/m2/d) of the standing stock of chlorophyll-a. As sedimenting OM passes through the main thermocline (53 m, FST-17: Table 32) only 6% of primary production (TOC) and 0.7% (1.6 mg/m2/d) of photic zone chlorophyll-a remains. Comparing the values of Gagosian et gl. (1980, 1983b) to those obtained here (Table 32) we find excellent agreement. That is, equating the 22.2 mg 'pheo pigment'/gC found in FST-16 to 2% chlorophyll-a survival allows us to calculate an expected survival, based on the FST-17 yield of 8.4 mg/gC, of 0.76% at 53 m(i .g. 22.2/11% = 8.4/X%, X= 0.76%). This is in excellent agreement with the value reported from on-board hydrographic data and allows us to state that differential decomposition of chloro phyll-a derivatives, as a total, has not occurred during the one year the samples spent in a frozen state. However, while the relative survival of chlorophyll derivatives in the present sediment traps (FST-16/-17) is as expected from the on-board hydrographic data, the absolute yield is not. That is, based on the above flux of "chlorophyll-a" (i.g. the sampling area (0.26 m2 ) and the time of deployment (7.04 hr. 0.2933d), we would 'expect' 381 and 195 total pheopigments in these traps. Analyses (Table 32) revealed only ca. 95 and 68 total pheopigments for the samples collected at 11 and 53 m, respectively. This amounts to about 25 and 35%, respectively, of the 'expected' yields. There is, however, no reason to suspect the results either on Table 32 or in Gagosian et gl. (1980, 1983). It is well known that day and night OM fluxes differ due to vertically migrating zooplankton (Herman, 1983; et gl., Parsons et gl., 1977; Riley and Chester, 1971; Sellner, 1981; Wakeham., 1983) and to spatial.and

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305 temporal phytoplankton patchiness (Hallegraeff, 1981; Harvey, 1934; Kreps and Verbinskaya, 1930; Marra et 1982; Parsons et . 1977). In fact, in the immediate area of collection the samples FST-16/-17 (Table 32) it is reported that day and night OM flux was vastly different (Gagosian et 1980, 1983a-b; Lee and Cronin, 1983; Volkman et 1983; Wakeham et 1983). This difference could be up to tenfold on a class-specific basis. That is, the night flux of sterols was lOx that of the day flux at this site, due mainly to the presence of vertical migrators and their activities (i.g., molting, fecal pellets: Gagosian et 1983b). Therefore, given the above, the values reported here for the pheopigments trapped in FST-16/-17 (Table 32) appear well in line with the chlorophyll dynamics of the Peruvian upwelling system. The e xact ratios of chlorophyll-a to the alternate phytoplankton chlorophylls (-b, -c1/-c2 ) are phenomena of species specificity (Allen, 1966; Jackson, 1976; Strain, 1958; Strain and Svec, 1966). E xamples of chlorophylls-b/-a ratios are 0 2-0.7 (Bjornland, 1982; Jeffrey, 1976) and for chlorophylls-c (c1/c2)/-a of 0.4-0.8 (Bidigare, 1989; Jeffrey, 1963). The fate of chlorophyll-b, an accessory pigment in green algae (Chlorophyta), was not directly observed during the present studies, other than by its absence. However, as chlorophyll-b has been suggested the precursor of choice for certain 3-H-3desmethyl-DPEP structures (viz. IUPAC, 7-nor-DPEP: Chicarelli and Maxwell, 1987). The reaction leading from chlorophyll-b to the '7-nor-DPEP' structure (Figure 91) must occur early on in the water column. That is, chloro phyll-b and/or its recognizable derivatives are not reported below

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CHLOROPHYLL -b H,_,-,,0 <;=a "D O CH3 'Phytyl 3-("7") nor-OPE P CHLOROPHYLL-c3 ,.CH3 HOOC Figure 91 Structural relationships between the chlorophylls b and -c3 and 7-nor-DPEP("J-desmetyl-DPEP":Fisher nomen clature). w 0 Ci'

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307 about 100-200 m in marine settings (Jeffrey, 1976; SooHoo and Kiefer, 1982a}. Given that chlorophyll-b is less prone to 'pheophytinize' than the 'a' pigment (Brown et gl., 1977; Hoyt, 1966b; Joslyn and Mackinney, 1938; Lamort, 1956; Mackinney and Joslyn, 1940}, it is possible that a more complete photo oxidative/oxidative destruction could occur. Specifically, conversion to pheopigments, notably pheophytin, deactivates the dihydroporphyrin nucleus towards oxidative destruction (Chichester and Nakayama, 1965; Hendry et gl., 1987; Hoyt, 1966b}. Therefore, for chlorophyll-b to survive into the realm of geochemical fossilization, one or both of the following need to occur. First, in moderately deep water {g.g., Z >150m), the removal of the auxochrome effect of the 3-formyl group must be occurring rapidly upon senescence and/or predation. That is, without the keto conjugation of that formyl group, the chromophore of a 3-desformyl-pheophorbide-b is the same as the a-series pheopigments. Secondly, perhaps chlorophyll-b [XIV] nuclei only survive in very shallow highly anoxic depositional settings. However, it is also quite possible (probable -?) that the recently identified chlorophyll-c3 (Bidigare et gl., 1990}, a 3carbomethoxy-derivative of chlorophyll-c 2 could very well be the precursor to the 7("3",Fisher)-nor-DPEP compounds (Figure 91}. The fate of the very prevalent accessory pigments chlorophylls c1/-c2 including -c3 and Mg-2,4-divinyl-pheoporphyrin-a5 (see Bidigare, 1989; Bidigare et gl., 1990}, in the marine water column also serves an enigma. That is, as shown during the current study of sediment trap samples (Table 32}, chlorophylls-c [XIX] do exist in the mixture of tetrapyrrole pigments found in water column detritus. However, as will be detailed in the following section, chlorophylls-c

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308 [XIX] are inexplicably absent from surface sediments, given that the water/sediment interface is well below the photic zone. Complexation with larger and/or forming macromolecular complex es (viz. 'protokerogens') may offer a partial explanation as to the apparent pre-and pre-/syn-depositional 'disappearance' of the chlorophylls-c [XIX]. The overall fate of the chlorophylls in fresh water and marine aquatic water columns, as gleaned both from the algal and sediment trap studies reported above and in the literature, is synopsized below. Phytoplankton cells can terminate their active existence in the euphotic zone in two, often overlapping manners. First, senescence and death can lead to the input of intact algal cells into the sediments. Cells following this route contain chlorophyll derivatives dictated by the chemistry of that particular species cytoplasm (g.g., acidity, chlorophyllase, etc.and external factors such as light and oxygen (viz. photooxidation). In these cases, pheophytins and pheophorbides dominate with major losses occurring via photooxidative/oxidative means of, as yet {cf. Hendry et gj. 1987), unknown mechanisms. The second and vastly more prevalent route involves the passage of phytoplankton cells through at least one, and possibly more, heterotrophic organisms. Typically, zooplankton (g.g. copepoda, euphausids, salps, anchoveta, etc.) feed on phytoplankton, living and/or senescent/dead, and mechanically disrupt the algal cell during mastication/digestion. Here, the combined action of zooplankton gut acids/enzymes and the remaining active components of the phytoplankton cytoplasm work to form mainly pheophorbides. Recently there is good evidence that loss of the 10carbomethoxy moiety, forming pyro-pheophorbide-a [VIII] from chloro phyll-a [I], occurs wi. dely at this stage as well {cf. Eckardt et gj.,

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1991; keely et gJ., 1988, 1990; King and Repeta, 1991; Prowse and Maxwell, 1991). At any rate, the vast majority of the chlorophyll 309 derivatives which enter sediments in a 'typical' fresh water or marine sedimentary environment do so as 'pheophorbides' (viz. no Mg2+, no phytol) and arrive in fast sinking fecal pellets. Sedimentary Alteration of Chlorophyll Derivatives Since it is impossible to clearly separate the biotic and abiotic alterations of chlorophyll in the water column (predepositional), we shall consider the 'geochemistry of chlorophyll' as encompassing the, essentially abiotic, changes which occur in the structure of chlorophyll-derivatives in the syn-and post-depositional sedimentary environment. However, even upon incorporation into sediment, biological alterations are still possible and are all but unknown for the tetra-pyrrole pigments. That is, the role of benthos, such as polychaete coprophages (Taghon et g]., 1984), other 'ilytrophic' [mud eating] organisms (Swain et g]., p. 164), or bioturbation in general is also unknown in these studies. Further, until sediments stabilize in an anoxic reducing condition, abiotic oxidation is also possible. Therefore, such phenomena as resuspension of surface sediments into a 'nephloid layer' (Feely et g]. 1973 and references cited) with prolonged exposure to water column oxygen could also lead to additional unknown alterations to the tetrapyrrole mixture. Ideally, for a geochemical investigation to be as straightforward as possible, the researcher would like a setting or a series of settings in which water column productivity is monotonous through time and both the water and sediment column chemistries respond only to the

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310 physicochemical pressures of sediment accumulation That is, as OM becomes buried it would undergo only the influences of directly analyz able changes. Such changes would include; smooth dewatering/compaction curves with time, linear pressure increases and constant heat flow. Such settings, for more than brief geologic glimpses, obviously do not exist. as the dynamic nature of tectonics would predict. Therefore, we must examine a series of snapshots and short features and attempt to edit and splice to yield the correct picture. Chlorophyll Diagenesis As given above, we (Baker and Louda, 1983, 1986; Louda and Baker, 1986) have defined the diagentic stages for chlorophyll geochemistry as the following: early-diagenesis, the oxidative alteration and/or reductive defunctionalization of pheophorbides and derived 'chlorins' (viz. dihydroporphyrins); mid-diagenesis, the aromatization of dihydro porphyrins to yield porphyrins; and late-diagenesis, the chelation of metals by porphyrins or in other ways the conversion of all tetrapyrrole pigments to the metalloporphyrin level. Early-diagenesis is presently the most complicated level and the least understood from a structural-mechanistic viewpoint. As such, early-diagenesis has received the most study herein and will dominate this section. Midand late-diagenesis are, in essence, two sequential apparently first order reactions occurring in direct response to the integrated effects of time and temperature. Herein, early diagenesis will be examined in three ways. First, we shall trace the survival/demise of chlorophyll derivatives and relate trends to productivity, paleoenvironment and post-depositional

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311 sediment physicochemistries Eh, pH, %water, temperature, etc.). Second, an examination of crude dihyroporphyrin polarity classes esters, acids and di-/tri-acid ['polar'] assemblages) will be made in an effort to reveal trends in the loss of phytyl esters and the fates of carboxylated pigments. Third, an effort is made to trace the sequential yet anastomotic defunctionalization of the dihydroporphyrins prior to the aromatization/chelation events of mid-/late-diagenesis. Pigment yield, the concentration relative to some 'independent' variable sediment weight, organic carbon, etc.), has been traced since the onset of geochemical/limnological studies (Harvey, 1934; Kozminski, 1938; Kreps and Verbinskaya, 1930; Trask and Wu, 1930; Treibs, 1936) and has laid the basis for paleolimnological studies (Daley, 1973; Daley et . 1977; Kemp and Lewis, 1968; Sanger and Crowl, 1979; Vallentyne, 1955, 1960). Throughout these present studies we have kept track of pigment quantitation and have converted it to a yield value using dry sediment weight, percent organic carbon and/or extractable organic matter (EOM, bitumen) as the comparators of choice. Here, yield will be discussed as representing the combined effects of initial pigment stock (viz. paleoproductivity) plus those factors which lead to pigment (g.g. predation, oxidation) or preservation anoxia, rapid burial). Specifics for selected site studies will be withheld for consideration during discussion of defunctionalization and diagenesis per se The concentration of an organic compound or class in sediments depends both upon its original supply and rate of removal. Removal here is used to infer the combined effects of actual destruction (viz.

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312 metabolism, abiotic oxidation, etc.) plus decreases in concentration within bitumen via incorporation into inextractable complexes (g.g. kerogen). Considering the overall domain of chlorophyll geochemistry, we shall delineate four main regions or events which affect the ultimate yield of tetrapyrrole pigments for any given point in that continuum. First, the actual supply and preservation of pigment to the surface sediment. Second, the rate at which surface sediments stabilize and become reducing. It is during this period, as we will show later, that stabilization of the tetrapyrrole nucleus also occurs and a portion of the original extractable pigment becomes hidden from analysis through geopolymerization/chemiabsorption reactions. Third, yields of pigment, then as metalloporphyrins, increase at the onset of petroleum generation due to release from bound states. Lastly, yields decrease during continued petroleum generation due to dilution and finally by thermal destruction. During the present consideration of chlorophyll diagenesis, we shall be concerned mainly with the first two events given above. That is, sedimentary chlorophyll derivative yields will be taken as reflecting the combined effects of supply from overlying waters and the environment of deposition, namely oxic versus anoxic, destructive versus preservative. Table 33 contains tetrapyrrole pigment yield data for 38 sediment samples comprising down-hole suites from three sites associated with the Peruvian upwelling system at 15$. These sites (SC4, SC6, BC5) are given in Figures 89 and 92. In this region the oxygen minimum zone (OMZ) intersects the bottom at depths spanning 100 and 500 meters. The Peru upwelling system was chosen as a study site since it forms a

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313 Table 33. Tetrapyrrole pigment yield data for surface to near-surface sediments collected beneath or near the Peruvian upwelling system at about 15S. SAMPLE 1 (depth,cm) SC4(02) SC4(2-4) SC4(47) SC4(7-10) SC4(10-13 ) SC4(13-16 ) SC4(16-19 ) SC4(19-22) SC4(22-25) SC4(2528) SC4(28-31) SC4(31-40) SC4(40-46) SC4(4652) SC4(52-54 ) SC4(5457) SC4(57-64) SC4(64-70) SC6(0-3) SC6(3-6) SC6(6-9) SC6(9-12 ) SC6(12-15 ) SC6(15-18) SC6(18-21) SC6(21-27) SC6(27-33) SC6(33-39) SC6(39-45) SC6(45-51) SC6(51-57) BCS(0-2) BC5(2-6) BC5(6-8) BC5(8-12) BC5(12-20) BC5(20-24) BC5(24-28) PERCEtvi WATER 79.0 69.5 63.9 70. 4 58.2 63. 7 70.3 65.5 63.0 60. 4 52. 0 63. 3 53.5 64.0 66.2 72.4 81.3 67.2 90.4 90.7 89.1 89.3 89.4 89.3 89.1 86.4 85.8 87.4 88.6 84.8 74.9 74.7 74. 6 80.0 78.6 73.3 65.5 67.5 ORGANIC3 CARBON,% 3.14 2.3 2.3 2 7 2.0 1.4 2.35 2.7 1.67 1.6 1.13 1.80 0.80 1.24 d n a 1.5 1. 79 2 .02 5.71 5.38 6.29 5.98 5.68 6.18 7.10 7.82 7.71 4.38 3.29 5.93 5.58 3 .38 3 .58 3 71 3. 78 4.04 3.29 3.02 TEI'RAPYRROLE4 PIGMENTS pg/g-sed. ,dry wt. 150. 1 54.8 36.9 40.7 20. 4 2.3 42.2 37.0 49. 4 8 3 5. 8 22.3 4.5 7.9 2.5 34.0 54.9 40.5 263.4 207.1 150.0 171.6 179.3 179.5 191.3 163.8 131.5 123.5 112.1 165.8 50.1 25.6 30. 8 28.6 26.8 24.0 17.4 16.2 5 P.Y.I. 47.80 23.83 16.04 15.07 10.20 1.64 18.00 13.70 29.58 5 .19 5.13 12.39 5.62 6.3 7 22.67 30.67 20.05 46.13 38.49 23.85 28.70 31.57 29.05 26.94 20.95 17.06 28.20 34.07 25.96 8.98 7.57 8 .60 7. 71 7 .07 5 .94 5.29 5.38 FOOTNOTES:(1)SC=Soutar Core,BC-Box Core:Sample splits as received from by heating at 106-108C for 18hours, repeated to constant dry weight. (3) Provided by Dr J .\-1. Farrington, \olHOI. (4)Determined as the sum of chromatographic fractions or as a crude extract assuming the major pigment to be pheophoribide-a(see text). (5)PYI =Pigment Yield Index.Defined as the tetrapyrrole yield in ug/g sediment,dry weight divided by the percent o r ganic carbon(See Louda et al. ,19 80)

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5 10 E 50 ---100 N DISTANCE OFFSHORE 70 60 50 40 30 OM Z (<0.1mL/L) I ro._ w 0 1----0. 5 314 10 \ I \ \ \ I I I \ Figure 92. Dissolved oxygen transect of the waters off Peru at about 15S.Sample locations(Table 33)indicated by asterisk.Dotted area indicates growth of Thioploca sp. (Redrawn from Henrichs and Farrington,1984 and data in Gagosian al. ,1980).

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315 modern active parallel environment to certain ancient systems Miocene Monterey Formation: cf. Graham and Williams, 1985; Isaacs and Petersen, 1987; Orr, 1986) which, over geologic time, have produced significant petroleum source rock accumulations. The sample suites investigated included a deep water oxic oceanic site (BC5: 15 25.9's x 76 08.5'W; Z = 5,380 m), a site in the middle of the present strong DMZ (5C6: 15 05.1'5 x 75 43.9'W; Z =268m) and an upper shelf site (5C4: 15 03.1'5 x 75 30.3'W; Z = 92 m). This last site (5C4) is characterized as being at the edge of the present OMZ and is influenced by fluctuating oxygen levels as the OMZ expands and contracts (cf. Henrichs and Farrington, 1984; Gagosian et 1980; McCaffrey et 1990). As given earlier, it is desirable to standardize pigment yield to the organic portion of sediments both on a dry weight basis. This then allows for better inter-sample, inter-site and intra-site comparisons as it disregards dilution by inorganic constituents and down-hole concentration due to dewatering/compaction. In the present study, the yields of tetrapyrrole pigments will be reported as both absolute pigment/ g-sediment, dry weight) and relative (re corg' dry wt.) values. In the latter case, the value determined is termed the Qigment tield index or P.Y.I. cf. Louda et 1980). The determination of P.Y.I. is simple and requires only the division of the absolute yield dry wt.) by the percent organic carbon (Corg' dry wt.) of the sediment. Examining the pigment yield data collected for the Peru sediments (Table 33), we find preservation trends in-line with what one might expect based upon the description of the three sites given above. That

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316 is highest yields and best down-hole preservation occurred within the strong 'permanent' DMZ beneath the region of highest surface productivity. Plotting the P.Y. I values in Table 33 versus sediment depth affords much easier inter-site comparisons (Figures 93a-b) Site BC5, the oxic (02 > 3.0 ml/L) oceanic 'reference' site, reveal s a rather monotonous down-hole trend. That is, organic carbon values range from 3.0-4.0 % and P Y.I. values f rom 5.3-8.6 are found. A slight trend for decreasing P.Y.I with depth is noticed Even though this site is characterized 'oxic,' due to the o xygen content of the near-bottom waters, it is evident that good downhole preservation of chlorophyll derivatives is occurring Further, the organic carbon c ontent of this oceanic s ite is from 3-10 times that routinely found for sediments deposited at such great depths (cf. Premuzic et gl., 1982; Riley and Chester, 1971). Though sedimentation rates are not available for site BC5 they must be considerably less than for the inshore sites. Therefore, on that basis it is not surprising to find a down-hole trend lacking the 'zig-zag' patterns related to environmental controls. That is, each 2-4 em slice at site BC5 probably incorporates a longer time span and all highs and lows in input will have been averaged, both by sediment mix ing and sampling bias. Having found that the P Y.I. value for detritus raining out of the productive region off Peru tends towards 50 (sec SC4(0-2) and SC6(0-3) : Table 33), we can but surmise that significant destruction of chlorophyll derivatives, as related to the overall carbon content, has occurred during the pre-and syn-depositional histories of these sediments. That is, P .Y.I. values average 6.8 for the f irst quarter meter (0-25 em) at BC5 as compared to 30.7 for the 'ano xic' site SC6. However, as stated, for an oceanic

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P IGMENT YIELD INDEX(P YI) 0 10 20 30 40 50 0 10 20 30 40 50 0 c./) L 10 (1) 20 +-' (1) E c 30 (1) u 2 40 f2 5 C? 50 m ::::> (j) I 60 BC5 I ________________ ......... ........... ........... SC4 b) Figure 93 D ownhole trends in the Pig m ent Yield Index(PYI) for three sites sampled beneath the Peruvian upwelling system at about l5S(See Table 23). (a) Sites BCS and SC6, (b) Site SC4. w ....... -....J

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site at this depth (Z = 5,380 m), there still exists significant organic carbon and respectable chlorophyll derivative preservation. 318 The two sites located on the continental shelf, SC6 and SC4 (see Figure 89), represent some of the best modern analogs to an embryonic marine source rock studied by the author. Here, diatomaceous sediments are rapidly accumulating (ca. 1 1 cm/y: Henrichs and Farrington, 1984) in an anoxic environment. Added to the detrital organic matter is the reformed OM due to the high activity of Thioploca spp. (Gagosian et gl., 1980; McCaffrey et gl., 1990), a white gliding filamentous member of the Beggiatoaceae (Gallardo, 1977; Sassen et gl., 1993). The sediments at sites SC6 and SC4 were olive-green diatomaceous.oozes containing about 1-2% carbonate carbon and were infiltrated to about 25 and 5 em, respectively, with grayish white Thioploca spp. filaments (Gagosian and J. W. Farrington, pers. commun.; and personal observations of the author). The description of the color of these chemosynthetic procaryotes has purpose That is, in one of the earliest reports on the presence of Thioploca spp. in the sediments off Peru Chile, the coexistence of Oscillatoria type cyanobacteria and the extraction of a chlorophyll derivative was reported (Gallardo, 1977). However, that sample was from a shallow site at 36S. The coincidence of the DMZ and the euphotic zone is a rarity in open marine situations and the more usual occurrence of Thioploca spp. is in deeper darker realms. In deeper waters, well below the euphotic zone, the usual color of Thioploca spp. is light grey to black. These colors being imparted by finely disseminated iron sulfides (Sassen et gl., 1993). Down-hole fluctuations in P.Y.I. values can be considered as due to the interplay of three main parameters. These being original

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319 supply, oxygen tension of the surfa c e sediments (viz pre-/syn depositional preservation or destruction) and post-depositiona l pigment loss to destruction or polymerization mechanisms. The first two factors act together to dictate the initial charge (viz. concentration, yield) of tetrapyrrole pigments to the surface sediments As will be discussed below, it has been concluded from the study of hundreds of deep sea sediments (see Table 34) that o xic post-depositional conditions, especially with significant bioturbation, leads to a rather rap i d and complete loss of tetrapyrrole pigments On the other hand, i f post depositional conditions are strongly anoxic, a period of relative pigment concentration stability follows. In this case, rapid pigment 'disappearance' occurs only after about 25-100 meters of burial and, as will be discussed for diagenetic reactions, relates mainly to the defunctionalization of these pigments and/or the i r i ncorporation into geopolymers. Therefore, while certain pigment destruction mechan isms are most likely active in such near surface (ca. 0-0.5 meter) anoxic sediments, the yield of c hlorophyll der i vatives appears to be governed mainly by original supply, as modified by the degree of syn depositional anoxia. E x amination of down-hole pigment yield data (Table 33) allows comparison of the trend for the strongly anoxic ('permanent DMZ') slope site (SC6: Figure 93a) with that for the upper slope site (SC4: Figure 93b) which e x periences fluctuating o x ic/ano xic conditions. Overall, y i elds at SC6 increase towards present time This may reflect a decrease in population density of the Peruvian anchoveta (cf. Rosenberg e t gl. 1983) or minor pigment destruction with depth or both. Given that the sedimentation rate (Pb-210) at site SC6 is about 1.1 em yr-1 from 0-27 em and about 0 3 em yr-1 below that

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Table 34 Tetrapyrrole pigment yield data for samples from DSDP/IPOD Legs 56 57, 58, 60, 61, 63, 64, 65, 66 and 71. DEPTII 1 GEOLOGIC1 PERCENT1 PERCEm-2 TETRAPYRROLES3 DSDP/IPOD SAMPLE NUMBER1 sub-bottom, ORGANIC 4 meters AGE CARBON WATER )Jg/gsed ,dry wt. P.Y.I. 4.0 L.PLEIST. 0.8 na 4 56-434 -1 5 3(15-29) 133.0 E.PLIOC. 0.5 na o. 0. 56-434 23-2(100 115) 208.0 PLIOC. 0.7 na o. 56-4348-15-1(92 -1 02) 419.5 PLIOC 0.6 na 0 o. 41.5 E.PLIOC. 0.4 na 1. 4. 56-435 13-2(100-115) 115.0 E.PLIOC. 0.7 na 56-436-7 4(125-150) 61.0 PLEIST. 0.6 na 0 56-436 -114(100-125) 99.0 L. PLIOC. 0.4 na 0 .12>': 0. 30.': 56-436 19-2(100-125) 172. 0 E.PLIOC. 0.2 na 0 0.60. : 56-436-24-1(130-150) 218.5 E.PLIOC. 0.2 na o. o 56-436 -311(115-140) 284.5 M.MIOC. 0.2 na 56-436 -363(125 -1 50) 335.0 M.MIOC. na na 57-438-5-3(125-150) 37 E.PLEIST. 1.1 35.4 1.05 0 .96 57-438A4-4(125-150) 47 L.PLEIST. 0 8 32.0 1.06 1.33 57438-10-4(125 150) 86. 5 PLEIST. 1.2 52. 0 0 .95 0 79 57-438A-18-2(125-150) 223.0 E. PLIOC. 1.0 47.0 2.20 2.20 57-438A-24-4(125-150) 283.0 E.PLIOC. 0 9 48. 0 0.64 0.71 57-438A-34-5(125-150) 379. 0 PLIOC/MIOC. 0 8 46.0 0 .60 0 75 57-438A-46-3(125-150) 492.0 E.MIOC. 1.1 38. 0 0 .11 0 .11 57-438A50-4(125-150) 532. 0 E.MIOC. 1.1 43. 0 0.03 0.03 57-438A55-5(125 -1 50) 581.0 E.moc. 0 7 36.4 0.01 0 .02 57-438A-65-5(100-125) 676. 0 M.MIOC. 0 8 25. 0 0.008 0 .01 57-438A-79-3(115 -140) 806. 0 M.MIOC. 0 8 45. 0 nd 0 57-439-9-4(115-150) 893. 0 M.MIOC. 0 6 33. 6 nd 0 57-438812-5(131-142) 9:36;0 0.8 22. 4 nd 0 57-439-21-1(140-150) 994.0 OLIGO. 0.4 18. 0 nd 0 57-439 -315(6-15) 1,093 0 OLIGO. 0.3 7.8 nd 0 w N 0

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Table 34 (cont'd). DEPIH,1 GFDl..OCIC1 sub-bottom D SDP/IPOD SAMPLE NUMBER meters AGE 57-440-5-6(0-20) 42.5 PLEIST. 57-440A-7-6(125-150) 138.0 PLEIST. 57-4408-3 5(125-150) 165.0 PLEIST. 57-4408-8-4(125-150) 211.0 PLEIST. 57-4408 12-4(125-150) 249. 0 PLEIST. 57-4408-17-5(1350150) 298.0 PLEIST. 57-4408-23-4(110-130) 354.1 L.PLIOC. 57-4408-34-3(125-150) 456.0 L.PLIOC. 57-4408-39-3(125-150) 504.0 E.PLIOC. 57-4408-46-3(125-150) 571.0 E.PLIOC. 57-4408 56-3(125-150) 664.0 PLIOC/MIOC. 57-4408-63-3(125-150) 722.0 L.MIOC. 57-4408-68-2(130-140) 778.0 L.MIOC. 58-442A-2-3(93-143) 12 HOLOC. 58-442A-7-3(100-140) 55 PLEIST. 58-442A-14-2(100-140) 125 PLEIST. 58-442A-28 2(100-140) 215 M.MIOC. 58-443-2-4(6-46) 15 L.PLEIST. 58-443-9-4(100-140) 75 PLEIST. 58-443-23-3(100-140) 205 L.t-1IOC. 58-443-34-2( 100-140 ) 325 L.MIOC. 58-443-43 -1(100-140) 400 M.MIOC. 58-444-2-4(94-134) 5 L.PLEIST. 58-444-7-5(94-134) 55 L.PLEIST. PERCENT1 PERCENT2 ORGANIC CARBON WATER 1.6 44.0 1.2 41.1 1.0 46.0 0 7 44. 0 1.2 36. 0 0.64 33.0 0.8 34.0 0.8 33.0 1.5 38.0 1.3 34.0 0.6 32.0 0.8 36.1 0.6 28.7 0 .40 48 0.30 38 0.19 42 0.10 35 0.41 55 0.10 53 0.10 46 0.07 43 0 .10 38 0.18 --0 .06 --TITRAPYRROLES3 )-lg/g-sed ,dry wt. 6.71 3 .77 2.53 0. 75 1.03 0. 72 1.84 0.79 2.12 1.84 0.27 0.30 0.09 0.33 0.09 0.006 nd 0.43 0.05 nd nd nd 0.08 nd P.Y.I. 4 .19 3 .14 2.53 1.07 0.86 1.12 2 .30 0.98 1.41 1.42 0.45 0.38 0.15 0 .84 0.31 0.04 0 1.05 0.53 0 0 0 0.48 0 4 w N .......

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Table 34 (cont'd). DEPlli l GEOLcxac1 PERCENT1 sub-bottom ORGANIC DSDP/IPOD SAMPLE NUMBER meters AGE CARBON 58-445-1-4(94-134) 5.5 HOLOC. 0.15 58-445-6-5(94-134) 50 L .PLIOC. 0 .09 58-445-11-2(100-140) 100 L.PLIOC. 0 .07 58-445-32 2(90-130) 299 E .MIOC. 0 .08 58-445-83 3(100-140) 780 M .EOCENE 0 .09 58-446-3 5(90 130) 20 MIOC. 0 .06 58446-9-4(90-130) 75 E.MIOC. 0 .05 58446-14 4(94-134) 120 M .OLIGO. 0.06 60-452-2-5(100-130) 16 QUATERN. 0.28 60-4532-3-(100130) 13 L.PLEIS T 0 .31 60-453-10-2(100-130) 86 L.PLEIST 0 .32 60-453-18-4(100-130) 166.5 PLIOC. 0.56 60-453-29-2(100-130) 268 E.PLIOC. 0.38 60453-35 3(100-130) 326.5 E.PLIOC. 0.33 60-460A-1-5(100 130) 7 0 QUATERN. 0.22 60-460-4-4(100-130) 34 L.OLIGO. 0 .10 61-4623 4(100-125) 25.0 PLEIST/PLIOC 0 .13 61-462-7 5(100-125) 64.5 L.PLIOC. 0.03 61-462 17-4(100-125) 158.0 E.MIOC. 0.03 61-462-49-4(100-130) 462.0 MAESTR. 0.05 61-462-54-2(120-140) 511.0 CAI'fPAN. 0 .03 61-462-59-1(120-140) 550.5 M.CRETAC. 0.03 PERCE:N'J? TETRAPYRROLES3 WATER wt. -<0.002 --nd -nd --nd --nd 52 nd 60 nd 45 nd 65 tr 57 0.002 75 tr 52 nd 20 0.002 42 tr 61 0 .09 56 nd --nd -nd -nd -nd --nd -nd P. Y I. <0.02 0 0 0 0 0 0 0 <0.001 0 .006 < 0 .001 0 <0.001 <0.001 0.43 0 0 0 0 0 0 0 4 w N N

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Table 34 (cont'd). DEPTI-1, 1 GEOLOGIC1 PERCENT1 PERCENT2 TETRAPYRROLES3 sub-bottom ORGANIC 4 DSDP/IPOD SAMPLE meters AGE CARBON WATER J:!g/g-sed wt. P. Y.l. 63-467-3-3(100-130) 19.5 QUATERN. 1.2 45.9 2.00 1.66 63-467-8-5(100-134) 70.0 QUATERN. 1.9 34.0 7.92 4.17 63-467-13-4(100-132) 116.0 L.PLIOC. 2.9 54. 4 14.58 5.03 63-467-18-5(100-130) 165. 0 L.PLIOC. 3.4 48.0 15.19 4.47 63-467-25-6(100-131) 233.0 L.PLIOC. 1.7 34.4 2.47 1.45 63-467-32-2(100-135) 293. 5 E.PLIOC. 2.9 34.2 2 .81 0 .97 63-467-36-2(100-133) 340. 5 E.PLIOC. 2.7 35. 8 6.12 2 .27 63-467-41-4(100-135) 382.0 E.PLIOC. 3.0 33.8 4 .44 1.48 63-467-48-2(100-132) 445.5 L.MIOC. 4.6 40.7 11.74 2.55 63-467 58-3(142-148) 542.0 L.MIOC. 4.4 20.6 5.73 1.30 63-467-63-2(118-125) 588.0 L.MIOC. 5.2 20.8 38.32 7.37 63-467-74-1(145-150) 691.0 L.MIOC. 2.9 25.4 39.13 13.49 63-467-85-4(100-130) 800.0 M.MIOC. 0.7 25.2 0 .48 0.69 63-467-91-2(100-107) 854.0 M.MIOC. 2.3 12.2 6.07 2.64 63-467-97-2(113-118) 911.0 M.MIOC. 1.9 15.3 1. 74 0.96 63-467-110-3(9-14) 1,035.0 M.MIOC. 0 9 8.2 2.42 2.69 63-471-3-2(100-130) 21.5 QUATERN. 0 8 59.4 0 .59 0.74 63-471-8-4(100-135) 72.0 L.MIOC. 1.1 56.4 4.15 3. 77 63-471-34-2(107-115) 316.0 M .MIOC. 0.7 26.4 0.37 0.53 63-471-50-2(100-125) 468.0 M.MIOC. 0.9 20. 2 0.16 0.18 63-471-69-3(125-150) 650.0 M .MIOC. 0.8 15.1 0.68 0.85 64-10G-3(3 9-5 9) 4.9cm RECENT 2.58 87.6 69.50 26.94 64-10G-5(7.9-9.8) 8.8cm RECENT 2.94 82.0 48.69 16.56 64-10G-7(11.8-13 8) 12.8cm RECENT 3.73 75.6 55.45 14.85 64-10G-10(17.7-19.7) 18.7cm RECENT 1.04 51.5 14.73 1 4.14 64-18G-I(1-10) 5 0 c m RECENT 3.03 83.6 51.79 17.1 0 64-18GV(100-17 0) 1.65 RECENT 2.13 76. 4 3.05 1.41 w N w

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Table 34 (cont'd). DEPTI-1 l GEOLOGIC1 PERCENT1 PERCENT2 TITRAPYRROLES3 DSDP/IPOD SA}WLE NUMBER1 sub-bot tom ORGANIC 4 meters AGE CARBON WATER dry wt. P. Y.I. 64-4742-3(120-150) 6 2 QUATERN. 2.2 49.5 6.95 3 .16 64-47417-6(100-125) 153. 2 QUATERN. 1.6 35.2 0 .50 0 .31 64474A-7-2(110-140) 223. 1 QUATERN. 1.4 37. 0 0 .24 0.17 64-474A 28-2(120-150) 413.2 QUATERN. 1.7 14.4 0 .03 0 .02 64-474A-32 2(120-150) 451.2 QUATERN. 1.9 19.6 0 .17 0.09 64-474A-41-3(120-150) 538.3 QUA TERN. 0 7 24.9 0.08 0.12 64477-5-1(120-140) 30.7 QUATERN. 2.6 61.6 21.02 8.08 64-477-7 -1(132-14 2) 49.7 QUATERN. 1.8 50.0 9 .76 5.42 64-477-16-5(58 88) 121.6 QUATERN. 0.7 35.8 nd 0 644 7720-1(115-135) 154.1 QUATERN. 1.4 20.0 nd 0 64-479 3-2(110-130) 15.0 QUATERN. 2.8 75.0 41.05 14.6 6 64-479-5-3(125-150) 35.6 QUATERN. 2.7 67.2 2 1 .94 8.12 64-479-7-5(110 140) 57.6 QUATERN. 2 7 64.0 28.48 10.55 64-479-9-2(115-140) 72.0 QUATERN. 3 0 65.2 8.40 2.80 64-479-13 1(110-140) 108.6 QUATERN. 2 9 66.8 9 .34 3 .22 64-479-15-5(110 140) 133.6 QUATERN. 2.9 63.6 16.48 5.68 64-479-17-5(120-150) 156.6 QUATERN. 2.8 67.4 15.40 5 .50 64-479-19-5(115-140) 171. 6 QUATERN. 2.7 50.8 7.13 2.64 64-479-22-5(110-140) 200.1 QUATERN. 2 6 44.8 1.89 0.73 64-479-27-4(120-150) 246.1 QUATERN. 2 5 48.8 1. 79 0. 72 64-479-29-5(120 -150 ) 268. 2 QUATERN. 2.6 47.1 1.53 0.59 64-479 34-5(110-140) 314.1 QUATERN. 3.0 46. 8 1.77 0.59 342.8 QUATERN. 2.8 61.4 0.93 0.33 64-479-394(110-140) 360 1 QUATERN. 3 5 53. 6 1.89 0.34 64-479 -431(120 140) 393.7 QUATERN. 2 6 40. 8 1.56 0 .60 64-479-47-4(110-140) 436.1 QUATERN. 1.2 24. 5 0.96 0 .76 w N

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Table 34 (cont'd). DEI?'lli l GEDLOGIC1 PERCENT1 TETRAPYRROLF.S3 DSDP/IPOD SAMPLE NUMBER1 sub bottom ORGANIC 4 meters AGE CARBON WATER pg/g-sed. 1dr;r: wt. P Y .I. I 64-4812 2(125-150) 7.6 QUATERN. 1.4 72.0 69.52 49.66 64-4818 2(110-140) 36.1 QUATERN. 1.7 47.2 21.75 12.80 64481-11-1(125 150) 47. 5 QUATh'RN. 1.1 43.4 22.09 20.08 64-481A-8-2(110-150) 111.1 QUATERN. 2.0 45.6 0.04 0 .02 64-481A-10 2(110 -1 50) 130.1 QUATERN. 1.3 41.5 0.04 0 .03 64-481A-13-6(0-14) 163.5 QUATERN. 1.4 30. 8 nd 0 64481A-22-4(122-150) 257.2 QUATERN. 2.5 49.4 0.29 0.12 64481A-24-5(110-140) 267.6 QUATERN. 1.3 31.6 0.02 0.02 64-481A-26-5(120-150) 286.7 QUATERN. 1.4 30. 8 0.3 0.02 64-481A-30-5(110-140) 324.6 QUATERN. 1.1 34. 9 0.08 0.07 65-482A-2-4(127-150) 11.5 QUATERN. 2.16 40.7 8.03 3. 72 65-482A-5-2(124-136) 41.5 QUATERN. 1.43 37. 6 0 .82 0 .57 654828-2 2(123-150) 56.2 QUATERN. 1.89 37.5 1.01 0.53 65-482C3 3(120-150) 67.7 QUATERN. dna 44.6 0.53 654828-5-2(125-150) 84.7 QUATERN. 1.86 35.5 0.25 0.13 65-4820-7-2(114-144) 131.1 QUATERN. 1.80 30.9 nd 0 66-48 7-2-3(120-140) 4.2 M-L.PLEIST. 2.04 48.5 1.98 0.97 66487-6-3(120 -1 50) 42.1 M-PLEIST. 1.19 49.1 1.32 1.11 66-487-9 6(120-150) 75.1 E-PLEIST. 0.93 47.5 0.27 0.29 66-488-2-2(120-150) 3 7 L-QUATERN. 1.84 36. 6 4.94 2.68 66-488-5-1(120 -1 40) 29.5 M-L.QUATERN. 2.38 37. 0 2.34 0 .98 66-488 -1 0 4(120-140) 81.6 M-L.QUATERN. 2.24 27. 3 6.33 2.82 66-488-19 4(120-143) 166.1 QUATERN. 1.92 21.6 3.84 2.00 66-488-29-4(120-140) 262.1 QUATERN. 1.67 18.3 0 .004 0.002 w N lJl

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Table 34 (cont'd) DEI!TI-1, 1 GFDLOGIC1 DSDP/IPOD SAMPLE subbottom meters AGE 66-490-2 -4(110-1 25) 13. 6 QUATERN. 66-4908-2( 110-1 35) 67. 6 QUATERN. 66-490-15-3(110-140) 136.6 QUATERN. 66490-2 1-5(125-1 40) 195.1 L.PLIOC. 66490-34 2(110-1 40) 305.1 PLIOC. 66-490-593(110-125) 534. 6 MIOC. 66492-2 -4 (110 -1 25) 9.1 QUATERN. 66492-5-4(110 140) 37.6 E.PLIOC. 66-492-8-2(110 -1 30) 63.1 L.MIOC. 66492-11-3(110 -1 40) 93.1 L.MIOC. 71-511-3-4(120 -1 50) 20.5 E OLIGO. 71-511-1 6 -1 (120 -1 50) 139 5 L.EOCENE 71-51131-5( 120150) 259. 5 CA!'IPAN. 71-5 11-37-1 ( 120-1 50) 310. 5 CAMPAN. 71511-46-2(120 -15 0) 397.5 CON./SANr. 71-511-58-3(120-150) 513. 0 BARR./APT. 71511-60 5(120-150) 535.0 BARR./APT. 71-511-625( 1 20-150) 554. 0 BARR. /L. JUR. 7 1-511-64-4(120-150) 571.5 L.JUR. 715 11-6 6 4(120 150) 590. 5 L.JUR. 71-511-68-2(120-150) 606. 5 L.JUR. 71-51 1 -70-3(120-150) 627.0 L.JUR. PERCENT1 PERCENT2 ORGANIC CARBON WATER 2 .11 33. 6 2 .62 25. 7 dna 29. 0 2 .48 29. 0 1.47 23.5 1.31 16.6 2 .40 32. 9 dna 42. 6 1.56 50. 8 dna 42. 5 1.02 45.1 0 .60 53. 3 0 .65 37. 2 0 .84 15.1 0 .37 27.4 0.31 35. 4 5 .28 26. 6 4 .29 30.8 5 .69 26.7 6 .25 31.8 3 .88 31.2 4 .91 27.4 TETRAPYRROLES3 1dry wt. i 1.5 1 1. 75 1.61 0 .55 0 .84 0.06 2.54 2 .21 2 .6 7 1.38 0 .045 0 .299 0 .022 0 .029 0.006 0 .012 2 .004 3 .382 2 .760 3 .781 1 .628 1 .588 P.Y I. 0. 72 0.67 0.22 0 .57 0.05 1.06 1. 71 0 .04 0 .50 0 .03 0.03 0 .02 0 .04 0 .38 0. 79 0 .49 0 .60 0 .42 0 .32 4 w N (1\

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Table 34 (cont'd). FOOTNOTES: 1) DSDP/IPOD sample number given as em.). Data as to location,depth,geologic age and percent organic carbon taken from the Initial Reports of the Deep Sea Drilling Project as follows:Legs 56-57(Scientific Party,1980),Leg 58(deVries Klein,Kobayashi et al.,1980),Leg 60(Hussong,Uyeda et al.,1981), Leg 61(Larson,Schlanger et al.,1981),Leg 63(Yeats,Haq et al.,1981),Leg 64 (Curray,Moore et al.,1982),Leg 65(Lewis,Robinson et al.,1983),Leg 66(Watkins,Moore et al.,1982),Leg 71 (Ludwig,Krashenninnikov et al.,1983). 2) Percent water determined on duplicate samples(ca.2-5 grams each)by heating to 106-108C for 18 hours,repeated until stable.Reflects wet,occluded and adsorbed water,excludes true waters of not analyzed. 3) Tetrapyrrole pigments reflect the sum of individua l pigment classes determined after separation. Summation of chlorin(viz.dihydroporphyrin) pigments foltowed separtion into non-polar(phytyl and other esters),mono-and di-/tri-carboxylic acid pigments(see Table IV).Summation of 'chlorins' plus porphyrins(i.e. free-base and metallo)was after separation into these classes(see Figures 21,23 and 27).Quantitation was via Beer-Lambert calculation using class-specific extinction coefficients as given in Table XVI.Tetrapyrrole yield data from:Leg 56(Louda et al.,1980),Leg 57(Baker and Louda,1980a),Baker and Louda,1980b),Leg 60(Baker and Louda,1981a),Leg 61(Baker and Louda{1981b),Leg 63(Louda and Baker,1981),Leg 64(Baker and Louda,1982),Leg 71(Baker and Louda, 1986b),Legs 65 and 66(this study:Louda unpublished data). 4) PYI is a figment rield Index previously defined by the author as "tetrapyrrole pigment in ug/g sediment---divided by organic carbon in percent dry weight"(Louda et al.,1980).Sediment weight is taken on a dry weight basis(Baker and Louda,1980a).Only the values for Leg are given with pigment yield calculated using sediment wet weight. nd =none detected,tr=trace. 11) Geologic Age abbreviations are: E=early,L=late,M=middle.,PLEIST.= Pleistocene,PLIOC.=Pliocene, MIOC.=Miocene,OLIGO.=Oli gocene,HOLOC.=Holocene,QUATERN.=Quaternary,MAESTR.=Maestrichian, CAMPAN.=Campanian,CRETAC.=Cretaceous,CON.=Coniacian,SANf.=Santonian,BARR.=Barremian,APT.= Aptian,JUR.=Jurassic w N -.....J

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328 (Henrichs and Farrington, 1984; Volkman et gl., 1983) one can calculate that the noticeable minima in P.Y.I. values at ca. 7 and 30 em corre-spond with sediment deposition during 1972 and 1951-3 A.D. respectively. These periods of lessened pigment presence correspond quite well with El Nino Southern Oscillation (ENSO) events reported in the literature (Graham and White, 1988; Ramage, 1986). Conditions favor-0 able for strong upwelling at 15 S existed during 1977-8 and, as sedi-ments were recovered during 1978, the datum for 0-3 em at both SC6 and SC4 would appear to represent an optimal or maximum yield for this environment. Turning attention to the upslope site SC4 (Figures 89, 92) we find that pigment yields (figure 93b) fluctuate widely with depth. Unfortunately, sedimentation rates for this site are unknown but, given its closer proximity to the coast, the influence of terri-gena-clastic input could be expected to become significant during ENSO events due to the accompanying increases in rainfall/runoff (cf. McCaffrey et gl., 1990). The marked minima in tetrapyrrole P Y.I. found at the intervals of 13-16, 25-31 and 40-52 em are taken here as indicating the combined effects of lowered initial input, possibly related to ENSO events, and more rapid pigment destruction as the depositional site became oxygenated. Likewise, the pronounced maxima at 22-25 and 57-64 em must represent deposition during periods of high upwelling induced productivity and into strongly anoxic bottom waters. Comparing these two sites, it is concluded that fluctuations in initial supplies of tetrapyrrole pigments obviously affect the absolute yields (i.g. pigment/g-sediment) of these pigments. However, it is the presence of absence of oxygen in the syn-and post-depositional environment that is mainly responsible for fateing the destruction or

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preservation, respectively, of the chlorophyll derivatives in near surface (ca. 0-1 m) sediments. 329 The pigment yield data obtained for the analyses of 167 deep sea sediments provided by DSDP/IPOD are given in Table 34. Sediment profiles on the order of 1 kilometer are included here though the majority of sample suites cover a range of about 4-500 meters sub bottom. Obviously, the depth and time span covered by these sample suites complicates the examination of pigment yield as a paleoproduc tivity-paleoenvironmental tool. That is, not only does initial pigment supply (productivity) and syn-/post-depositional oxygen tension (envi ronment) become important in determining down-hole trends but a whole host of geologic/geochemical parameters now enter the picture. Examples include; sea level fluctuations, plate tectonics (latitude, longitude changes), uplift/subsidence, heat flow and others. The reader is refered to reports by the author in the Initial Reports of the Deep Sea Drilling project, as cited in the footnotes of Table 34, for coverage of the geologic parameters of these samples. Herein, emphasis is on overall trends. Figure 94a-c contains the down-hole plots of P.Y.I. for selected DSDP/IPOD sites (Table 34). Sediments from the oceanic plate east of the Japan Trench (56-436; Z = 5,240 m), the Shikoku Basin (58-442; Z = 4,639 m: 58-443; Z = 4,372 m: 58-444; Z = 4,843 m), the Daito Ridge (58-445; Z = 3,377 m), the Daito Basin (58-446; Z = 4,952 m), the Mariana Basin (60-452; Z = 5,858 m), the Mariana Trough (60-453; Z = 4,693 m), the inner (arc-side) wall of the Mariana Trench (60-460; Z = 6,452 m) and the Nauru Basin (61-462; Z = 5,181 m) all reveal a total demise of tetrapyrrole pigments with increased sediment depth/age

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( PYI) 0 --I I I I 0 I I 200 E ........ f2 400 f-0 m I (l) ::> l/) 600-I f-a... w oaoo 0 I 6. \OOO (a PIGMENT YIELD INDEX ( py I ) 0 5 10 0 5 10 ( b (c Figure 94. Plots of tetrapyrrol e pigmen t yield versu s sub-bottom depth for various D S DP/IPOD sites. (a) Solid circes = 58-442A, 58 -444, 58-445, 60 -452, 60-453, 60 460A. Open circles = 57-438 Ope n triangles = 57 440 (b) 64 -479. (c) 63 467 .(see Table 34). w w 0

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331 (Figure 94a). It is noted here that the original concentrations of both chlorophyll derivatives and total organic carbon {TOC) were exceedingly low (see Table 34). All of these sites occur in the western extension of the north Pacific gyre and, as such, receive mainly pelagic to hemipelagic sediment from strongly oligotrophic waters. In each of these cases, the combination of low sedimentation rate, well-oxygenated water columns and long transport time/water column residence, due to depth (Z > 45,000 m), has allowed tetrapyrrole pigment destruction to proceed in well oxygenated environment. Each site yielded purpurins and chlorins, pigments derived via the oxidative cleavage of the isocyclic ring in the phorbide nucleus of chlorophyll (cf. Hynninen, 1973, 1979), which further indicates oxic depositional environments {cf. Louda and Baker, 1986). These processes are covered more fully later in text. Turning now to yield trends which reveal the preservation or 'fossilization' of chlorophyll derivatives we examine Figure 94b. Plotted here are the P.Y.I. versus depth data {Table 34) for DSDP/ IPOD site 64-479 and samples from site lOG, collected during Leg 64 site survey operations (see Baker and Louda, 1982, 1986a). This plot we consider to represent a 'type-specimen', for the tetrapyrrole pigment yield profile in a sediment column which leads to preservation and the eventual production of metallo-porphyrins. This profile will be revisited during discussion of diagenetic reactions. However, for yield consideration, it can be noted that the period of most rapid pigment 'loss' (g.g. 0-150 m sub-bottom) corresponds to phorbide defunctionalization reactions. Loss is placed in semi-quotes to infer the possibility that not all of this 'loss' is due to pigment

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332 destruction. That is, it is during this period of defunctionalization (early diagenesis) that the author proposes the incorporation of tetrapyrroles into proto-kerogen structures takes place. Once the aromatization (mid-diagenesis) stage is achieved these pigments attain much greater overall stability and loss due to macrocyclic breakdown mechanisms decreases. 'Loss' at this stage due to gee-polymerization reactions has also nearly ceased as the tetrapyrrole periphery has been stripped of its reactive functional groups. Stabilization of down-hole pigment trends resulting in the generation of free-base and metallo porphyrins has been observed during the author's studies {Table 34) for DSDP/IPOD sites 56-434 (mid-slope terrace, Japan trench; Z = 4,515 m: Louda et gl., 1980), 57-440 (mid-slope terrace, Japan trench; Z = 4,515 m: Baker and Louda, 1980a), 63-467 (San Miguel Gap, California border lands; Z = 2,128 m: Louda and Baker, 1981), 63-471 (deep sea fan at base of continental slope, western Baja California, Mexico; Z = 3,101 m: Louda and Baker, 1981), 64-479 (Guaymas basin slope middle of DMZ; Z = 747 m: Baker and Louda, 1982; Louda and Baker, 1986) and certain on-shore sediment/shale sequences to be covered during diagenetic reaction discussion. To date, the best example of anoxic ('reductive') chlorophyll diagenesis is the profile studied from the DMZ site in the eastern slope of the Guaymas basin, Gulf of California. These samples (viz. pigment isolates) are still under study and will continue to provide insight into chlorophyll diagenesis for years to come. The generation of rich sediments with good-to-excellent preservation of chlorophyll derivatives in the marine environment is intimately linked to the development of strong anoxia in the syn-and post-depositional stages The development of sedimentary anoxia can

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333 occur not only in direct relation to well defined OMZ's associated with upwelling induced regions of high productivity but also through the intervention of less subtle geologic occurrences, specifically mass movement or turbidite deposition. Overall, the Pacific yields higher TOC values in its eastern (ca. 1.5%, dry wt.) versus its western (ca. 0.7%, dry wt.) boundary sediments (see Premuzic et gl., 1982; Summerhayes and Gilbert, 1982). Therefore, as a rule, the sediments off Japan are low in TOC due both to the lack of upwelling induced productivity and the presence of well 9xygenated bottom waters (Summerhayes and Gilbert, 1982). However, even in sediments with lo w TOC values (g.g. 0.2-0.8%) we find significant tetrapyrrole preservation (P.Y.I.) values 2-5: Table 34) for DSDP/IPOD sites 56-434, 56-435 and 57-440, all of which were deposited as turbidite impoundments in the mid-inner slope of the Japan Trench. Thus, even though bottom waters are oxic, rapid burial through redeposition processes can lead to preservation of non-refractory (i.g. 'metabol i zable' and/or 'labile') OM constituents. The more common, or at least the most commonly hel d, scenar io for the accumulation and preservation of organic matter is that of deposition out of a highly productive after column into bottom waters and sediments with well defined, strong and persistent anoxia (cf. Demaison and Moore, 1980). During the present studies, sediment suites deposited, for the most part, from such environments include (Table 34) the San Miguel Gap of the California borderlands (63-467), Gulf of California, Guaymas Basin and Slope s ites (64-10G, 64-18G, 64-479, 64481), the Falkland Plateaus (Jurassic to early Cretaceous strata: 71-511) and, of course, the Peru upwelling sites (Table 33). In essence;

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334 ali of these sites received hemipelagic sediments with large amounts of autochthonous dominated organic matter deposited into or through a strong OMZ. Strong oxygen depletion is common along the western American (eastern Pacific) margin from Oregon to Chile (Kester, 1975). The down-hole pigment yield profile at OSOP/IPOD site 63-467 (Figure 94c) serves here as an example of the use of chlorophyll derivatives as a paleolimnological or paleobotanic tool (see Daley, 1973; Kemp and Lewis, 1968; Vallentyne, 1954, 1955). However, in contrast to the limited Holocene time span discussed in those reports, here application is e x tended to the Miocene, and with other samples (71-511) to the Jurassic. Examination of Figure 94c reveals two distinct regions of enhanced pigment preservation, one very marked in the middle/late miocene strata and another in the latest Pliocene. In a past report, the author has detailed certain paleogeographical and organic geochemical arguments to link these strata at the San Miguel Gap with the more eastern deposition of the Monterey and Mud Pit shales, or their time equivalents (Louda and Baker, 1981). Since that time, many additional reports have linked these strata to the upwelling induced high productivity deposition of organic matter at DSDP/IPOD 63-467 during time sequences corresponding to the formations cited above (g.g. Curiale et . 1985; Graham and Williams, 1985; Isaacs and Petersen, 1987; Summerhayes, 1981). This profile, Figure 94a, remains as the best example of chlorophyll derivative yield data being used for paleolimnological purposes with ancient sediments. Taking into account all of the chlorophyll derivative yield data, discussions and conclusions above, we now offer an overview or synthesis of these phenomena as they apply to a marine/aquatic water

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335 column and pre'oil-window' sediments Figure 95 i s the overall pigment yield trend as suggested here. In the water column the pigment maximum i s at or just below the region of highest productivity (i.g. Cfixation). Above this region, chlorophyll concentrations are kept lower by photooxidative processes. Beneath the zone of maximal ('living') chlorophyll concentrations several mechanisms act to decrease tetrapyrrole pigment abundance. Cellular se n escence and intercellular acid/enzymatic action lead to chlorophyll bleaching, destruction or conversion to pheopigments. Herbivory accounts for much of the change from chlorophyll (-a [II]) per se to the pheopigments, pheophytin-a [IIa] and pheophorbi de-a [VIa]. At the sa m e time, zooplankton initiate the rapid deposition of tetrapyrrol e pigment containing detritus by packing their excreta into fast sinking, r e l ative to a single free phytoplankton cell, feca l pellets. The net result of cellular senescence, mixed layer, photoo xidation and predation is the near total destruction of chlorophyl l and its derivatives by the region around the first major pycnocline known as the remineralization maximum. The net results of the above leads to pigment array s which average chlorophyll-to-'pheopigment' ratios of >10 and <<0.5 for the euphotic mixed layer ('epipelagic' zone) and the aphotic remainder ('mesopelagic' zone, and deeper) of the water column, respectively. As depth i n the water column increases, and therefore time since 'death', chlorophyll-to-pheopigment ratios continue to drop. At the time of deposition, or shortly thereafter, chlorophyll per se is absent. Indicated in Figure 95 is a near bottom increase in the concentration of chlorophyll derivatives (dotted line). This i s to allow for situations in which a nephloid layer (see Feely et }., 1973) and/or

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N :L 0 CHLOROPHYLL plus DERIV ATIVES )J9/liter L 0 5 10( 5 ) 15 20(10) 2 5 ru E z 2 ::> _j ou 0:: r w 1-1,000 ,-... N ......... I 1-n.. w 0 a 101 100 ,-... E 2 0 1-1-0 CD I co ::> Vl I 1-n.. w 0 1,000 0 0 \ \. '\ CHL/PHE0<<0. 5 NL defunctionalization geopdymerizat ion aromatization chelation 100 200 300 L ru """" c E \J (/) TETRAPYRROLES ( )Jg /g-sed. ,dry wt.) 10 20 30 50 PY. I 336 Figure 95. S ummary of pigment yield trends with depth i n the water column and underlying sediments for a marine anoxic environment. Abbreviations:CHL= chlorophyll,PHEO= pheopigments,PZ= photic zone, PYC= first major pycnocline, RMM= remineralization oxygen min imum zone, NL= nephloid layer(if present).Asterisk= sediment trap samp les(Table 32).All values are only illustrative.

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337 turbidity currents exist. That is, any strong physicochemical gradient or event could lead to an increase in particulate organic matter (POM), and therefore chlorophyll derivative concentration (i.g. per Liter sea water). Once deposited, tetrapyrrole pigment yield (g.g. P.Y.I ) would follow down-hole profiles much like that reported for DSDP/IPOD site 64-479 (see Figure 94b) in anoxic conditions or for the western Pacific sites (Figure 94a) in oxic environments. The pigments and reactions characteristic of these preservative or destructive conditions, respectively, are covered later. The preceding discussion of pigment yield dealt with the quantitative aspects of chlorophyll diagenesis. Underlying changes in yield or quantity are the qualitative aspects of pigment reactions, reaction pathways and the environmental pressures responsible for these. During the first portion of these studies, as reported in Chapter 3 chromatographic methodology was developed and perfected which allowed for the facile split of sedimentary 'chlorins' (viz. dihydroporphyrins) into three distinct polarity classes. These classes are non-polar esters plus hydrocarbons, mono-carboxylic acids and polar di-/tri carboxylic acids. As these polarity classes are isolated during column chromatography over microcrystalline cellulose, they may also be functionally identified as 'Cell-1', 'Cell-2' and 'Cell-3', respectively. In line with the precepts developed during the present studies (see Baker and Louda, 1986a; Louda and Baker, 1986), it was hoped that a simple chromatographic split would provide a modicum of paleo-envi ronmental information as follows: Phytyl esters, most notably pheo phytin-a [IIa], are thought to enter sediments mainly in intact phyto plankton which have escaped predation and also to indicate pre-and

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338 syn-depositional anoxia which inter alia eliminates aerobic life forms. The mono-carboxylic acid phorbides, such as pheophorbide-a [VIa] and pyro-pheophorbide-a [VIII], are believed to form in the water column and sediments during herbivory by primary and secondary consumers. Thus, such pigments would be a measure of predation pressure and partially of oxic conditions in pre-depositional settings. The polar di-and tri-carboxylic acid chlorins and purpurins are known to form only via the oxidative cleavage of the isocyclic ring of phorbides. These pigments would then indicate syn-and post-depositional aerobic conditions. While the author still maintains that the above generalizations are valid, certain complications to the above do exist. First, not all of the polar pigments in 'Cell-3' are o xidatively formed derivatives. That is, the chlorophylls-c [XIX] and their derivatives contain the highly polar acrylic acid moiety chromatograph into 'Cell-3' and must be separated out, when present, and either included in 'Cell-1' or considered alone. Second, certain zooplankton, such as salps or certain species of copepodes, prey on phytoplankton and excrete chlorophyll derivatives entirely or predominately, respectively, as pheophytin (Hallegraeff, 1981). Therefore, care must be exercised when inferring low predator pressure for the water column which deposited sediments dominated by pheophytins. Lastly, the very recent identification of phorbide steryl ester in recent surface sediments from the Black Sea (King and Repetta, 1991), in the Miocene Marau shale (lacustrine) of Brazil (Prowse and Maxwell, 1991) and from a variety of samples, including a diatom bloom (Eckardt et 1992), has certainly altered the original hypothesis on the meaning of

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339 'Cell-1' pigments, as a whole. These constraints in mind, we shall now briefly examine various Cell-1/-2/-3 distributions. Table 35 contains the preliminary polarity class distributions determined on 13 selected or pooled samples of near surface sediments from beneath the Peruvian upwelling zone (see Table 32). Overall, very little difference was observed for sediments deposited into either known oxic (BCS) or anoxic (SC6) bottom waters. Therefore, using these preliminary chromatographic data to interpret the depositional environments present for the sequence of sediments known to be laid down in fluctuating anoxic/oxic conditions is of little validity. Records were kept for numerous deep sea sediments obtained from the DSDP/IPOD program and a selection from sub-bottom depths less than 300 meters is given here as Table 36. As for the near surface sediments from Peru, these data of chlorophyll derivatives fluctuate widely among the defined polarity classes Certain geochemical events further complicate the interpretation of preliminary tetrapyrrole pigment polarity class analyses E xamples of such factors include in-sediment esterifications, decarboxylation and geopolymerizations All of these would increase the apparent importance of the non-polar 'Cell-1' fraction. As a conclusion to this portion of these studies, it is suggested that paleoenvironmental interpretations with tetrapyrrole pigments in immature sediments be limited to considerations of yield and the presence or absence of chlorin and purpurin acids. In pre-or syn depositional settings, the relative importance of pheophorbides (phorbide mono-acids: 'Cell-2') can aid in establishing predation pressure Therefore, a high yield of chlorophyll derivatives and a low

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340 Table 35. Preliminary classification of chlorophyll derivatives isolated from surface to near-surface sediments in the Peruvian upwelling zone at about 15S. SAMPLE(1 ) PERCENTAGES PHYTYI.ATED DI-/TRI-ACIDS (deJ2th2cm) "CEI.l-1" "CEI.l-2" "CEI.l-3" SC4(0-:-2) 36.2 31.0 32.8 SC4(16-19) 2.0 82.2 15.8 SC4(19-28) 38.4 50.2 11.4 SC4(31-40) 27.5 39.2 33.3 SC4(52-54) 10.8 68.6 20.6 SC6(0-3) 17.1 55.7 27.2 SC6(6-15) 44.1 34.1 21.8 SC6(33-45) 38.5 34.6 26.9 SC6(51-57) 29.2 37.6 33 2 BC5(0-2) 14.3 46.9 38.8 BC5(2-12) 32.6 40.0 27.4 BC5(12-20) 32.9 37.8 29.3 BC5(20-28) 25.0 42.1 32.9 FOO'INOTES : 1)Samples analyzed here were used as received from WHOI(Table 34 ) or pooled using similar yield stratification as a factor. 2)Separation of chlorophyll derivatives via microcrystaline cellulose chromatography and quantitation via electronic absorption spectroscopy is described earlier in text(see Tables 4 and 16).

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341 Table 36 Preliminary classification of chlorophyl l derivatives isolated from various deep sea sediments(1). SAMPLE(1 ) DEPTH,m., (1) MOLAR PERCENTAGE(2J sub-bottom CEll.1 CEll.2 CELL3 57-4405 6 42.5 23 53 24 57-440A7 6 138. 0 42 58 nd 57-4408 3 5 165. 0 1 8 52 30 57-44088-4 211. 0 47 45 8 57-4408-124 249. 0 3 82 15 57-4408-175 298.0 30 63 7 58-442A2 3 12.0 39 ----[6 1]-----60-460A1 5 7 0 35 -----[65]-----634673-3 19. 5 17 53 30 63471-3 2 21.5 14 86 nd 63-4718 4 72. 0 19 49 32 64-477 5 1 30. 7 18 43 39 64-479 3 2 15.0 22 47 31 64-4795-3 35. 6 26 28 46 64-4797 5 57.6 15 34 51 64-4799 2 72.0 10 44 46 64-479-13-1 108.6 5 47 47 64-481 8 2 36. 1 18 54 28 64-481 11-1 47. 5 22 40 38 64-10G-10 (4 .9cm) 40 39 24 65-482A2-4 11.5 15 51 34 65-482A-5-5 41.5 22 47 31 65-4828 2-2 56. 2 19 43 38 66-4872 3 4 2 25 38 37 66487-6-3 42.1 42 31 27 66-4 87-9-6 75.1 10 38 52 66-488-2-2 3 7 42 39 18 66-4885-1 29. 5 13 62 25 66-488-10 4 81.6 30 36 34 66488-19-4 166.1 29 34 37 66-4902-4 13.6 14 38 48 66490-8-2 67.6 11 41 48 66-49015-3 136.6 10 41 49 66492-2 4 9.1 2 69 29 66492-5-4 37.6 9 40 51 66-4928 2 63.1 22 3 1 47 66492-11-3 93. 1 10 3 1 59 FOO'INOTES: 1) DSDP/IPOD Sampl es and depth given previo u s l y in Tabl e 34. 2) See fdotnote 2 in Table 3 5

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342 abundance of oxidatively generated chlorin and purpurin acids can be taken as indicating strong syn-and post-depositional conditions beneath a highly productive water column. Likewise, very low yields and elevated percentages of the chlorin and purpurin acids indicate syn-and post-depositional oxicity but tell little of the water column productivity. Intermediate cases, which are the majority, lead to difficult paleoenvironmental interpretations. Overall, the best conclusions will be reached only when chlorophyll derivative data is added to other organic geochemical data bases (see g.g. Hunt, 1979; Tissot and Weltz, 1978). The essence of chlorophyll geochemistry lies in the individual reactions and reaction pathways taking place during early diagenesis. The premier organic geochemical pathways were, of course, the suggested conversion of chlorophyll-a [I] into DPEP [XXXVIII] and heme (g.g. [LIV], [LV] into etioporphyrin-III (i.g. "ETIO": [LII]) by the late Professor Alfred Treibs (1936). Structurally, these compounds were compared in the first chapter (Figure 1). The reaction pathways which Treibs suggested (1936) were based upon his knowledge of the in vitro chemistry of the tetrapyrrole pigments, both in the literature of the day (g.g. Conant et gJ., 1931a-b; Tswett, 1906a-c; Weigett and Noack, 1932; and Stoll, 1913; Wilschke, 1914) and as a member of the Hans Fischer group in the Technische Hochschule in Munich, Germany (g.g. Fischer and Orth, 1937; Fischer and Stern, 1940). Though a great many studies have taken place since Treibs' time and variant theories for the generation of 'petroporphyrins' have been proposed (cf. g .g. Baker, 1969; Baker and Palmer, 1978; Dunning, 1963; Dunning and Moore, 1957; Hodgson et gj., 1967; Keely et gj., 1990), it is the contention

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343 of the author that, after 58 years, all of Treibs' postulates (1936) are in essence correct. Therefore, the Treibs' scheme for the geochemistry of tetrapyrrole pigments, both initiating from 'chlorophyll' and 'heme', is taken as the framework on which all results can be placed. That is, all chlorophylls (a, b, c1 c2 c3), bacteriochlorophylls (a, b, c, d, e), hemes (a, b, c, d), cytochromes and other more minor biotic tetrapyrroles can theoretically be mentally and/or geochemically processed via the Treibs' scheme to yield a very wide suite of prod ucts All other routes will be considered herein as modifications or side reactions to the Treibs' scheme (cf Baker and Palmer, 1978) and with each example we shall endeavor to reveal how that actually is the case. When considering biotic sources for geologic tetrapyrroles one must not be limited only to chlorophyll-a [I], but must consider all of the alternate isomers as well. The majority of the known well characterized chlorophylls were described earlier (Figures 7-9). To date, the chlorophylls-c (-c1/-c2:[XIX]; Figure 7) remain an enigma. That is, they are obviously present in marine and fresh-water phytoplankton (Figure 87), often at high levels a/c = 2.38 for Aureococcus spp.: Bidigare, 1989), and were shown to be present in oceanic debris (viz. seston) as collected in sediment traps (Table 32). However, the chlorophylls-c (XIX] and their recognizable derivatives have yet to be confirmed in sediments collected below the photic zone. Chlorophyll-b [XIV], also known to be present in marine phytoplankton of the phylum Chlorophyta Eutreptiella gymnastica; chlorophyll-al-b = 1.56: Bjorkland, 1982. cf. Jeffrey, 1974, 1976, 1980), or its recognizable derivatives (XV], (XVII], [XXVIII]) have also been lacking in

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344 aphotic sediments. Jeffrey {1976) reports chlorophyll-b/-a ratios of 0.2, 0.5 and << 0.05 at 5, 100 and 200 meters, respectively, in north Pacific gyre. One can but speculate as to the fates of chlorophylls-b and-c in the aquatic depositional environment. Chlorophyll-b, or more properly pheophytin-b and/or pheophorbide-b, requires but the reduction of the 3-formyl moiety (7-formyl in revised nomenclature: see Bonnett, 1978) to yield a chromophore (viz. 3-methanol-3-desformyl-pheophorbide b or 3-methanol-3-desmethyl-pheophorbide-a: cf. [XVIII]) indistinguishable through routine electronic spectrometric assay from many chlorophyll-a derivatives. Likewise, the complete loss of the formyl moiety, as formyl a methanol, leaves an open B-position at C-3 (C-7, revised) and also does little to the UV/VIS spectrum at the 'chlorin' (viz dihydroporphyrin) stage. Given the shear abundance and ubiquity of the chlorophylls-c in the vast majority of marine depositional environments, it appears highly unlikely that these pigments fail to enter the tetrapyrrole pool in sediments. Recent identifications of certain geologic porphyrins with a 'rearranged' or 'out-of-place' isocyclic ring, relative to true DPEP [XXXVIII] structures, has lead to the suggestions that these arise via a condensation between the carbon of the 7-acrylic acid moiety and the C-10 site on the original isocyclic ring {Figure 96: see Bareham et al 1990; Ocampo et gl., 1984, 1985a). In fact, structure 8 in Figure 96, a requisite intermediate, has been identified as aNi-complex in Messel oil shale (Ocampo et gl., 1985a). On a purely phenomenological basis, it is suggested here that the chlorophylls-c may react with proto-geopolymers during the very earliest part of diagenesis and, being removed from the more easily analyzed bitumen, become 'hidden'. The reasons for such an argument

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( A ) -COOC H 3 HOOC R3 OOH ( 8 ) R2 -co.l ( c ) Figure 96. Cyclization and rearrangement of the isocyclic ring in chlorophyllsc Chlorophylls-c structures g iven in Figures 7 and 91. R3 in structure ''B'' can be COOH o r H (Callot al. ,1984). CA= Central A toms(2H,Ni or VO). w V1

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346 are as follows: the chlorophylls-c very likely do exist in sediments; given that they are metallo-(Mg2+) or free-base (-Mg2+, + 2H} porphyrins, their UV/VIS spectrum (see Appendix A [XIX]) would be hard to overlook; and they appear to be absent from the bitumen of early diagenetic stage sediments. A variety of photoautotrophic anaerobic bacteria exist and contain unique suites of chlorophylls, all of which were reviewed in the Introduction (see Figures 8-9). The chlorophylls of the Chlorobacteri aceae, green sulfur bacteria, yield 'early' derivatives with a pheo phorbide-a [VIa] to mesopyropheophorbide-a [X] like chromophore. Therefore, without analysis to the structural level (g.g. RPHPLC, NMR, XRD}, such compounds are likely to be overlooked during routine analysis. Recently, a few structures for geologic porphyrins thought to derive from selected 'Chlorobium' chlorophylls (bacteriochloro phylls-d} have been described (Bareham et . 1990; Callot et 1990; Ocamp et . 1987; Wolff et 1984}. To date, extant envi ronments supporting significant populations of Chlorobium spp. are only eutrophic meromictic ponds (Caraco and Puccoon, 1986; Gophen et . 1974) and certain sulfur lakes (Ehrlich, 1981}. However, the distribution of extinct Chlorobia and environments which could support same are all but unknown. The purple sulfur and non-sulfur bacteria, Thiorhodaceae (Chromatiaceae) and Athiorhodaceae (Rhodospirillaceae}, respectively, are more ubiquitous in modern sedimentary environments, as will be shown below (cf. Ehrlich, 1981; Stanier et . 1970). A variety of sediments were analyzed during a preliminary search for alternate (viz. "non-a") chlorophylls in modern sedimentary environments. The first sediment,

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347 serving as a source for 'known' bacteriochlorophyll-a (viz. bacteriopheophytin-a [XXI]), was a marine littoral bacterial mat collected in Tampa Bay {27 52.3'N x 82 35.7'W). Discussion of this sample can be found in Appendix A and is presently here solely to reveal the breadth of environments in which bacteriochlorophyll-a can be found. A sandy oxic high-medium energy beach environment is hardly conducive to the preservation of tetrapyrrole pigments {cf Gillan and Johns, 1980) and such is not claimed here. The presence of bacteriochlorophyll-a in a 37 fresh water/terrestrial peat accumulation in semi-tropical Florida {26 18'N x 80 15'W) has previously been demonstrated by the author {Palmer et gl., 1982). In that case, a mean pheophytin-a to bacteriopheophytin-a ratio of 65:35 was found, with no apparent down-core trend in that ratio. However, after only 0.5 meters of burial yields were decreased 3 to 8 fold. Here, the decrease in yield without an alteration in the ratio of pheophytin-a/bacteriopheo phytin-a is taken to indicate that there was neither preferential destruction of either form nor was there a discernable conversion of the bacterial (viz. 3,4 7,8-tetrahydro) to 'regular' (viz. 7,8-dihyro phorbide chromophore, as suggested for sedimentary environments (Baker and Louda, 1986; Keely and Breveton, 1986). Surface sediments from Big Soda Lake, an alkaline meromictic lake near Fallon, Nevada (Figure 88, were e xtracted and analyzed for chlorophyll derivatives. The results revealed the presence of both pheophytin-a [IIa] and bacteriopheophytin-a [XXI] in concentrations of 67. 5 and 9.2 (wet wt.), respectively. On a molar basis, the pheophytin-a to bacteriopheophytin-a ratio was found to be 7 .4:1. Significant preservation of carotenoids, indicating strongly anoxic

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348 depositional conditions (cf. Baker and Louda, 1982), was also noted. Big Soda Lake has been permanently stratified s ince 1907, has mean and maximum depths of 26 and 64 meters with a permanent chemocline (H2S) at 34.5 meters and a strong seasonal oxy-cline (Z0 ) at 20-28 meters (Cloern et gl., 1983; Kharaka et gJ. 1984). In the summer, purple sulfur bacteria (Ectothiorhodospira vacuolata) blooms in the anoxic l ayer above the chemocline. Winter biomass is dominated by diatoms in the "mixolimnion" (Cloern et gl., 1983) which can be observed micro scopically within the surface sediments in good condition, incl uding intact chloroplasts (Oremland et gJ. 1982). Big Soda L ake, though lacking a trona (HC03-, C03=) nature in its bottom waters, could be a small scale analog to the deposition of certain sequences (g.g. l ean oil shales) allied with the Green River formation (cf. Hunt et gJ., 1954; Smith, 1983; Wolfbauer, 1973). That is, during periods of maximal aereal expansion, the Green River formation includes true algal lacustrine facies as opposed to the very organic rich kerogen/carbonate laminated oil shales one usually associates with that formation (see Wolfbauer, 1973). A modern environment which may be considered as an analog for the deposition of highly organic rich shales (Hatcher et gl., 1983: cf. Burgess, 1987) is the brackish paralic basin called Mangrove L ake on the island of Bermuda (Figure 88, "ML"). Mangrove Lake is described as a small (300 x 400 m) depression within lithified Pleistocene carb onates and having a maximum depth of about 20 meters. Presently, the water depth is about 2 meters and overlays an e xtensive deposit of anoxic gel (sapropel). Underlying the gel and lining the bed rock is about 2 meters of peat which grades from fresh water to marine origin

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349 as the lake evolved (Hatcher, 1978; Hatcher et . 1977, 1982, 1983). Geochemical data pertinent to the present study of chlorophyll diagen esis in three samples from Mangrove Lake are given in Table 37. These samples, with depth, c onsisted of olive-brown, dark-brown and pinkish gels. However, all of these samples correspond to the upper redbrown" gel layer described in the literature (cf. Hatcher et 1982). Extremely high TOC values are present in the dried gel (50-60 % : Table 37) and range from 40-70 % for the entire lake (Hatcher et 1983). lhe organic matter in the gel has been characterized as primarily aliphatic algal remains with a strong addition of microbial compounds. and a lesser terrestrial fingerprint (Hatcher et . 1982, 1983). Examination of the pigment profile for the three strata studied here (Table 37) we find both the input of aerobic phytoplank t on/algae and of anaerobic, probably purple sulfur, bacteria. Several depth related trends can be discerned from these data. The ratios of chlorophyll a derivatives to bacteriochlorophyll-a derivatives and of carotenoids to total chlorophyll derivatives react in an inverse manner, the first decreasing and the latter increasing A maximum for all pigments is found at about 2 meters depth in the core. P.Y.I. values are actually low when compared to anoxic open marine/upwelling sedimentary environments P.Y.I. = 15-50: Tables 33-34). At the time of coring, strongly anoxic conditions with pervasive hydrogen sulfide (8-15 m mol/L) extended to the gel surface. The overlaying waters, whose salinity fluctuates with rainfall (S = 32.5-33.5%), have minor exchange with the sea and are oxygenated (cf. Hatcher et . 1982, 1983). On the basis of the above and the pigment data collected (Table 37), one can speculate as to the origins of the various

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Tabl e 3 7 Geochemical data for brackish-to-marine sapropel from Mangrove SAMPlE(!) DEP'IH SUB-(1 ) PERCENr(1 ) weight(2 ) PERCENT(!) ORGANIC GIL-a+ a-rLa CAROTS DESIGNATION SURFACE( em} WATER CARBON CAROTS CHL-a BACTa BACTa BACTa a-rL+BACT ML82( C1 +C6) 0 -95 93. 5 49. 2 96. 3 209. 9 96. 5 306. 4 2 .12 0 .31 6 2 ML82(C11) 190 -210 91.5 54. 1 448.4 244.6 1 89.1 433. 7 1.17 1.03 8 0 ML82(C18) 330 350 90. 5 58.9 163 7 44. 7 57.8 102 6 0.89 1.60 1.7 ( 1)Sampl es dep t h of collection,percent s water and organic carbon supp lied by D r s P A Hatcher a n d E .Spiker o f U S Geologica l Survey,Re s ton,Va.(U S A ) (2)Yields of carotenoids and ch l orophyl l de rivatives determined following over m icrocrystalline cell u lose(see Tab l e IV and associated text).Carotenoids determined using E1 = 2500(Louda,1978 :cf.Davies,1965). Chlorophyll derivatives de t ermined using extinction coefficient s as given XVI.Abbreviations;CAROTS = carotenoids,CHL-a=chlorophyll-a derivatives(viz.pheophytina [IIa] and p h eophorbide a [VI]),BACT-a = bacterioph eop h y t in-a[XXI J plus bacteriop h e ophorbide a (3) P Y I = pigment yiel d index.P .Y.I.= tetrapyrrol es in ug/g sedimen t,dry wt. divided by percen t organic carbon,dry wt. ( c f.Louda al. ,1980). w l.l1 0

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351 chlorophylls. Chlorophyll-a [I] most notably pheophytin-a [IIa] and pheophorbide-a [VI], most likely stems from phytoplankton in the short (ca. 2 m) water column plus any benthic forms (g.g. cyanobacteria) inhabiting the water/gel interface. Within the anoxic gel itself, and actually imparting the reddish to pink colors, a significant population of purple sulfur bacteria appears to be active. In order to more precisely establish the pigment ecology of this get (sapropel) forma tion it would be necessary for one to perform rapid, perhaps on-site, analyses. That is, the loss of magnesium from the chlorophylls, the 'pheophytinization' reaction, is extremely facile (Holden, 1976) and is known to be first-order with respect to acid (i. g. H+: Joslyn and Mackinney, 1938; Mackinney and Joslyn, 1940). So facile is the loss of Mg2+ from chlorophyll that, in order to estimate "living" chlorophyll, much time and effort has been spent on the development of methodology which will not result in the artifactual production of 'pheopigments' (see g.g. Holden, 1976; Parsons and Strickland, 1963; Parsons et gj. 1977; Rai, 1973; Richards and Thompson, 1952; Yentsch, 1965; Yentsch and Menzel, 1963). It is the contention of the author that at some point in the upper 2 meters of this sapropel a living population of anaerobic photoautotrophs, specifically purple sulfur bacteria, was sampled. The preservation of pigments stemming from the activities of anaerobic microbiota has the advantage of anoxia, by definition, during all phases of deposition Chromatographic analysis of the pigments in the sample from 1.2-2.1 meters sub-surface (ML82 (C11)) revealed that the chlorophyll-a derived population had experienced about 30% dephytylation (i.g. pheophytin-a [IIa]/pheophorbide-a [VI] 70:30) while only about 4% of the bacteriopheophorbides. An increasing carotenoid-to-

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352 tetrapyrrole ratio (CAROTS/CHL+BACT): Table 37) with depth in core ML82 indicates two separate yet congeneric phenomena. The first and largest increase in the relative, but also absolute, abundance of carotenoids appears to stem primarily from their presence as accessory photo-gathering pigments in the, as suggested above, living population of purple sulfur bacteria. Chromatographic analyses allowed preliminary identification of three main types of carotenoids, as the author has defined elsewhere (Baker and Louda, 1982). Specifically, these three types are carotenes, oxo-and oxy-carotenoids. The first type contains only C and H, while the second and third groups are CHO compounds. In a somewhat parallel classification, carotenoids can also be physicochemically split into hypophasic (H) and epiphasic (E) types based upon their behavior to partition between n-hexane (or petroleum ether) and 90% aqueous methanol (Foppen, 1971; Krinsky, 1963; Louda, 1978; Petracek and Zechmeister, 1956). In sediments, H/E values are typically low, indicating a predominance of the epiphasic carotenes, and decrease with burial depth (Baker and Louda, 1982; Fox et gl., 1944; Schwendinger and Erdman, 1963). This was at first thought to be due to a preferential destruction of the oxy-/oxo-carotenoids (Fox et gl., 1944; Schwendinger and Erdman, 1963). However, it is now agreed that dehydration reactions, decreasing the hypophasic carotenols and concurrently increasing the epiphasic carotenes, are responsible for the observed trend (Baker and Louda, 1982; Repetta and Gagosian, 1983). In general, living photoautotrophs and marine zooplankton contain carotenoid mixtures which yield H/E values between 4 and 14 (Vallentyne, 1960). In the present study, sample ML82 (C11), the 2 meter sample (Table 37), was found to contain an extremely high

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353 concentration of carotenoids with a carotenol to carotene ( -H/E) value of 3.2. Overall, an active stratum of anaerobic bacterial photosynthesis appears to be present in the near surface sapropel of Mangrove Lake. The extension of the above, albeit limited, studies on the impor tance of bacteriochlorophyll-a to geochemistr y in general may be a bit premature. However, certain analogies between sapropel accumulating environments and oil-shales do e xist and such correlations have been suggested (Bradley, 1966, 1970; Burgess, 1987: cf. Hunt, 1979; Tissot and Welte, 1978). Therefore in light of reports on the structures of certain geoporphyrins and the rather wide distribution of bacteriochlorophyll-a in nature, as described herein, it is appropriate to call this compound a precursor in the overall scope of tetrapyrrole geochemistry. To reiterate, studies given herein report bacteriochlorophyll-a derivatives, primarily bacteriopheophytin-a [ X XI], from a marine littoral habitat, a marine paralic basin, a fresh water lake and a fresh water/terrestrial peat. In each case, bacteriochlorophyll-a derivatives were as, or slightly more (viz. ML82(C18), Table 37), abundant than accompanying chlorophyll-a derivatives. That is, bacteriochlorophyll-a, in depositional environments conducive to the growth of purple sulfur (and non-sulfur, ?) bacteria, can be not only a significant but actually the major b iotic tetrapyrrole pigment serving as precursor material for eventual geoporphyrins. Going on the above, we now turn to bacteriochlorophyll-a itself, drawn here as bacteriopheophorbide-a (Figure 97) in order to describe potential early diagenetic reactions and to meld it into the overall Treibs' scheme. Several early diagenetic reactions are possible when

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""' '\: 2 H NH N-+H20 I -CHp :.oH AA (A/B/C) (2-ACETYL-OESVINYLPHFDPHORBIDE-a) (BACrERIOPHIDPIIORBIDE-a) / / ''TRUE"DPEP (2-DESVINYL-PHFDPIIORBIDE-a) -H 2 0 (A) AA / (PI-IfDPIIORBIDE-a) (A) -CH4 (B/C) 2 H ) ....--I I (C) 2-NETIM.-DESE.1liYL-DPEP ("ABEL'>ONlTE") H (B) (2-DESVINYLPHEOPHORBIDE-a) -0130/1 +2H (B) H )-NH N'' PHEOPHORBIDE-a) (C) )-N11 PIIIDPHORBIDE-a) (B) -2-H-DESETHYL-DPEP ( "3-nor-DPEP") (B) -C02 ""' NH N-(2-CARBOXY-DESVINYLPHFDPHORBIDE-a) (B) 1 )-Nu (2-f'ORNYL-DESVINYLPIIEDPHORBIOE-a) ("PHEXlPHORBIDE-d") Figure 97. Potential alteration of bacteriopheophorbide-a during early diagenesis and 'expected' DPEP series porphyrins. w V1 p..

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355 considering rings I and II, the 'upper' or 'northern' half of the molecule. First, the rather facile oxidation (viz. didehydrogenation, aromatization) of the 3,4 bond in ring II most likely would occur and yield the nucleus of a chlorophyll-a derivative, save the 2-acetyl moiety. Second, the straight forward reduction of the 2-acetyl group and the subsequent dehydration of the resultant 2-a-hydroxyethyl (cf. [XXii]) substituent would yield pheophorbide-a [VI] per se. Thus, 'Route A' in Figure 97 would yield a compound indistinguishable, except possibly isotopically (i.g. o13C: Bareham et gl., 1989, 1990; Popp et gl., 1989), from chlorophyll-a [I] derivatives. Alternately, it is intriguing to consider the reactions within 'Routes B' of Figure 97. Two different, albeit theoretical, routes will take the 2-acetyldesvinyl-pheophorbide-a derivative to a 2-H-desvinyl-pheophorbide-a structure. The first route requires only the elements of water and produces acetic acid (or acetate) which could then serve as substrate for methanogenic archebacteria (g.g. CH3COOH CH4 + C02 or CH3COOH + 4H2 2CH4 + 2H20: Ehrlich, 1981 and references cited). Alternately, reduction and methyl loss could result in a 2-formyl-desvinyl pheophorbide-a structure. This structure is also identical to "pheophorbide-d" thought to be present in certain red algae (cf. Holt, 1966; Jackson, 1976). Arguments were presented in the Introduction here which point to chlorophyll-d. and therefore its derivatives, as being a potential isolation artifact. Once formed, the 2-formyl moiety could be eliminated directly, through the 'methanol' form after reduction or decarboxylated following oxidation. The result of all 'Routes B' leaves a 2-H-desvinyl-pheophorbide-a structure and provides a perfect, though only speculative, precursor to 2-nor-C30-DPEP (revised

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356 nomenclature: 3-nor-C30-DPEP) which is a known geoporpyrin (Fookes, 1983; Sundararaman and Bareham, 1991). Further, in a recent report (Sundararaman and Bareham, 1991) it was even suggested, from other evidences, that the 2-nor-C30 DPEP structure was preferentially formed in low pH ('acidic') paleoenvironments, not unlike the lacustrine environments reported here. Lastly, 'Route C' in figure 97 yields a 2methyl-desvinyl-pheophorbide-a structure via reduction of the acetyl cleavage products. This structure, as well as previously suggested vinyl cleavage products (Louda and Baker, 1986), serves as the requisite precursor to the porphyrin, 2-methyl-desethyl-DPEP (alt. 3-nor-C31-DPEP: Fookes, 1983a; Storm et gl., 1984). Many of the studies performed by the author on the early diagenesis of chlorophyll have been reported in review (Baker and Louda, 1986a) and specialty (Louda and Baker, 1986) reports. Here a synopsis of these studies will be provided and newer data added as appropriate. Throughout the remainder of this work when the words 'chlorophyll' and 'chlorophyll derivatives' are used the '-a' form [I] is to be assumed. However, it should be remembered that all of the chlorophyll (see Figures 7-9), heme-type (see Figures 10-11), and other (Figure 12) tetrapyrrole pigments can be 'processed,' either theoretically and/or geochemically, through Treibs' scheme reactions. The Pleistocene diatomaceous sediments deposited within the DMZ of the Guaymas Basin slope in the gulf of California has provided the best and most complete study of the overall diagenesis of chlorophyll to date. That is, the defunctionalization of phorbides, aromatization and chelation reactions which comprise early-, mid-and late-diagenesis, respectively, are all present. It should be reiterated (cf. Baker and

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357 Louda, 1986a; Louda and Baker, 1986) here that even though these stages are sequential they also overlap. Pigments reported here were extracted, chromatographed, spectrometrically analyzed and derivatized as given in Chapters 2 and 3. Chromophore identification, deta i led previously, consisted of recording the UV/VIS spectrum of the native pigment before and after treatment with sodium borohydride for conju gated ketone detection, and the in vitro copper (63C U2+) derivative, also before and after treatment with NaBH4 While this is only a tentative identification method, and does little to describe the alkyl substituents, it does pinpoint the chromophore. The chromophore, in these cases consists of the tetrapyrrole nucleus (porphyrin or 7,8-dihydroporphyrin = 'chlorin') plus several conjugated auxochromes (g.g. vinyl, keto, aldehyde, carboxyl). Preliminary descriptions of these pigments have been reported (Baker and Louda, 1980a-b, 1981a-b, 1982, 1983, 1984, 1986a-b; Baker et gl., 1987; Louda and Baker, 1981; 1986, 1987, 1990; Louda et gl,. 1980; Palmer et gl., 1982) and only the salient features plus newer data will be given here. As the author has suggested previously (Baker and Louda, 1986a; Louda and Baker, 1986), chlorophyll diagenesis can be divided into two main and divergent routes. These can be considered as (1) anoxic/ reductive/preservative and (2) oxic/oxidative/destructive. Route 1 leads to the preservation or 'fossilization' of tetrapyrrole pigments as metallo-porphyrins. Route 2 leads mainly to the destruction of pigment, though minor survival of severely altered pigments as etioporphyrins has been considered (Baker and Louda, 1980b, 1986a; Barwise, 1987; Louda and Baker 1981, 1986). Currently we maintain that the formation of pyro-pheophorbides (g.g. [VIlli]) through the loss of the

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358 10-carbomethoxy moiety is a, if not the, key fating reaction which leads chlorophyll derivatives towards preservation and away from oxidative destruction (Louda and Baker, 1986). That is, it is well known from the in vitro chemistry of chlorophyll derivatives (g.g. Fischer and Stern 1940; Hyninnen 1979a-b; Pennington et gJ. 1964, 1967a-b) that the addition of oxygen at the C-10 position, the so called 'allomerization' reaction (cf. Willstater and Stoll, 1913), only occurs with phorbides containing an intact C-10 carbomethoxy (methyl formate) moiety Pigments tentatively identified from deep sea sediments and fitting into the anoxic pathway include pheophytin-a [IIa], pyro pheophytin-a [III], and a series of decarbo x ylated pheophorbides. That is, thus far work has progressed mainly on the 'CELL-1' or non-polar pigments from these (DSOP/IPOO) sediments. All fractions are saved and the 'CELL-2' mono-carboxylic acids, most notably the pheophorbides per se, are to be studied next. There now seems to be three distinct series of non-polar phorbides That is, there are the phytyl esters ("pheophytins"), a minor amount of alkyl (viz. decarbo xylated) pheo phorbides (Louda and Baker, 1986) and the recently identified pheophor bide steryl esters (Eckardt et gJ., 1992; King and Repetta, 1991; Prowse and Maxwell, 1991). Following here are described many of the pigments isolated from DSDP/IPOD Leg 64, Guaymas Basin, sediments. Preliminary reports on these studies have appeared (Baker and Louda, 1982; Louda and Baker, 1986) which cover certain aspects of these studies. Pheophytin a [IIa] is perhaps the most common, or at least most easily recognized, chlorophyll-a [I] derivative encountered. Almost

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359 without exception, sediments in the earliest stages of diagenesis will contain pheophytin-a [IIa]. Pheophytin-a [IIa] is found in the non polar "Cell-1' fraction described earlier (Table 4). The electronic absorption spectra (Table 38) of pheophytin-a [IIa] e xactly matches the authentic pigment (cf. Table 10) as does the 'oxy-deoxo' (00) derivative 9-00-pheophytin-a [V]) obtained following treatment with sodium borohydride The copper chelates of both the native pigment and the 9-oxy-dexo derivative (cf. [LXI], [LXII]) also match the authentic pigments. In.many cases, more definitive or selective chromatography (LPHPLC) over silica gel results in two peaks for pheophytin-a [IIa]. These we tentatively identified as pheophytin-a [IIa] per se and pyro pheophytin-a [III] for the slightly more and less polar forms, respectively (Louda and Baker, 1986). In certain cases the 'pheophytins-a' area of the normal phase silica LPHPLC chromatograms show evidence of three components. In these cases, the third and most polar pheophytin a type is most likely 10-hydro x y-pheophytin-a (see Appendix A [IIa]), also known as pheophytin-a-allomer (Louda and Baker, 1986: cf. Hynninen, 1979a-b). Pheophytin-a [IIa] and pyro-pheophytin-a [III] have been more definitively identified, using 20-COSY NMR and FAB-MS, from the contemporaneous sediments of an eutrophic lake in Cumbria U.K. (Keely and Maxwell, 1990a-b, 1991; Keely et gj., 1988). Compounds with the e xact chromophore of mesopyropheophorbide-a [X] were isolated from the Pleistocene and Pliocene diatomaceous oozes recovered between 72 and 246 meters sub-bottom (in situ T = 15-31C) in the anoxic slope of the Guaymas Basin, Gulf of California. The UV/VIS spectrum (Table 38) of this compound matches that of an authentic pigment ([X], Table 10) as do the spectra of borohydride reduction

PAGE 388

Table 38. Electronic absorption spectra of selected tetrapyrrole isolate s from marine sediments. TENTATIVE IDENTIFICATION(1 ) -Pheophytin-a Mesopyropheophorbide-a!DC1 bide-a-1 DC 1 1 DC 1 -9-0D-Mesopyropheophorbide-a-1DC1 -7,8-Dihydro-OPEP series.(=DOMPP-a-1DC1 ) 1Cu-7, 8-Dihydro-OPEP series. -Purpurin-18 -Chlorin-p6 -Cyclopheophorbide-a enol *CuOioxydideoxo-cyclopheophorbide-a Cyclomesopheophorbide a enol ideoxo-c yc lomesopheophorbide -a Mesorhodin-1DC1 or Pyrrorhodin #1 Mesorhodin-'DC or Pyrrorhodin #2 FOOINOTES: COMPARE 10( 2 ) WAVELENGTH OF MAXIMAL ABSORPTION,nm(3 ) [COMPOUND] SO RET IV III II I [IIa] 410 505 535 610 667 [V] 396 500 ---596 651 [LXI] 421 505 547 603 650 [LXII ] 406 510 (540) (580) 622 [X] 406.5 498.0 532. 0 599.5 658.5 [XI ] 396.5 496 (520) 584 646. 5 [LXIIIa] 420 ----595 643 [LXIV] 403 (500) --(585) 616 [XI] 396.5 500 --588 646.5 [LXIV] 402.0 510 ----614.5 [XIII] 390 0 497 524 585 640.0 [LXV] 395.5 494 523) 564 606 [XXVI] 407 505 541 693 696 [XXVIIa] 400 499 535 612 671 ----358,423 452 --625 686.5 -----399. 5 ---598 651 ------404.5 ----(585) 625 ---354,407.5 (450) 587 634 678.0 ----396 495 525 (580) 643 [XLI] 405.5 509 549 585 636 [XLVI] 402 508 544 586 636 (!)Nomenclature used herein is described in the "Introduction",see also "Appendix-A".OD= oxy-deoxo derivative obtained via reduction of conjugated carbony-l moieties with sodiwn borohydride."DC", refers to isolation of the geologic pigment as a decarboxylated analo g of the structure given( s ee text).Native isolates indicated by derivatives by asterik(*). (2)Compound nwnbers given as Roman nwnerals refer to the synthetic standards given in Appendix A. (3)Spectra recorded in ethyl ether solvent.Wavelengths given as integers are to be taken as lnm. Wavelengths given as real nwnbers are to be taken as 0.2nm. w "' 0

PAGE 389

361 product (cf. [XI]) and the copper chelates of each (cf. [LSIIIa] and [LXIV], respectively) with their known counterparts. Upon normal phase LPHPLC over silica, this compound eluted well before the pheophytins-a given earlier. In order for a mesopyropheophorbide-a pigment to exhibit less polarity than pyropheophytin-a [III] it would need to lack the polarity imparted by the propionic acid carboxyl moiety. That is, on the present LPHPLC system, mesopyropheophorbide-a ME [X], elutes after pheophytin-a [Ila]. Further, it is reported that mesopyropheo phorbide-a [X] occurs esterified to a wide variety of sterols in aquatic sediments (Echardt et gj., 1992; King and Repetta, 1991; Prowse and Max well, 1991). These compounds, called NPC's (non-polar chlorins) by King and Repetta (1991) could explain the non-polar nature of the mesopyropheophorbides isolated here. This may indeed by the case in very recent sediments. However, in deeper, more nature sediments (g.g. Pilocene, ca. 25-30C) these persist and by that level of maturity most of the phorbide esters (i.g. pheophytin-a) have been lost. Therefore, we shall retain a tentative classification of these non-polar phorbides as '7-ethyl-7-desprop io-', indicated as 'DC' ('decarboxylate') in Table 38. An alternate line of evidence points to an early, perhaps even biotic in some cases, decarboxylation of 7,8-dihydroporphyrins That is, free-base porphyrins of the DPEP series arise from a phorbide pool in the bitumen and are entirely alkyl in nature. Several isolates of compounds with a chromophore very nearly the same as 9-oxydeoxo-mesopyropheophorbide-a [XI] have been found (cf. Louda and Baker, 1986). The spectrum of the native pigment and its in vitro copper derivative (Table 38) closely match those of the authentic pigments ([XI], [LXII]; Table 10). The native pigment failed to react

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362 wfth sodium borohydride and the absence of a conjugated carbonyl, as suspected, was shown. Given more detailed analyses, the proof of the 9-00-mesopyropheophorbide-a [XI] structure in b itumen would be of e x treme importance. That is, the o x y-deo x o (00) functional (viz 9-hydroxy) could serve as a requisite i ntermediate in a potential pathway for the loss of 9-keto substructure during the geochemical evolution of OPEP-series porphyrins (cf. Baker and Louda, 1986; Baker and Palmer, 1978; K eely et gl., 1990). Given as Figure 98 is the suggested reduction of a 9-keto-phorbide yielding the 9-o xydeox o, or 9-hydro x y (132 -hydroxy: revised nomenclature), substructure. The hydro x yl functional would then become amenable to additional reactions, includ ing incorporation into ether linkages with kerogen or dehydration. In the latter case, as shown in Figure 98, dehy dration would yield the strained cycloetheno structure which would most likely be rapidly (concurrently -?) reduced to the c ycloethano ring As given above, the isolates in question a r e believed to be decarbo x ylated forms of 9-o xydeox omesopyro pheophorbide-a (cf. [XI]). Several very small quantities (g g. < 10 ng) of a pigment with band I absorption at 651 nm and a Soret at ca. 396 nm were also found in sediments from OSOP/IPOO Leg 64 sediments (Baker and Louda, 1982). As such, this pigment mimics the chromophore of 9-00-py r opheophorbide-a [IX] and 9-00-pheophorbide-a [VII] (Table 10). Further, it would also be close to the chromophore of the deox opheophorbid es-a, though knowns for these are lacking (cf. Fischer and Stern, 1940). Additional investigation of this pigment would requi r e e xtra OSOP/IPOO samples (g.g 64-479-22-5: Baker and Louda, 1982), and this is unlikely.

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[X] H3CH H' HN [XI] [XII] ,, 0 ... R ... ... CH 3 +2H +2H H H H H Figure 98. Suggested mechanism for the removal of the 9 -keto moiety from chlorophyll-a derivatives.Shown are portions of the following compounds;[X]=mesopyropheophorbide-a, [XI] = 9-oxydeoxo-mesopyropheophoribde-a, and, [XII] = deoxomesopyropheophorbide-a. 363

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364 Oecarboxylated analogs of deoxomesopyropheophorbide-a [XII] ('DOMPP-a') are very nearly always present in sediments approaching or within mid-diagenesis. That is, as we have defined the stages of chlorophyll diagenesis (Baker and Louda, 1983, 1986), mid-diagenesis encompasses the aromatization of 7,8-dihyroporphyrins (i.g. phorbides, chlorins) to yield porphyrins. As isolated, these geochemical pigments are present in the non-polar chromatographic fractions and e xhibit HClnumbers of about 5-7. Thus, these phorbides are decarboxylated (7-R-7despropio: R = -H, -CH3 -CH2CH3 -CH2 CH2 CH3 ) pigments. Based upon the world distribution of free-base porphyrins, the products of these (DOMPP-a) precursors, it is suggested here that the most common form is likely to be 7-ethyl-7-despropio-DOMPP-a (cf. [XII], [XIII]). Whether these pigments are isolated from deep sea sediments or ancient marine shales (g.g. Pliocene/Miocene Sisquoc shale, this study) they give a carbon number distribution (g.g. C28-C33) which mimics co-isolated free-based porphyrins (Figure 99). Going on their similarity to DPEPseries porphyrins, these decarboxylated DOMPP-a [XII] pigments (cf. [XIII]) may be alternately termed a 7,8-dihydro-DPEP series. The electronic absorption spectra of the native pigments (Table 38) and their in vitro copper chelates match authentic DOMPP-a [XII] (and 7-PDP-DOMPP-a [XIII] and Cu DOMPP-a [LXVIa], respectively (see Table 10). The above list of pigments encompasses all of the structures required to move chlorophyll through the straightforward Treibs' scheme and to generate porphyrins of the ('true') DPEP-series. Concurrent with the present studies, structural proof has appeared in the literature for the following tetrapyrrole pigments; pheophytin-a [IIa], pyro pheophorbide-a (III], pyro-pheophorbide-a [VIII ] meso-

PAGE 393

>t-'tf) z w r-:-0 w N _j
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366 pyropheophorbide-a [X], chlorophyll-a [I] and pyro-chlorophyll-a (Keely and Maxwell, 1990a-b; Keely et S}. 1988, 1990). All o f t hese structures were from the contemporaneous sediments of an e u trophic lake and, as such, had experienced little 'aging'. However, the p r ovision of structural proof for these pigments in a sedimentar y setting i s indeed a breakthrough The allusion above to 'ageing' is meant t o infer that, as diagenesis progresses, a spread of carbon numb ers occurs That is, examination of the mass spectral distribution for the decarbo xyl ated DOMPP-a (alt. 7,8-dihydro-DPEP) series reveals that, as ide from the C32 (decarbo xylation) and C33 (reduct ion) members whic h are 'expected' by Treibs' scheme geochemistr y a certai n amount o f d eal kylation (C28-C31, -4 to -1CH2 ) has also occurred (Figure 99). It rem ains f or future studies to unravel the timing and mechanism of this well documented earl y diagenetic dealk ylation (cf. Baker and Palmer, 1 9 7 8; Baker and L ouda, 1983, 1986a). That is, is this spread o f c a r bon numbers due t o: (1) the input of several variant forms of chloroph y l l ; (2) ver y early diagenetic, possibly biotically mediated, reac t ions; ( 3 ) lo w tem perature thermo cataly t i c pro c esses; or, (4) a combinat ion o f t hese ? These pigments described above were all isolated f ro m the non polar pool of tetrapyrroles. As given earlier, a la rge amount, often the majority, of the early diagenetic pigments are present as carbo x ylic acids As work on this nex t group of pigments is sti l l underway only the briefest mention will be made. Many of the same chromophores identified herein are possessed in the several pheophorbide-a pigments i solated by the Bristol group ( K eely and Max well, 1990ab ; K eely et gj., 1988, 1990), as given above. As stated, the aut hor feels that many parallel diagenetic steps occur in the various pigment pools;

PAGE 395

367 alkyl (i.g. decarboxylated), carbo xylic acids, steryl and phytyl esters and the as yet unproven bound (g g kerogen) state. Aside from the phorbides one might expect in geologic samples, according to the direct precepts of the Treibs hypothesis (Treibs, 1936: cf. Baker and Palmer, 1978), several other tetrapyrrole pigments have been isolated from early diagenetic staged sediments. Throughout all of these studies a series of chlorin and purpurin acids have been linked with o xic depositional conditions and rapid pigment loss (see Louda and Baker, 1982, 1986). Many of these pigments ('Cell-1' and 'Cell-2' fractions: Table 4) resemble chlorin acids reported from the oxidative degradation of chlorophyll (g.g. chlorin-e4 isochlorin-e4 chlorin-e6 [XXIII], phyllochlorin, etc.: Fischer and Stern, 1940) but, to date, have not been isolated in pure enough form and/or quantity enough for further tests. True HPLC and RP-HPLC, will aid this work. However, two forms of chlorin acids are consistently present and in sufficient quantities so as to suggest them as major components in oxically deposited sediments These compounds have been tentatively identified by the author as purpurin-18 [XXVI] and chlorin-p6 [XXVIIa] in a variety of deep sea sediments (see Baker and Louda, 1980a-b, 1981a-b, 1982, 1984, 1986a; Louda and Baker, 1981, 1986; Louda et g]. 1980). The example of an isolate of purpurin-18 [XXVI] given here is from a grayish olive green silty clay recovered from 21.5 meters sub bottom at a 3.1 km deep site west of the Baja, California borderlands. In this case, Figure 100a, the 'Cell-2' fraction (Table 4), conta i ning the mono-carboxylic acids, exhibits a distinct shoulder at about 690 nm in the UV/VIS spectrum. Application of second derivative technique (Figure 100b) to this spectrum reveals the positions of the major

PAGE 396

1 .0-,--------------, 0 8 w 00.6 z -0 II Jl I a::
PAGE 397

369 absorption maxima. Of particular note are maxima at 541 and 696 nm, corresponding excellently with the positions of bands III and I (cf. Table 10) of authentic purpurin-18 [XXVI]. Subsequent separation and purification of this fraction lead the isolation of purpurin-18 [XXVI] (Figure 101, solid trace) and the tentatively identified chlorin-p6 [XXVIIa] (Figure 101, dashed trace). There is little doubt in the author's mind that these pigments are as claimed, especially given the unique nature of the purpurin-18 spectrum and the data collected on authentic tetrapyrroles (Appendix A). However, work continues on these isolates and structural studies are planned Previously we isolated and tentatively identified a 'chlorin' with band I absorption at 686.5 nm as a diketo phorbide, possibly a or 2-formyl pheophorbide-a (Louda and Baker, 1986). Additional work on this pigment, the isolation of a closely related chromophore with band I absorption at 678 nm and, most notably, correlation of this pigment ('chlorin-686') with the reports of a synthetic 7,10-cyclopheophorbide a enol (revised : 132 173-cyclo pheophorbide-a enol: Falk et gJ., 1975; Isenring et gJ., 1975), which were previously overlooked. The present compound ('chlorin-686': cf. Louda and Baker, 1986) was isolated from Recent to Pleistocene diatomaceous oozes recovered at 7.6 (64-481-2-2), 30. 7 (64-477-5-1) and 35.6 (64-479-5-3) meters sub-bottom at the DSDP/IPOD sites given (see Baker and Louda, 1982). The electronic absorption spectrum for the native pigment (Table 38: Figure 102) is an exact match for that reported for an in vitro known preparation of 132,173-cyclopheophorbide-a Treatment of the isolate ('chlorin-686') with NaBH4 lead to a severe reduction in the extent of UV/VIS 'fine structure' (Figure 102) and shifted the Soret and band I

PAGE 398

1 .0-,--------:----------. 0.8 <( m 25 0 4 (j) (]] <( 0.2 670.5 f't I \ I \695. 5 :-....V' I 0 350 550 750 WAVELENGTHJnm 370 Figure 101. Electronic spectra of purpurin-18 (solid) and chlorin-p6(dashed) isolated from DSDP/ IPOD sample 64-481A-2-2.

PAGE 399

1 '0 -r-:::3:-:9:-::9:-. 5::-----r-------r------r-----.. 0.8 0.6 w u z <{ CD 0::: 0 (/) CD 0.4 <{ 0 2 {\ \ \ 686. 5 651 (\ I \ 371 350 450 550 650 750 WAVELENGTH(nm) Figure 102. Electronic absorption spectrum of 132,173 -cyclopheophorbide-a enol(solid) and the dioxy-dideoxoderivative(dashed) obtained via reduction with sodium borohydride.Solvent= ethylether.Sample DSDP/IPOD 64-481-2-2(See Table 38).

PAGE 400

372 absorptions to the blue by ca 23 and 35 nm, respectively (Table 38). An hypsochromic shift in band I position of this magnitude is well within known (cf. Table 10) and e x pected shifts for the reduction of 2 conjugated carbonyl moieties on a phorbide skeleton Insertion of copper (II) provided the copper (II) dio x ydideo x ocyclopheophorbide-a (Table 38) with Soret and band I absorptions at 404.5 and 625 nm, respectively. The spectra of the reduced compound and its copper derivative are highly reminiscent of the spectra obtained for 9-o x yde o x opyropheopho r bide-a [IX] and its copper chelate (cf. [LXII]). Given that 'chlorin 686' can now be tentatively identified as 132,173-cyclo pheophorbide-a, potentially formal via a dehydration-cyclization reaction (Figure 103), the UV/VIS of the reduced pigment should mimic that for the 9-oxydeoxo pheophorbides-a ([V], [VII], [IX]), and it does. This structure (Figure 103: 132 173-cyclopheophorbide-a enol) has attained important status in the geochemistry of tetrapyrroles as a precursor(s) to $everal porphyrins with a 7,y-(15,17-:revised) cyclobu tano moiety (Bareham et gl., 1990; Callot et gJ, 1990; Chicarelli and Maxwell, 1986; Chicarelli et gj., 1984, 1987; Fookes, 1983b; Ocampo et gl., 1987; Wolff et gl., 1984). The enol itself has been isolated from the ("freeze-dried") tissues of a sponge (Porifera; Keratosa: Darwinella oxeata) where its presence was suggested as being formed from dietary chlorophyll-a (Karuso et gj., 1986). The notation above that the sample was "freeze dried" (Karuso et gl., 1986) was made in order to point out the potential for artifact formation with lyophilization. That is, dehydration artifacts have been associated with the freeze drying of sampies in the past (Van De Meent et gl., 1977) and, as the cyclopheophorbide-a enol in question must be formed via a

PAGE 401

R -H 0 2 ...._ --Figure 103. The dehydration of pheophorbides-a to yield cyclopheophorbide-a enols. R=vinyl;pyropheophorbide-a(left); 132,173 ,cyclopheophorbide-a enol(right).R=ethyl; mesopyropheophorbide-a(left); 132 ,173-cyclomesopheophorbide-a enol(right). w -......1 w

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374 dehydration reaction, it appears prudent to question the 'natural' occurrence of it in the sponge. As far as we can tell, this appears to be the first, albeit tentative, identification of a cyclopheophorbide-a enol in sediments. Additional, structural, studies are planned. A second compound, 'chlorin-678' (cf. Baker and Louda, 1982), possessing spectral (= chromophore) characteristics akin to 132,173 -cyclopheophorbide-a enol has also been isolated from deep sea sediments (samples 63-471-8-4; Louda and Baker, 1981 and 64-474-2-3; Baker and Louda, 1982). This pigment yielded an electronic absorption spectrum (Table 38) as given in figure 104. As noted above, the 'fine structure' or overall qualitative appearance is e x tremely reminiscent of that found for the 132,173-cyclopheophorbide-a enols, both authentic (Falk et gl., 1975; Isenring et gl. 1975) and geologic (Figure 102). In the present case, the major absorption maxima, the Soret and band I, are hypsochromically shifted about 16 and 8 nm, respectively, as compared to the 132,173-cyclopheophorbide-a-enol data. Such shifts are consistent with the removal of vinyl configuration during the change from a pheophorbide to a mesopheophorbide (cf. Figure 3). In order to more fully test the possibility that this compound ('chlorin-678') was the meso analog of 132,173-cyclopheophorbide-a enol, reduction with sodium borohydride was performed. As usual, the course of the reaction was monitored and, prior to completion, band I split into three maxima. These peaks were located at 678, 658 and 646 nm. At completion, band I absorption was at 643 nm with a Soret peak at 396 nm. Further, the satellite Soret bands typical of the native p igment had disappeared Going on the above, after comparison to the data gathered on authentic pigments during these studies, it was concluded that the isolate was a

PAGE 403

375 I U ....,......----------....------.-----., 0 8 0 6 w u z <{ (]) 0:: 0 If) (]) 0 4 <{ 0 2 407.5 (450) 67 8 0 350 450 550 650 750 WAVELENGTH(nm) Figure 104. Electronic absorption spectrum of 132,173 -c yclomesopheophorbide-a enol(tentativ e ).Solvent=ethy l ether.Sample DSDP/ IPOD 63-471-8-4(See Table 38).

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376 132,173-cyclomesopheophorbide-a enol. That is, the borohydride reduction test produced, in turn, chromophores equalling mesopyropheo phorbide-a ([X], I = 656-658 5 nm) and 9-o xydeox omesopyropheophorbide-a ([XI], I= 642 nm: Table 10). Another set of geochemical compounds are pigments which we previously called 'chlorin-636-, again the numerical designation indicates the position (nm) of band I absorption (Baker and Louda, 1980, 1982; Louda and Baker, 1981). The compounds to be discussed do not include a compound with absorption at 636 nm from Gulf of California sediments (DSDP/IPOD Leg 64; Baker and Louda, 1982), as that pigment remains enigmatic. However, two forms of 'chlorin-636', one from Japan Trench sediments (57-438A-4 4 and 57-438-10-4: Baker and Louda, 1980) and the other from the Baja California borderlands (63-471-8-4: Louda and Baker, 1981), have a high potential for being very significant in tetrapyrrole geochemistry. The UV/VIS spectra of these pigments are given in Table 38 and Figures 105a-b. Prior to the accumulation of the electronic absorption data given earlier (Tables 10 and 11: see Appendix A), the preliminary classification of these pigments was made (Baker and 1980; Louda and Baker, 1981) solely on the order of visible band extinction. Classification of a chromophore on the basis of visible band order, described in detail earlier in tex t is a time honored method (see g.g. Buchler, 1978; Gouterman, 1978; Stern and Wenderlein, 1926b; Treibs, 1973; Weiss, 1978). However, as much of the original work on the visible spectroscopy of tetrapyrrole pigments (g.g. Fischer and Orth, 1937; Fischer and Stern, 1940) occurred before measurements in the violet to near ultra-violet (g.g. A < 475.nm), the application of Soret to visible band ratios was unknown. Re-

PAGE 405

09 0.8 07 0 6 w 0.5 u z (tj 0:: 0 (f) co
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378 examination of these data during the present study allow for a better identification to be made. Direct comparisons of these pigments with authentic meso-rhodin-IX [XLI] and pyrrorhodin-XV [XLVI] (Table 10 and Appendix A) revealed several similarities. First, the ratio of Soret to band I absorption (ca. 402/636 nm) is 9-10:1. This is exactly on the order of the known rhodin series and is both too low for other porphyrins and too high for true 'chlorins' (viz. 7,8-dihydroporphyrins and 3,4,7,8-tetrahydroporphyrins: see Table 10). Second, the HClnumber (see Table 3 and associated text) was found to be about 7. This also is much more porphyrin, rather than chlorin, like. Third, the visible band orders (I> IV>III>II; Figure 104a and I>II>IV>III; Figure 105b), while found with chlorins, are also reminiscent of the 'porphyrin rhodins'. The term rhodin here is meant to refer only to the porphyrin structures and in no way connects to b-series chlorophyll derivatives (see Chapter 1). At this juncture, the only spectral dilemma remaining during the comparison of these geologic pigments with the authentic knowns is that while the visible band orders are closest to the rhodins with a conjugated ketone (j.g. [XLII], [XLVII]: Table 10), the actual positions of band I maxima are nearer the parent rhodins (j.g. [XLI], [XLVI]: Table 10). As both mesorhodin-IX [XLI] and pyrrorhodin-XV [XLVI] are formed from compounds derived ultimately from protoporphyrin-IX [LIII], it appears possible that a remnant vinyl moiety could be present and account for the proposed bathochromic shift in band I position. Though the exact structures of these compounds are unknown, it appears quite reasonable to suggest here that both are congeners of mesorhodin-IX [XLI] or pyrrorhodin-XV [XLVI]. If the geochemicals are akin to mesorhodin-IX [XLI], lacking a B-H, then they

PAGE 407

379 must be decarboxylated forms as the HCl-number and chromatographic behavior reveals. Going on the supposition that the 'rhodin-636' identified above is actually a rhodin structure, then it becomes beneficial to consider this compound(s) as diagenetic intermediates. Presented as Figure 106 are the combined known and suggested syntheses for the rhodins derived from mesoporphyrin-IX [LV] and pyrroporphyrinxv [LX]. Bold arrows in Figure 106 delineate reactions well documented in vitro (cf. Fischer and Orth, 1937; Fuhrhop, 1978). This series of reactions begins with the heme pigment protoporphyrin-IX [LIII]. During the in vitro preparation of the rhodins ([XLI], [XLVI]), both vinyl groups are first reduced to ethyls. Above, the potential for a vinyl-rhodin was mentioned. Though not shown, if the reader mentally moves a vinyl retaining compound through the scheme, such a structure can easily be envisioned. The known rhodins, mesorhodin-IX [XLI] and pyrrorhodin-XV [XLVI] are formed from the dehydration-cyclization of mesoporphyrin-IX [LV] and pyrroporphyrin [LX], respectively. In both cases, two isomers result, depending upon which propionic acid moiety was cyclized (mesorhodins-IX [XLI]) or removed (pyrroporphyrins [LX]). The literature studies stop at the rhodin stage. That is, the reported structures retain the conjugated keto moiety. During the present work sodium borohydride was routinely and extensively used as an in vitro analytical probe for conjugated carbonyl moieties. This reaction was extremely facile with the known rhodins and produced the expected 'oxy deoxo' derivatives ([XLII] and [XLVII]: Appendix A and Table 10). As part of Figure 106 (light arrows) it is suggested that a straightforward reduction of the ketone, dehydration of the intermediate hydroxyl and reduction of the 'cyclopropen' moiety would yield a purely

PAGE 408

+4H -CH2CH2COO PROTOPORPHYRIN-IX(LIII) --N IN-{ J. /A.f' H +2H AND fiOOC PYRROPORPHYRIN-"2''(LX) +2H ISOMERIC C31 CYCLOPROPANOPORPIIYRINS(l #leta-H ) Figure 106. The cyclization of and pyrroporphyrins-XV[LX] to yield isomeric pseudo -DPEP compo unds( CAP') with a cycl opropane moiety. w 00 0

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381 alkyl cyclopropano-porphyrin. The first step is extremely facile and the resulting oxydeoxo-compounds ([XLII], [XLVII]) are produced. The remainder of these pathways (Figure 106), the reduction of the cyclopropen derivatives and the isolation of the four individual isomers of the cyclopropano-porphyrins was the thrust of a Master's thesis research project at Florida Atlantic University (Guteirrez, 1986). However, these compounds, either through the oxydeoxo-rhodins ([XLIV], [XLVII]) or corresponding verdins ([XLIII], [XLIV]), proved difficult to reduce after the introduction of a double bond (i. g. verdin stage) and became superaromatic ([XLV]) instead. In spite of this in vitro failure to produce a cycloprano-porphyrin, though direct syntheses with the condensation of pyrroles and tetrahydroindoles has been reported (Lash, 1989; Lash et . 1990), a route similar to that suggested (Figure 106) must be operative in sediments. That is, a variety of geoporphyrins bearing the cyclopropano (m,B-propano: Lash, 1989) moiety have been identified from sediments, shales and oils (Callot et 1990; Chicarelli et . 1984; Keely et 1990; Wolff et 1984). The last 'chlorin' to be discussed is actually a complex of more than 4,000 daltons, as shown via GPC over Sephadex LH-20. This complex yields an electronic absorption as given in figure 107. Repeated attempts to purify a variety of isolates of this complex from an assortment of deep sea sediments has proved futile. That is, this macromolecular complex remains intact as a single fraction during GPC, CC over silica and alumina and RP-LPHPLC. The electronic absorption spectrum reveals the presence of not only a chlorin-type chromophore but the asymptotic absorption decreasing from the ultra violet into the

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382 1.0 -.-------------., 0 9 396 0 8 0.7 0.2 0 1 0. 0 350 450 550 650 750 A., nanometers Figure 107. Electronic absorption spectrum of 'CHLORI N -660' ,suggested as being a macromolecular complex.Dashed trace in the visible region is for this isolate following treatment with sodium borohydride.

PAGE 411

383 red is typical of complex sedimentary organic matter, such as asphaltenes or proto-kerogen and humates. This pigment is typically found in early diagenetic sediments and may represent the incorporation of tetrapyrrole pigments into protokerogen. Analytical derivatization of complex 'chlorin-660' with sodium borohydride reveals the presence of a single ketone conjugated to the chlorin macrocycle. This is noted from the hypsochromic shift in band I absorption of 12 nm (Figure 107: cf. Table 10). The preceding discussion has covered results obtained from the analyses of samples within the period of organic evolution known as early diagenesis. As given, this phase of tetrapyrrole geochemistry includes the defunctionalization of phorbides, the oxidative cleavage of phorbides to yield chlorins and certain cyclization reactions which can generate phorbide -like porphyrins from biotically derived heme-type pigments. The remainder of tetrapyrrole diagenesis (cf. Baker and Louda. 1983) involves (1) the aromatization of 7,8-dihydroporphyrins, the phorbides and chlorins, to yield free-base porphyrins during the period called mid-diagenesis and (2) the chelation of metal cations (esp. Ni2+) which forms metallo-geoporphyrins in late diagenesis. These processes and the usual' order of these reactions are the results of past (see Baker and Louda, 1980s, 1982. 1983, 1984, 1986a-b; Louda and Baker, 1981, 1986; Palmer et gl . 1982) and present studies. That the order of the exact timing of these reactions is dependent upon geologic constraints is evidenced by the identification of nickel mesopyropheo phorbide-a from the Eocene lacustrine messel oil shale (Prowse et gl . 1990). Therefore, the arbitrary division of tetrapyrrole geochemistry

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384 into discrete phases or steps should, as has been stated previously (Baker and Louda, 1983, 1986a), be viewed only as a convenience. In actuality, tetrapyrrole geochemistry is a continuum and the exact order of events will be ruled by the history of physical forces imposed on the pigments. In the present work mid-diagenesis will refer to that period in tetrapyrrole evolution when free-base porphyrins are the dominant sedimentary pigments and are being generated from their precursor 7,8-dihydroporphyrins. Figure 108 is the profile of aromatization derived during the study of the Guaymas basin pigments (cf. Baker and Louda, 1982, 1983) and is presented here as reference. The aromatization reaction, a didehydrogenation, is shown as Figure 109. Though the structures shown are alkyl pigments, an analogous reaction is observed for defunctionalized phorbide deoxomesopyropheophoride-a [XII]) and their corresponding porphyrins (i.g. deoxophylloerythrins [XXXVII]). A limited homology of OPE-series porphyrins (C28 to C33) was isolated from DSDP/IPOD sample 63-471-34-2 collected from the Baja California borderlands (Louda and Baker, 1981). One isolate of _a rhodoporphyrin (cf. [LIX]) from the Gulf of California sediments has been obtained. The UV/VIS of this pigment is given in Figure 110. This geochemical rhodoporphyrin, a porphyrin with a 'rhodofying' (electron withdrawal) group conjugated to the macro cycle, may represent a porphyrin derived from purpurins/ chlorins generated during early diagenesis. Additional study is planned, though the visible band order (III>IV>II>>I) is quite distinctive. Aromatization of free-base 7,8-dihydroporphyrins (phorbides, chlorins) would not be predicted for mixtures containing metallic

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Dl HYDRO PORPHYI N S D I H Y ORO PORPHYRINS 100 80 60 %40 20 0 100 80 60%40 20 0 100 I I I I I I I I I I 13.2 0 7.9 1 0 20 Vl Vl300 u t 100 0 L.. w' +' 20 u Cl) Cl) ...... 0::: E 0 Cl) 30 :::> w 1-200 0::: E 0::: ...... 30 0 w +' +' 0 ...... a._ .0 0::: 0 40 2: I w -? 700 w .0 a._ .0 1-300 2: :J 50 w Ill I 401-1-I a._ 1-900 .!::1 a._ w \ 0 1,100l '-, T6o (b) I I I I I I I I I 500 0 20 40%60 80 100 0 20 40 % 60 80 100 PORPHYRINS PORPHYRINS figure 108 Downhole profiles for the tetrapyrrole aromatization reaction.Dihydroporphyrins calcul a t e d as the s u m of all phorbides,chlorins and purpurins. 'Porphyrins' is the s u m of free-base and metallo-porphyrins. (a) B lack Sea, DSDP site 42B 380A (calculated from data in Baker e t al.,1978). (b) This study,DSDP/IPOD site 64 479 Gulf of California(cf. Baker and Louda,1982,1983). w CX> V1

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0.6 w u z <( 0 tJ) co <( 0.2 350 450 550 650 WAVELENGTHJ nm 387 Figure 110. Electronic absorption spectrum of mixed 'rhodoporphyrins' isolated from DSDP/IPOD sample 64-47929-4(See Table 38).Solvent =ethyl ether.

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388 cations, such as sediments. That is, all past in vitro work points to chelation of metals (i.g. Cu2 > Zn2 > Mn2 > > > > Cd2: Fleischer et gj., 1964; Longo et gj., 1978; Schneider, 1975) being favored over aromatization (Eisner and Linstead, 1955). Therefore, as the generation of free-base porphyrins is the geologically overwhelming fate for phorbides and chlorins (cf. Baker and Louda, 1983, 1986a-b; Louda and Baker, 1986; Verne-Mismer et gj., 1990) mechanisms to remove or at least reduce metal cation availability must be operative. Here we of course infer the participation of sulfides, as has been well studied for the question of Ni2+ versus (V0)2+ dominance (see Barwise, 1990; Fish et gj., 1987). That is, it is suggested here that the formation of metal sulfides (MS) decreases the availability of chelatable metal cations to a concentration so low that the constraints of mass action begins to favor the intramolecular reaction of aromatization over chelations. Were this not the case, then metallochlorins should dominate geologic samples and free-base porphyrins should be absent. Such is not the case and free-base porphyrin generation is favored over metallochlorin formation. Further, given that metallo chlorins aromatize more easily than free-base chlorins (Eisner and Linstead, 1955), the existence of nickel mesopyropheophorbide-a in a shale (Prowse et gj., 1990) is indeed unique. The period of mid-diagenesis typically involves the generation of alkyl (decarboxylated, defunctionalized) free-base porphyrins. However, as we proposed on the basis of a much more limited data base (Baker and Louda, 1986b, cf. Blumer and Omenn), there appears to be a redox equilibrium established at this stage (Figure 111). Given as Table 39 are all of the porphyrin to 7,8-dihydro DPEP (cf. [XIII] or

PAGE 417

-2 H .. +2H 7,8-DIHYDRO-DPEP (=7-EilfYL-7-DESPROPIO-DOMPP-a) (see cmpds.(XII),(XIII)) -2H + Ni DPEP(XXXVIII) Ni:DPEP(XC] Figure 111. Suggested overall diagenetic trend for the aromatization and chelation reactions,incorporating the observed equilibrium reaction at the aromatization step. w 00 \.0

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Tab l e 39. o f to free-base porphyr1ns, as found 1n deep sea sediments. SUB-BOTTOM(1) f1) DEP'IB GEOLOG C ORGANic(1) in situ(1) 390 (2) SAMPLE(1) meters AGE CARBON r;-uc PH:H2 /7,8-DiH-PH: H 2 71-511 -605 535.5 B.-A. 5.28 40.3 6.1 (3) 511-625 554.0 BA/L.J. 4.29 41.0 6. 8 511-64 4 571.5 L.JUR. 5 .69 43.0 7.8 511-66 4 590.5 L.JUR. 6 .25 44. 4 6.2 51168-2 606. 5 L.JUR. 3.88 45. 6 4.8 511-703 627.0 L.JUR. 4 9 1 47.1 8.3 634678 5 70:0 QUAT. 1.9 8 9 (ALK) 38. 0 (ACID) 2.4 467-13-4 116.0 U.PLIO. 2.9 11.8 (ALK) 3.5 (ACID) 3.2 467-18 5 165.0 U .PLIO. 3 4 14.9 (ALK) 10. 7 (ACID) 1.5 467-256 233. 0 U.PLIO. 1.7 19.2 21.7 467-32-2 293.5 L.PLIO. 2.9 23.0 27.3 46736-2 340. 5 L.PLIO. 2.7 26.0 38.2 46741-4 382.0 L.PLIO. 3.0 28.6 81.0 467-48 2 445.5 U .MIOC. 4.6 32.6 28. 9 46758-3 542.0 U .MIOC. 4.4 38.6 113.0 46763-2 588. 0 U.MIOC. 5.2 41.5 53.0 467-74-1 691.0 U.MIOC. 2.9 48. 0 21.2 63-471 8-4 72.0 MIOC. 1.1 11.5 12.7 64-477 7-1 49.7 HOI.OC. 1.8 50.0 36. 0 64-479-13-1 108.6 PLEIST. 2 9 18.3 7.7 479-155 133 6 PLEIST 2 9 20. 7 4.6 47919-5 171.6 PLEIST. 2 7 24.3 5.1 47922-5 200.1 PLEIST. 2 6 27.1 9.9 47934-5 314 1 PLIOC. 3 0 38.0 37. 0 479-39-5 360.1 PLIOC. 3 5 42.4 63.7 479-431 393.7 PLIOC. 2.6 45.6 82.2 479-474 436. 1 PLIOC. 1.2 49.7 88.0 FOOTNOTES: (!)SAMPLE numbers given are in DSDP/IPOD format of LEG-SITE-CORE-SECTION. Sample,geologic age,depth of burial,organic carbon and in sit u temperatures are taken LEG et a l .,1983),LEG b1\Yeats,Haq et al., 1981),LEG 64(Cur r ay,Moore et al. 198 ) (2)Ratio of free-base porphyr insTPH:H2 ) t o 7,8-dihydro free-base (7,8-DiH-PH:H2 ) determined spectrophotometrically as given in 'RESULTS-I:". (ALK)= alkyl pigments,(ACID)= mono carboxy lic(propionic-?)acid (3)Data from Leg 71 samples reported previously(Baker et al.,1986b).

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391 7,8-dihydro-DPE [XII] ; DOMPP-a) ratios determined during the present work. It can be noted that for three samples from the San Miguel Gap (63-467-8-5, -13-4, -18-5: Table 39) free-base porphyrins acids have been isolated since our original study (Louda and Baker, 1981) of that site. The behavior of the free-base porphyrin carboxylic acids mimics the alkyl pigments in that a redox equilibrium between them and their precursor 7,8-dihydro analogs exists. That these pigments are congeneric is more than apparent from the mass spectral distribution of co isolated 7,8-dihydro DPEP-and DPEP-series porphyrins (Figure 99). The exact physicochemical controls on the position of equilibrium are unknown at present but the interaction of Eh, pH, sediment lithology, temperature, pressure and OM type appear as a reasonable estimate. Sediments of similar age, depth, lithologies and temperatures found at the Falkland Plateau site (71-511: Table 39) revealed an average ratio of DPEP-to-7,8-dihydro-DPEP of 7:1 (Baker and Louda, 1986b). Data from the Leg 63 San Miguel Gap sediments, which vary greatly in depth, age, OM and lithologies (cf. Louda and Baker, 1981) reveal a wide scatter, probably correlated in unknown ways to sediment and organic facies differences. Examination of the nearly continuously anoxic accumulation of diatomaceous oozes from the Guaymas Basin slope in the Gulf of California (64-479) reveals a trend which one might predict. That is, at the onset of aromatization (64-479-13-1 through 64-479-22-5) the ratio is low (5-10) and as time-temperature (viz. organic maturity) increases so does the extent of aromatization (porphyrin-to-7,8-dihydroporphyrin; ca. 40-90+: Table 39). To reiterate, the 'stages' of tetrapyrrole geochemistry overlap. Therefore, sometime near the middle to end of the aromatization phase,

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392 chelation of nickel begins (Figure 111). This issues in the stage of late-diagenesis. The period defined (Baker and Louda, 1983) as late-diagenesis encompasses, at least for bitumen/extractable tetrapyrroles, the chelation of nickel (II) cations. As yet, the chelation of vanadyl has been unobserved and the phenomenology of vanadyl porphyrin generation is covered in the next section ('catagenesis'). To date, we have been fortuitous enough to have studied only one sampl e suite which includes the complete profile of chelation. That is, only one suite (63-467: Louda and Baker, 1981) reveals the change from 0 to 100% nickel porphyrins, at the expense (100 to 0%) of free-base porphyrins. This profile, corrected for the epigenetic copper highly dealkylated etioporphyrins (Baker and Louda, 1983, 1984; Louda and Baker, 1981), is presented as Figure 112. Aside from this one complete chelation profile, a number of partial, due to sampling and/or sediment constraints, profiles have been investigated. Such profiles i nclude the Black Sea (Baker and Palmer, 1979; cf. Baker and Louda, 1983), the Guaymas Basin (Baker and Louda, 1982), the Falkland Plateau (Baker and Louda, 1986b) and the wildcat well drilled into the Monterey and Rincon formations of the Transverse ranges, onshore California, discussed below. A sample suite consisting of nine shales and a juvenile petroleum from between the fifth and sixth shale down-hol e was provided by Mobil Oil Research and Development. These samples are considered more fully during the following section. However, the uppermost two shales, from (#1) 4140-4500 and (#2) 5820-5940 feet, revealed the completion of nickel chelation. Further, in shale #2, vanadyl porphyrins were not

PAGE 421

393 0/oFREE-BASE PORPH Y RINS 100 50 0 0 4 5 10 0 50 100 0/o METALLOPORPHY R I NS Figure 112. Downhole plot for the chelation reacti on (Ni) as observed for DSDP/IPOD site 63 467 in the San Miguel Gap,California borderlands(Louda a n d Baker, 19 8 1 )

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394 detectable and this behavior, in true oil prone shale sequences, reinforces trends found for deep sea sediments still under their depositional environments. The mass spectral distributions of the free-base porphyrins and their precursor/equilibrium products, the 7,8-dihydro-DPEP series, from the uppermost (#1) shale at this site were given earlier (Figure 99). The nickel porphyrins isolated from shale #1 were found to have a distribution of DPEP-series porphyrins (C27 to C33: 10/35/80/97/100/92/6) nearly identical to the free-base porphyrins (C27 to C32: 5/18/51/69/100/72). The prejudice towards nickel (II) found for the formation of metalloporphyrins from free-base porphyrins in sedimentary bitumen is most likely controlled by sulfide (Barwise, 1990; Baker and Louda, 1986a; Jacobs and Emerson, 1982; Jacobs et gj. 1985; Kremling, 1983; Lewan, 1984; Lewan and Maynard, 1982). Jacobs and co-workers (1985) have shown that "Nickel is unique in it solubility behavior among the metals investigated. Processes controlling the solubility of nickel appear to be largely independent of the redox state ... Examinations of pH-Eh stability fields for the wide variety of metals in S-0-H systems (g.g. Cu-Fe-S-0-H, etc.) given in Garrels and Christ (1965) reveal that as and HS-are sequestered by Fe2+ and Cu2+, the Ni2\aq.> field enlarges to more negative Eh values. Therefore, given the above, it is not surprising that nickel (II) porphyrins dominate sedimentary bitumen The competition between Ni2+ and (V0)2+ is covered in the next section. The above has considered only nickel alkyl porphyrins, mainly of the DPEP-series. However, both functionalized and/or carboxylic acid Ni porphyrins also exist. Ni porphyrin acids, present as a limited

PAGE 423

395 pseudohomology (C28-C33), were isolated from a middle Miocene claystone off the coast of Baja California and previously described (63-471-34-2: Louda and Baker, 1981). Therefore, the participation of nickel deoxophylloerythrin(s) [LXXXVI] in chlorophyll geochemistry is shown. Previously, we reported decarboxylated nickel phylloerythrins [LXXVIII] in marine asediments (Baker and Louda, 1982; Louda and Baker, 1981) and based those tentative identifications on chromatographic behavior (i.g. decarboxylated aspect) and electronic spectra (a-band = 582 nm: Table 11, cmpd. [LXXVIII]). During the present continuation of those studies, additional isolates have been purified and mass spectral analyses made. Shown as Figure 113a is the electronic absorption spectrum of an isolate of nickel (II) phylloerythrin. React io n of this pigment with sodium borohydride yields a positive test for the presence of a conjugated ketone as the spectrum converts to that typical of a metallo-alkyl porphyrin (cf. Table 11). Certain amounts of decarbox ylated phylloerythrins can be shown to be present in a variety of freebase porphyrins, by the appearance of a band at about 595 nm (viz. Cu PE, [LXXX]: Table 11) following in vitro analytical chelation with copper. Work on the free-base phylloerythrins cont inues and will be published elsewhere. Returning to the nickel (II) phylloerythrin(s), the mass spectral distribution of one such isolate (64-479-39-4) is given as Figure 113b. During mass spectral analyses the best ion current, at 14 and 70 eV, was attained at a probe temperature of about 285-310C. This is a bit hotter than required for the purely alkyl nickel porphyrins (Louda and Baker, 1990) and could be expected on the basis of the presence of the carbonyl function. The mass spectrum obtained reveals the presence of C28 to C32 decarboxylated Ni

PAGE 424

09 ,....--------------., 08 0 7 06 W05 u z <( cO 0.4 0:: 0 lll m03 <( ( a) 586.5 ( b ) 0.2 480 500 520 mjz 540 560 0.1 28 29 30 31 32 CARBON NUMBER OOI I I I 1 350 450 550 650 WAVE LENGTH,nm Figure 113. Electronic absorption (a) and mass (b) spectra of decarboxylated nickel 'phylloerythrins' isolated from a Quaternary diatomaceou s ooze recovered in the Guaymas Basin,Gulf of California(Sample 64-479-37-5, Table 34). w ...0 "'

PAGE 425

397 phylloerythrins. Using the C32 pseudohomolog as an example, the mass at 546 m/z represents 58Ni(II) 9-oxo-DPEP (548 m/z 60Ni). That is, the decarboxylated (7-ethyl-7-despropio: ?) analog of Ni PE [LXXVIII]. Since the conjugated carbonyl ( =0, 16 Da) replaces two hydrogens (2H, 2Da) the net result is a mass of 14 Daltons, the same as the nominal mass of a methylene equivalency. Therefore, the carbon numbers for the decarboxylated nickel phylloerythrins are one less than given at each nominal mass in Table 19 for the Ni DPEP series. As far as can be told, this is the first definitive proof of the nickel phyl loerythrins, as decarboxylated pigments, in geologic samples. Late-diagenesis is taken as being completed when only nickel porphyrins exist or vanadyl porphyrins begin to 'appear' (cf. Baker and Louda, 1983, 1986a). Tetrapyrrole Catagenesis We previously classified tetrapyrrole catagenesis as encompassing the 'appearance' of vanadyl porphyrins and the maturation of both nickel and vanadyl porphyrins (Baker and Louda, 1983). The investiga tion of hundreds of deep sea sediments had revealed most clearly that vanadyl porphyrins, and presumably their precursors, are not part of bitumen until sufficient time-temperature stress has elicited their release from a bound or hidden state (see Baker and Louda, 1983, 1986ab; Baker et gj., 1977, 1978a, 1987; Louda and Baker, 1981, 1986). In the case of Miocene aged diatomaceous clays we found 'release' of vanadyl porphyrins to occur when in situ temperatures of 60-70C were attained (Louda and Baker, 1981). To date, the only direct formation of vanadyl porphyrins from free-base pigments in the bitumen, akin to

PAGE 426

398 nickel chelation, has been observed for the late Jurassic b lack shales recovered from the Falkland plateau (Baker and Louda, 1986b). In that case, namely a long-term low-temperature setting, vanadyl chelation was found at about 45-50C. It must be reiterated that this one case was for sediments aged on the order of 150, versus ca. 15 (Miocene, Leg 63: Louda and Baker, 1981), millions of years. A factor of 10 in time could certainly lead to diagenetic-catagenetic differences. Examination of shales and petroleum crudes, to be discussed below, during these studies lead to the compilation of an overal l maturational trend for the vanadyl porphyrins. This trend, which the author has published previously, is shown here as Figure 114. The mass spectral distributions are given here, somewhat prematurely, to serve as a reference for the remainder of this section. In general, Figure 114 traces the evolution of vanadyl porphyrins from their appearance as a DPEP-dominated array of limited carbon number spread, through sequential maturation including the addition of higher carbon numbers and a switch from OPEP (alt: 'CAP') to ETIO-dominance, to a point where the dealkylation of the remaining ETIO-compounds destroys all pigment. A sample suite from a wildcat well ("WCA") drilled into the Sisquoc, Monterey and Rincon Formations of the Transverse ranges of California (see g.g. Cram, 1971; Curiale et . 1985; Isaacs and Petersen, 1987; Orr, 1986) was provided by Mobil Oil Research and Development. The yields of bitumen (viz. extractable organic matter = EOM), nickel-and vanadyl-porphyrins from these 9 shales and a juvenile petroleum recovered from the microfractured region between shales 5 and 6 are given in Table 40. Shale number 1 was found to contain both free-base and 7,8-dihydro-porphyrins, the mass spectral distributions

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(-) d e I I I I I f t I \ \ : t .;. t :' : . .. I : r ... \ 1. : .. \ /:.: .. . \ I I h I I I I I I 40 (+) 24 28 CARBON NUMBER 399 I I I I . I I I I I I I 36 40 Figure 114. The evolution of vanadyl porphyrins in oil shales and petroleums of marine origin as traced with mass spectral histograms of DPEP(solid) and ETIO(dashed) series pigments(Baker and Louda,1986a).

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Table 40. Geochemical data for nine shales and a juvenile petroleum from a test well (Wildcat A= WCA) drilled in coastal California(!). SHALE AGE / (l) NUMBER FEET FORMATION % EOM(l) 1 4140 E.PLIOC. /-:0 .312 2 5820 L. MIOC. /SISQ 0 .325 3 6150 L.MIOC./SISQ. 0.186 4 6510 MIOC. /MONT. 1.433 5 6630 MIOC. /MONT. 0.872 OIL ---------(100.0) 6 7170 MIOC./MONT. 3.900 7 7350 E .MIOC. /RIN. 0. 700 8 7470 L.OLIG. /RIN. 0 .700 9 7980 EOCENE/---0 .288 FOOTNOTES: TETRAPYRROLE PIGMENT OONCENTRATION(2 ) ug wt. ug / g EOM MOLAR RATIO PH:H2 Ni:PHs VO:PHs Ni:PHs VO:PHs Ni:PH/VO:PH 2.96 0.41 nd trace 1.93 nd nd 2 .35 + nd 2 3 1 0.24 nd 5 .91 7.98 (nd) ------nd 7.52 97.81 nd 5.80 28.98 nd 2.44 8.39 nd 1.54 1.18 130.4 592.8 1,267.0 161.0 677. 8 127.4 192.7 830.5 348. 0 674.8 nd nd ++ 16.8 914. 3 939. 6 2,506.0 4,147.8 1,198.4 516. 9 100/0 100/0 >99/<1 91/9 43/57 12/88 7/93 17/83 23/77 57/43 (l)Samples,depth of burial to top of interval,geologic age/formation and weight percent extractable organic matter0%EOM) data supplied by W L.Orr and the Mobil Research and Development Corporation. Wildcat "A" test well is also described in Orr(1986). (2)Tetrapyrrole pigment yields calculate d using millimolar extinction coefficients of 6 .54, 34.8 2 and 26.14 for free-base(PH:H2), nickel(Ni:PHs) and vanadyl(VO:PHs) porphyrins,respectively (see Table 16). 0 0

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401 of which were given earlier (Figure 99). Including the occurrence of the free-base porphyrins in shale #1, this sequence reflects the end of late-diagenesis (shales 1-3) and the initiation of catagenesis (shales 3-9), as defined here and elsewhere (Baker and Louda, 1983) for tetrapyrrole pigments. The parallel with overall organic maturity (cf. Hunt, 1979; Tissot and Welte, 1978) is obvious in that a juvenile petroleum has been produced and is in the primary stages of migration (W. L. Orr, pers. commun.). Table 41 contains the mole percentage distribution of nickel and vanadyl porphyrins and selected mass spectral parameters obtained on these pigments. The lack of data on the nickel porpryrins for the WCA oil and shales #6-9 stems from a current inability to purify small amounts of these pigments from 'oil-window' bitumen (see Figure 23 and associated text). This sample suite serves quite well as a case study for the onset of tetrapyrrole catagenesis as it pertains to the release of vanadyl porphyrins from a bound and/or occluded state. Examination of yields taken on the basis of EOM reveals a tremendous release of vanadyl porphyrins (i.g. 17 to 4,200 pg/g-EOM: Table 40) during the onset of catagenesis. Further, it can be deduced from examination of the mass spectral distributions (Table 41) that mid-range carbon number (C28-C35) vanadyl porphyrins appear first (A.I. = 0.19, shale 5) with the addition of highly alkylated forms (A.I. values = 0.5 to 0.7, shales 6 to 8) later. This is graphically apparent with a display of the mass spectral histograms for the down-hole evolution of vanadyl porphyrins in the "WCA" well (Figure 115). The "WCA" oil is shown out of sequence in order to better visualize the shale pigment changes.

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Table 41. Mass spectrometric indices determined for the nickel and vanadyl geeporphyrins isolated from nine shales and a juvenile petroleum recovered from a test wel l ( 'Wildcat A ) in coastal California(!). SHALE(l) NICKEL PORPHYRINS VANADYL PORPHYRINS NUMBER % DPEP A. I. X % DPEP A.I. X 1 <100.0 0.01 508.0 2 80. 5 0.03 512.8 3 80.1 0 .02 515.1 4 62.3 0 .01 506.7 dna dna dna 5 74.4 0.04 511.2 84.0 0.19 530.0 (OIL) dna dna dna 84. 5 0.59 534.8 6 dna dna dna 91.3 0.50 527.6 7 dna dna dna 90.3 0.51 535. 2 8 dna dna dna 88.6 0. 72 537.9 9 dna dna dna 87.8 0.46+ 535. 0 FOOTNOTES: (1)Samples and sample descriptions given in Table 40. (2)Percentage DPEP(% DPEP),alkylation index(A.I.) and weighted average mas s(X) are de scribed in Table 23 and associated text. "dna = data not available. .f:--0 N

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S H ALE 5 SHALE 6 SHAL E 7 500 m/ z 600 700 26 28 30 32 34 36 38 40 42 44 CARBON SHALE B SHALE 9 O IL/TAR 500 m/ z 600 700 --,---,.---,--,--..--.---.--r--r-r-----,r---r---r--r-r--.--.--.-,---, 26 2 B 30 32 3 4 36 38 40 42 44 NUMBER Figure 115. Average d low voltage (4.5-6.0 eV) mass spectra of the vanadyl porphyrins isolated from a downhole sequence of shales and an inte r calated juvenile petrole u m in a wildcat tes t well, onshore California. Arrow indicates d epth s e qu ence. Asterisk indicates that t h e petroleum (FRL-7431: Table 44) was recovered in the microfractured zone between shales 5 and 6 "(Tables 40-41). 0 w

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404 Even though the depth of this sequence extends to about 2,500 meters sub-surface, the vitrinite reflectance maximizes only in the range of 0.3-0.4 R0 (W. L. Orr, pers. commn.). The generally accepted onset or 'window' of oil generation occurs at about R0 = 0.5-0.6 (Hunt, 1979; Tissot and Welte, 1978). However, as more data is analyzed, it becomes increasingly clear that the use of vitrinite reflectance, of tremendous .versatility in coal forming environments, with oil prone sediments and shales must be modified for different organic facies (Orr, 1986; Saxby, 1982). Pertinent to the present studies is the concept of more highly labile type II OM due to the presence of very high amounts of heteroatomic (NSO) linkages, most notably sulfur (Orr, 1986; Orr and White, 1990). Orr (1986) has suggested that type II (cf. Tissot and Welte, 1978) kerogens with S/C values above 0.04 be classified as type-liS. This type of OM forms the bulk of the oil-prone material in the Monterey and allied formations of California (Isaacs and Petersen, 1987; Milner et gj., 1977; Orr, 1986). Recent molecular studies on other classes of biomarkers have revealed a strong propensity for the formation of sulfur-OM linkages at very specific sites. That is, nearly all data shows the formation of bulk OM-S-biolipid linkages at the site(s) of original functional moieties in the biolipid (Kohnen et gj., 1991, 1992; Richnow et gj., 1992; Rowland et gj., 1993; ROllkOtter and Michaelis, 1990). Given the above, it is possible to speculate as to the possible formation of OM-S-tetrapyrrole linkages. Figure 116 contains an idealized situation in which a tetrapyrrole might be sequestered into the bulk OM (viz. humates, proto-kerogen, kerogen). Several direct linkages (g.g. ester, ether) are possible with original functionalities (i.g. propionic acid, reduced ketone,

PAGE 433

)/ ))./,; l / / ./ -::-/' -405 Figure 116. Postulated incorporation of a chlorophyll derivative into a &eopolymeric state,such as kerogen. Pheophorbide-a[VIaJ is outlined by a dotted line.CA = central atom(s)(H,H or VO),R=alkyl.Highly stylized representation of kerogen follows ideas in Behar and Vandenbrouche(1986).

PAGE 434

406 respectively). Further, these could be (1) $-substituted to yield this analogs, (2) directly S-linked, as at the origi nal v i nyl ( s) and (3) alkyl bonds could form (cf. Quirke et gj., 1980a) via reaction at vinyl moieties. As stated, such processes (Figure 115) are ent irely speculative. However, the sequestering of tetrapyrrole pigments into a non e xtractable state early in diagenesis and the involvement of sulfur explains two enigmatic facets of geochemistry. First, vanadyl porphyrins 'evolve' in a non-e xtractable state until the rmal stress sufficient to just begin petroleum generation has been endured Second, there is a well-defined yet hardly a direct mathematical, relationship between Sand V in sedimentary OM and petroleums (Aizenshtat et gj., 1979; Barwise, 1990; Baker, 1969; Beach and Shewmaker, 1957; Dunning et gl., 1960; Eglinton et gj., 1980; Erdman and Harju, 1963; Fish and Komlenic, 1984; Fish et gj. 1984; Hodgson et gj. 1967; Nissenbaum et gl.' 1979). Differences in source OM, the amount of heteroatoms (esp. S) and the evolution of tetrapyrrole pigments in oil shales can be found from the study of a limited suite of samples from the Bakken formation. Here (Figure 117, Tables 42-43) five samples from the Lower Bakken and E xshaw formations of the western Canadian basis (Leenheer, 1984) were analyzed for nickel and vanadyl porphyrins (Tabl e 42). The vitrinite reflectance values ranged from 0.48 to 1.1. In the case of the Bakken shales, a maturational profile for the vanadyl porphyrins was found to parallel that found for the "WCA" well descr ibed above. That is, first generation of mid-range carbon numbers of DPEPdominated pigments followed by the more highly alkylated types with increased maturity However, in the case of the Bakken shales equivalent stages of VO-

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NICKEL Sample 6362 I I I 6486 none detected I I II II I I I I I I I 24 28 32 36 CARBON Figure 117. Low vo ltage(4.5-nickel and vanadyl porphyrins aged oil shales of the Bakken ETIO(da shed) series envelopes VANADYL II ,,.,. I 0.97 .AI -....,, 24 28 32 36 40 44 NUMBER 407 6.0 eV)mass spectra of the isolated from Mississippian formation.DPEP(solid) and are delineated,as indicated.

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Table 42. Geochemical data including metalloporphyrin concentrations for Mississippian aged shales of the Bakken Formation. SAMPLE(l) PERCENT METALLOPRPHYRIN OONCENTRATIONS(3 ) DESIGNATION dry weight ug / mg/gFDM (Depth of R(l) C ( 1 ) EOM(2) b urial,feet) l) org Ni:Phs VO:PHs Ni:PHs VO:PHs 2650 0 .48 7.70 0 .73 7.8 113. 9 1.08 15.66 6362 0 .65 13.03 1.88 94.3 269. 9 5.01 1 4.33 6182 0. 77 13.46 1. 79 83. 2 249. 8 4 .65 13.96 6486 0 .97 1.35 0 .42 nd 1.3 nd 0.31 8318 1.1 1. 74 0.23 nd 0 2 nd 0 .09 --------------FOOTNOTES: (!)Samples suppl i ed by Mrs.M.J.Leenheer of Cities Services Company(Occidental Petroleum Inc.).Sample designations,mean vitrinite reflectance in oil(R) and weight percent orgnaic carbon(C ) taken from Leenheer(1984). 0 (2)Weight percent extracable oranic m2tter(EOM)determined gravimetrically after evaporation of extract at 45 C in vacuo(ca.26 in.Hg). (3)Yields if nickel porphyrins(Ni :PHs) and porphyrins(VO:PHs) calculated using millimolar extinction coefficients of 34.82 and 26.14,respectively(see Table 16). +--0 CX>

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Table 43. Mass spectral characterization of nickel and vanadyl porphyrins isolated from shales of the Bakken Formation. SAMPLE(!) MEAN(l) (2) DESIGNATION VITRINITE MASS SPECTRAL INDEX (Depth of REFLECfANCE NICKEL PORPHYRINS VANADYL PORPHYRINS burial,feet) (R ) % DPEP A I. X % DPEP A I. 0 2650 0.48 70.9 0 .06 507. 9 82.2 0 .1 5 6362 0 .65 67.0 0.12 500.2 65.3 0 .43 6182 0. 77 62. 2 0.18 506.8 72. 8 0.46 6486 0 .97 ----------3.5 0 .1 7 FOO'INarES : x 530.1 527. 3 530.5 506. 6 (1) Samples provided by Mrs.M.J.Leenheer Samples are described in Leenheer(1984) (2) Mass spectral indices are described in detail in text(see Table 23). .J:' 0 "'

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410 porphyrin maturation (viz. % DPEP, A.I. values: cf. Tables 41 and 43) are found at R0 values of 0.3 to 0.4 higher than found for the Monterey type II-S (Orr, 1986) sources. In the present case, the Bakken formation, also of marine anoxic deposition, is characterized as type II OM with low sulfur (0.1-1.0%) and high exinite contents (Price et gl., 1984; Thode, 1981). As such, the maturation of OM, with respect to the onset of intense oil generation, in these ancient rocks is somewhat suppressed as compared to the World means for type-II OM (cf. Saxby, 1982; Tissot and Welte, 1978). Barwise and Park (1983) suggested the direct use of percent DPEP as a proxy for %R0 Figure 117 is a comparison of the %DPEP versus %R0 plot which they published for various North Sea sediments and Kimeridge oil shales and the same type of plot generated for the present Bakken shale data. In the case of the Kimeridge shales (Figure 118a) and allied sediments, 50% DPEP values were attained at a %R0 of about 0.5 and a deadline for the DPEP series can be estimated at about 0.7. Similar data interpretation for the Bakken formation (Figure 118b) reveals 50% DPEP occurring at about 0.85 %R0 with the deadline pushed to over 1.0% R0 The deflection in the trend line for the Bakken shales to over 1.0% R0 was added to allow for the presence of vanadyl porphyrins, though at trace levels, in a shale at %R0 = 1.1 (Table 42). The relationship between %5 and vanadyl porphyrin enrichment, as well as an investigation intb the amount of petroleum metals contained in the nickel and vanadyl pigments, has been studied during the analyses of 27 petroleum crudes. These samples, arbitrarily arranged in order of increasing 0API gravity, are listed in Table 44. These data include 8 confamial oils from the Paleozoic reservoirs of the Big

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411 (a) 100 (b)100 8 0 80 60 60 0 a_ w a_ 0 40 -/-40 20 20 v o a Ni 0 0 0 2 0.4 0.6 08 1.0 0.2 0.4 0.6 0.8 1 0 / a R. "loR. Figure 118. Plots of percent DPEP versus vitrinite reflectance(R ) a) Kimmeridge shales and allied sediments,as redrawn Barwise and Park(1983), b)Bakken shales from the Western Canadian Basin(See Table 42).

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Table 44. Bulk petroleum parameters, elemental and porphyrinic metals and metal ratios for petroleum crudes from the Monterey Formation and the Big Horn Basin. (1) (1) (1) (1) YIELD(ug/g-oil) 2 ) CONCENTRATION AS METAL(ug/g-oil) ELEMEI'H'AI(I)--PORPINIUNIC{3) PERCENT(4) Ni / v (S) PORPHYRINIC FRL-No. FIELD(NAME) 0API VO:PHs Ni:PHs V Ni V/(V+Ni) V Ni V/(V+Ni) V Ni 'IUI'AL: PORPH. 7431 8061 7768 7071 7042 7050 7044 4907 8067 7072 7377 7073 8062 3735 4936 7771 8515 7077 3726 3729 4938 3915 8519 3922 3919 3145 3464 FOCYIN
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413 Horn Basin ('BHB'), a biodegraded terrigenoclastic source oil (FRL-4907) and 18 petroleum crudes sourced from the Miocene Monterey formation and allied rocks (see Orr, 1986). Certain subsets of oils exist in this compilation, in that oils from different depths within the same well/field are present (g.g. 7071, 7072, 7073, 7077: South Elwood field; etc.). The first conclusion which can be drawn from the data in Table 44 is that not all, and in fact only a moderate amount, of the nic kel and vanadium in petroleum crudes is contained in identifiable porphyrin complexes. Examination of the "Percent Porphyrinic" columns reveals that porphyrins contain only 14.2 (R = 5.3-27.5) and 9.2 (R = 1.0-17.8) percent of the total vanadium and nickel, respectively, contained in these petroleum crudes. Previously, much effort was expended on destroying the metalloporphyrins in petroleum (Erdman et . 1956a-b, 1957, 1959) due to beliefs that all of the metal was in porphyrins (Skinner, 1952). However, closer inspection revealed that only 3-10% was accountable for with intact identifiable metalloporphyrins (Dunning et . 1960). Due primarily to past practices in petroporphyrin analyses, detailed in "Chapters 1 and 2" metal contents and, therefore, assumed metalloporphyrins (viz. Ni PHs/VO PHs) ratios were reported only from elemental (AA) analyses. This not only destroyed data on the partitioning of Ni and V in bitumen but drastically altered porphyrin distributions by combining the structures originally with each metal. Since the report of Dunning et (1960) very little attention was given this problem until the mid-1980's (cf. Baker and Louda, 1986a; Filby and Branthaver, 1987). Certain inroads into describing the non porphyrin complexes of both vanadium and nickel (see Fish and Komlenic,

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1984; Fish et g]., 1984, 1987). The data given here on the percent porphy r i n i c V and Ni i n petro leum crudes is the f irst in that format since the time of Dunning and co-worker's {1960) suggestion. 414 The relationship between high sulfur contents and vanadium enrichment in sedimentary OM was introduced earlier in this section. The landmark application of known Eh-pH stability fields (g g Garrels and Christ, 1965) to the study of vanadium and nickel proportionality in sediments, shales and oils by Lewan {1984; Lewan and Maynard, 1982) went far towards providing a reasonable e x planat ion fqr V versus Ni enrichments and also lead to paleoenvironmental interpretations based on V/{V+Ni) ratios (g.g. Barwise, 1990; Curiale, 1987; Quirke, 1987; Sundararaman and Bareham, 1991). In general, current thought revolves around the fact that as sulfate reduction reaches intense levels, the amount of HS-/52 -becomes so high as to hinder the availability of Ni2+ and V4+, as {V02+), becomes the dominant metal in c orporated i nto porphyrin and other complex es Lewan {1984) has defined three depositional regimes (I-III) which would lead to OM with V/(V+Ni) values of < 0.1 and either 0.1=0.9 or > 0.5 when associated with low or high S OM. The current study i ncludes the analyses of many high S p etroleums and shales and serves well to e x emplify these relationships. G iven as F i gure 119 in a plot of weight percent sulfur versus the individual yields of nickel and vanadyl porphyrins. As the relationships found in this type of plot is bound to vary with sampling bias/data base and a strict mathematical relation is not forwarded here, no attempts at precise curve fitting (least squares, regression, etc.) were made. Rather, the Ni/S and V/S lines were added solely to point out the overall trends in a most general manner. However, it i s quite clear

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SULFUR I 0/o (wt.) 0 1 2 3 4 5 6 7 8 9 _J 100 200 0 500 CJ) --z 0:: >-I Cl.. 0:: 0 Cl.. 01,000 _j _J <{ 1-w 2: NiPH:: VO PH=!:::. 415 Figure 11 9 Plot of nickel(solid circles) and vanadyl(open triangles) porphyrin concentrations versus weight percent sulfur for twenty-five petroleum crudes(See Table 46).

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416 that as S contents increase, vanadyl porphyrin enrichment occurs. Higher S signals increased paleo sulfate reduction which, in turn, required large amounts of metabolizable autochthonous) organic matter and created strong anoxia. However. it is felt that a more direct S-porphyrin relationship exists and that this may include linkage to the bulk Om (Figure 116). The nickel porphyrins, which we have shown to evolve in the bitumen, show little if any relationship to 5-contents. Rather, the slight increase indicated in Figure 119 may be more directly tied to a higher level of organic preservation, which would include chlorophyll derivatives, in the pre-and syn-depositional environment of the eventual source rock Examination of the Ni and V contents in both the total oil and the porphyrinic sub-set, and the ratios determined, as a sour c e correlation tool (Barwise, 1990; Branthaver and Filby, 1987; Cusiale, 1987) allows us to consider the organic source and paleoenvironment According to the classifications of Barwise (1990) and Curiale (1987), who use the ratios N i/V and V/(V+Ni), respectively, the vast major ity of the oils reported in Table 44 are indeed from anoxic marine sources with high sulfur contents All of these conclus i ons were inter alia known. However, application of these conclusions to an oil or oils of unknown source may prove beneficial in the future. As far as the author can tell, this is the first report which studied both the total-elemental and porphyrinic subsets of Ni and V in petroleum. In accord with the percentage of total Ni and V found in porphyrin complexes, 9.2 and 14.2 % respectively, slightly lower Ni/V (higher V/(V+Ni)) values were determined for the porphyrinic subset of the total metals This concept will need to be remembered when c omparing ratios.

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417 All of the metalloporphyrins isolated from the petroleum crudes given in Table 44 were subjected to mass spectral analyses. Publication of the Ni-and VO-porphyrins in a more indepth manner is planned and will be in cluded in a larger overall biomarker study of the Monterey. However, the mass spectral descriptive indices given for the nickel (Table 45) and vanadyl (Table 46) porphyrins are presented as a prime data base for the investigation of the catagenetic maturation of these pigments. In order to show the underlying catagenetic evolution of these pigments, two types of plots were devised. First, a plot of vanadyl porphyrin concentration versus the percent DPEP (alt. CAP) of each isolate was made for all of the high-S marine sourced samples studied (Figure 120). This includes the "WCA" shale sequence (Tables 40, 41), numerically ordered by depth, and all of the Monterey and Big Horn Basin oils (Tables 44-46). Interpreting Figure 120 it is suggested that vanadyl porphyrins of a high % DPEP are some of the first released compounds during the onset of petroleum generation. That is, tracing the concentration of VO porphyrins on a basis reveals that, in the shale bitumen (= EOM = "oil"), the amounts of these pigments undergo dramatic increases (shales 5-7) as catagenesis begins. Next (shales 7-9 and oils) continuing petroleum generation dilute the concentration of metalloporphyrins. Maturation, including the eventual entrance into metagenesis, next leads to a switch from DPEP-to ETIO-series dominance. The data from the Bakken shale study (Tables 42-43) were left off the plot in Figure 120 on purpose That is, even though the trend lines would be unchanged, the extremely high yields (i.g. 14,000-16,000 T a ble 42) would have lead to a

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Table 45. Concentration and mass spectrometric charac-terization of nickel porphyrins i n twenty-six petroleum samples. SAMPLE(!) OAPI(l) YIELD(2 ) MASS SPECTRAL INDICES(3 ) (FRL#) GRAVITY ug/g-oil % DPEP A I. X 7431 4.0 127 dna dna dna 8061 9.6 118 81.2 0.16 514.0 7768 9.6 29 dna dna dna 7071 9.7 48 67.8 0.58 528.2 7042 11.8 81 62.7 0 .35 516.8 7050 11.9 57 59. 2 0.15 513. 5 7044 1 4 4 61 63. 6 0 .07 496.3 4907 14.7 101 72.2 0 .12 512.5 8067 16.7 100 57.7 0 .10 508. 2 7072 16.8 32 54. 6 0 .62 520.3 7377 16.8 118 72.7 0.09 511.0 7073 17. 1 40 76.8 0.89 503.1 8062 17.5 127 73.0 0.23 516. 8 3735 18.5 5 dna dna dna 4936 20.5 66 58.1 0 .10 511.1 7771 20.8 82 56.1 0 .43 520. 2 8515 21.4 91 dna dna dna 7077 21.5 32 77. 7 0.40 524. 6 3726 21.7 nd 3729 23.1 10 dna dna dna 4938 23.7 56 62.1 0.10 508.2 3915 26. 6 nd 8519 31.7 43 dna dna dna 3922 31.9 nd 391 9 33.2 nd 3145 34.0 nd FOO'INOTES: (!)Samp les and 0API gravity values supplied by W.L. Orr of Mobil Research and Development(See Table 44). (2)Quantitation made a millimolar extinction coefficient of 34. 82(See Table 16 ) nd" =none detected. 418 DPEP),alkylation index(A.I ) and the weighted average mass(X)were determined from the averaged and isotopically corrected normalized low voltage(6-8eV) mass spectrum .Calculation of these indices is described in Table 23 "dna"=data not available,impure isolate et cetera.

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419 Table 46 Concentration and mass spectrometric charac-terization of vanadyl porphyrins in twenty-six petroleum samples. SAMPLE(l) OAPI (1) YIELD(2 ) MASS SPECTRAL INDICES(3 ) (FRL#) GRAVI'IY ug7g-oii % DPEP A I. x 7431 4 0 940 84.5 0.59 534.8 8061 9.6 986 84.5 0 .32 525.5 7768 9 6 230 42. 5 1.00 538.6 7071 9.7 487 76.3 0.54 537.7 7042 11.8 340 83. 7 0.48 531.5 7050 11.9 243 67. 6 0.13 516. 4 7044 14.4 282 74. 8 532.2 4907 14.7 63 46.5 0.11 515. 4 8067 16.7 1,010 81.5 0.50 530.1 7072 16.8 297 75. 8 1.02 539.9 7377 16.8 959 78. 0 0.35 526. 4 7073 17.1 365 70.8 0.86 537.0 8062 17.5 1,494 83. 0 0.54 530.7 3735 18.5 140 44.4 0 .94 540. 1 4936 20.5 126 70. 2 0 .34 526. 2 7771 20.8 207 66.2 0.69 534.8 8515 21.4 605 76. 3 1.00 542.2 7077 21.5 248 66. 7 0.65 534.2 3726 21.7 so 13.8 0 .36 520.2 3729 23.1 200 51. 2 0.66 537.5 4938 23.7 258 69 .7 0 .25 526.4 3915 26. 6 20 7.4 0 .11 500. 6 8519 31.7 156 75. 7 0.94 541.3 3922 31.9 13 4 8 0.12 505.6 3919 33.2 10 44.4 1.60 555.9 3145 34.0 21 7.4 0 .02 508.4 FOOTNOTES: (1)Samples and 0API gravity values supplied b y W .L. Orr of Mobil Research and Development(See Table 44). (2)Quantitation made using a millimolar extinction coefficient of 26.14(See Table 16). (3)Perc entage DPEP(% DPEP),alkylation index(A.I.) and the weighted average mass(X) were determined from the averaged and isotopically corrected normalized low voltage(4 5-6 eV)mass spectrum. Calculation of these indices is described in Table 23.

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420 0/oOPEP 0 20 40 60 80 100 0 0 --. --g 1JOOO MATIJRATION --. <( _j 0 u z 0 c.J ........_, ..... V) 2 2JOOO E-t5 8 3 -........_, 0 ..... Q 1 et: 0 :::0:: w w I m Ol 2: --I 3JOOO z CL .. w 0 _j > 4: Ol I (j) --7 Figure 120. Cross plot of vanadyl porphyrin concentration and percent DPEP series for a series of maturing oil shales and twenty-three petroleum crudes(See Tables 40,41,42,43). N umbers indicate the shales fro m a wildcat well("WCA")as given in table 40.

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. visual minimization of % DPEP changes by forced alteration of the plot's aspect ratio. 421 Another method for the investigation of vanadyl porphyrin catagen esis and, by inference, its host's thermal maturation history is to cross plot two mass spectral parameters for each isolate. In this case (Figure 121), a plot of %-DPEP (alt. %-CAP) versus the alkylation inde x (A. I .), allows a closer e x amination of early to mid catagenetic changes. The vanadyl porphyr ins which f irst appear during the onset of catagenesis are of relatively high % DPEP and low A.I. As more highly alkylated pigments are released from a bound state, the A.I. value increases with little or no change in % DPEP. During later catagenesis dealkylation ensues, decreasing the a.I. value, and the switch from DPEP to ETIO dominance occ urs. This plot,% DPEP vs. A.I., is suggested as a potentially important means by which to a s sess the l evel of maturity for petroleum and shales in which vanadyl porphyrins contain 7 0 -95% DPEP. That is, the M S inde x % DPEP (alt. D/E or CAP/ETIO) is a valuable, widespread and accepted method for the assess ment of petroleum and shale maturat ion (see Baker and Louda, 1983, 1986a-b; Baker et gl., 1967, 1987; Barwise, 1982; Barwis e and Park, 1983; Burkova et gl., Didyk et g]., 197 5a-b; Louda and Baker, 1981, 1986; Morandi and Jensen, 1966; Sundara raman et gl. 1988 ; Van Berkel et gl., 1989). However, it is only one dimensional. The introduction of a second dimension (vi z A.I.) allows greater latitude in the application of % DPEP maturational assessments. Consider, for e x ample, that the major ity of the vanadyl porphyrins plotted in Figure 121 are of about 70-90% DPEP, with a c onsiderable number falling in the 80-90 % r ealm. The plot of % DPEP versus A.I. allows one to d i s c ern that one

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PERCENT 'DPEP' 0 20 40 60 80 10 0 X w 0 z z 0 0 2 0.4 0 6 I-<( 0 8 _j >-_j <( 1 0 1. 2 422 Figure 121 P lot of percent DPEP versus the a lkylation i ndex for vanadyl porphyrins isolated from shales(solid circles) and petrol eum crudes(open circles).Stars indicate samples treated by hydrous pyr o lysis(350/2d. )

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423 sample with 85% OPEP is a bit more mature than another with a similar %0PEP value due to a higher A.I. value (Figure 121: Tables 41, 43, 45). Attempts to perform usable plots of % OPEP versus A.I. for nickel porphyrins have revealed little, since the extent of a l kylation is limited for these pigments. The latter stages of catagenesis encompass the switch from OPEPto ETIO-series dominance and dealkylation of both series (Baker and Louda, 1983, 1986a; Louda and Baker, 1986). This switch in series dominance is easily visualized upon inspection of maturation profiles using mass spectral data. In Figure 114 this switch occurred between oils d and e. Inspection of mass spectral inoices in tabular (Tables 41, 43, 45, 46) or plotted (Figures 118, 120-121) forms also affords rapid identification of this 'switch' by finding the 50% OPEP (0/E = 1.00) point. Since the beginnings of 'modern' tetrapyrrole geochemistry, defined here as coinciding with 'routine' methodologies for porphyrin mass spectral analyses (cf. Baker, 1966; Baker et gl., 1967; Thomas and Blumer, 1964), a variety of potential explanations for var iant OPEP-toETIO ratios and trends in same have appeared. Baker et gl. (1967) suggested that a high or low 0/E value indicated a marine or non-marine source, respectively. Baker (1969) reviewed the situation and offered early oxidative opening of the isocyclic ring as a method by which original ('immature') 0-to-E ratios could be set (cf. Corwin, 1960) and thermal/thermocatalytic cleavage as a route to decrease that value (cf. Morandi and Jensen, 1966) during subsequent maturation. It became increasingly clear that a rather direct relationship between increasing organic maturation and decreasing OPEP/ETIO values (alt. % OPEP)

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424 existed. The concept of a thermal/thermocatalytic 'crossover' grew and was investigated (Baker and Palmer, 1978; Burkova et . 1980b; Didyk et . 1975a-b). More recently, the ideas that metallo-DPEP-type pigments are destroyed at a faster rate than the (pre-existing) ETIOseries (Barwise, 1987; Barwise and Park, 1983) and that metallo-ETIOs are released from kerogen in greater abundance, relative to the DPEPtypes, and "dilute" the DPEP-series (Sundararaman et . 1988) have appeared. Therefore, not only the ultimate source of the etioporphyrins in sediments but the methods by which they become the dominant tetrapyrrole pigments in more mature bitumen, especially petroleum, requires additional study. In an initial attempt to study the maturation of metallopor phyrins, primarily the crossover fro m DPEP-to-ETIO dominance and aspects of porphyrin-kerogen interactions, a small series of hydrous pyrolysis experiments were performed. Hydrous pyrolysis (cf. Comet et . 1986; Eglinton et . 1986; Engel et . 1986; Haering, 1984; Lewan et . 1979; Rowland et 1986; Schiefelbein, 1983) has been confirmed as a superior method for the modeling of thermal organic maturation when compared to anhydrous pyrolysis, which tends to generate an inordinate amount of artifacts (see Almon and Johns, 1977; Comet et 1986; Jurg and Eisma, 1977; Rubi nstein and Straus z 1979; Tissot and Vandelbrouche, 1983). Certain related studies do e xist in the literature, but these usually involved 'anhydrous' pyrolysis One of the first thermal maturity studies was the heating vanadyl etioporphyrin-I (C32E, 543 amu) at 420C by Yen and co-workers (1969) Recalculation of those data during the present work show the products to be C21 to C29 ETIO-series with a maximum at C25 and a weighted

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425 average mass of 450.5 m/z. Thus, thermal dealkylation altered the average carbon number of this VO-ETIO from 32.00 to 25.39. Similar thermal maturation studies revealed the decarboxylation and dealkylation of Cu (II) mesoporphyrin-IX (Casagrande and Hodgson, 1971, 1974) and the dealkylation with minor alkylation ('transalkylation') of vooctaethylporphyrin (Bonnett et ] . 1972, 1978) or VO-etioporphyrin-I (Eglinton, 1972). Perhaps the most classic study along these lines was that of Didyk and associates (1975a-b) in which the VO-porphyrin rich bitumen from the Cretaceous La Luna (Mara) source rock (Venezuela) was heated (0-385 h. at 210C) on bentonite. They reported pigment destruction (i.g. lowered yield), dealkylation and a dramatic lowering of the DPEP-to-ETIO ratio. A similar experiment but with synthetic VODPEP on an oil-free sandstone by Burkova and co-workers (1980) lead to about 95% total destruction and, significantly enough, about 3-5 % conversion to VO-etioporphyrins occurred. Vanadyl etioporphyrin treated similarly (10 hrs., 220C) was found to survive almost completely intact (Burkova et ] . 1980). This study strongly supports the suggestions of Barwise (1987) that a faster rate of DPEP distraction, relative to ETIO, is at the root of lowered DPEP-to-ETIO ratios paralleling overall organic maturity. In the first thermal study performed here, the nickel porphyrins in Green River oil shale obtained either by soxhlet extraction or supercritical water-methanol treatment were compared (cf. McKay, 1984; McKay and Chong, 1983; McKay et ] . 1983). The native bitumen of a sample of Green River oil shale (Anvil Points, Colorado: U.S. D.O.E. sample KRG2351; 37% OM) was ground and extracted for 48 hours with a benzene-methanol azeotrope. The e xtract and porphyrin yield data are

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426 given in Table 47. Three classes of nickel porphyrins could be identified during chromatographic analysis These were Ni alkyl porphyrins, Ni-porphyrin acids akin to Ni-DPE [LXXXVI] and Ni-phylloerythrins (Ni-PE [LXXVIII]). The nickel alkyl porphyrins were analyzed via MS and were found to consist solely of C28 to C33 Ni DPEP-series pigments, maximizing at C31 (Figure 122). The second sample was the experiment and consisted of the organic extract of an identical shale aliquot which had been treated with water and methanol under supercritical conditions. In this case, shale, water and methanol (1:3:3, w/w/w) were heated at 375C for 1 hour with a resulting pressure of 27.6 M Pa (4057 psi) being generated (J. F. McKay, pers. commun.). Subsequent analyses (Table 47) revealed a 7.3x increase in EOM and about 3.3x as many Ni porphyrins per gram of shale as via soxhlet extraction. The concentration of Ni-porphyrins in the bitumen increased only slightly (1.2 x ) with the overall increase in total yield being primarily linked to bitumen release. Aside from the qualitative aspects, the most pertinent changes occurred in pigment quality. Following supercritical treatment all pigment was present only as nickel alkyl porphyrins (Table 47) of the DPEP-and ETIO-series. Therefore, this thermal treatment lead to decarboxylation and loss of the ketone moiety (NiPE) as definite reactions. Further, the ETIOseries is now present and in a significant amount. However, as the yield of Ni-DPEP series porphyrins actually increased (8.3-14.4 NiD/g-shale), little can be concluded as to the source of the ETIOseries. That is, in this case, conversion of Ni-DPEP to Ni-ETIOporphyrins, or the release of pre-e xisting Ni-ETIO porphyrins, or both could have taken place. Though inconclusive as to the DPEP-to-ETIO

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427 Table 47. Comparison of extractable organic matter and nickel porphyrins derived from Anvil Points Green River oil shale by exhaustive soxhlet extraction and by treatment with supercritical methanol-water(!). PARAMETER TOTAL ORGANIC MATTER(TDM) EXTRACTABLE OM (EOM) [ EOM I 1DM ] xlOO NICKEL PORPHYRINS YIELD: ug/g-shale(2 ) YIELD: ug/g-EOM CLASS (molar percentages)(3 ) SOXHLET(l) EXTRACTION 37.3wt.% 2.7wt. % 7.2% 8 2 114.6 Ni alkyl porphyrins 68.8% Ni OPE-type (cf.[LXXXVI]) 12.3% SUPERCRITICAL(l) METHANOL-WATER 37 .5wt. % 19.5wt. % 52.3% 26.9 (3 3x) 137.9 (1. 2x) 100.0% nd nd Ni PEtype (cf.[LXXVIII]) 18.9% MASS SPECTRAL ANALYSIS OF Ni ALKYL PORPHYRINS(4 ) % DPEP A I. x FOOTNOTES: < 100.0 0 .015 517.1 53.7 0.052 501.8 (!)Samples of the Anvil Points Green River oil shale(Fischer Assay= 66 gal./ton;37.3 weight percent total organic matter)were supplied by J.F.McKay,as were the soxhlet and supercritical extracts. Soxhlet extraction was with benzene/methanol(60:40,v/v) for 24 hours.Supercritical treatment(cf.McKay gl.,1983) was with methanol/water(l;l,v/v) at 375C for 1 hour and an end pressure of 27. 6 MPa. (2)Yield of nickel porphyrin s calculated by summing all individual classes ,using extinction coefficients given in Table (3)Mass spectral indices were calculated as given in Table 2 3 from the averaged and isotopically corrected low-voltage(6-8eV)mass spectrum

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(a) 24 CARBON 32 NUMBER 428 Figure 122. Mass spectra of the nickel porphyrins isolated from Anvil Points Green River oil shale by (a) soxhlet or (b) treatment with supercritical methanol-water(See Table 47).Solid=DPEP series,dashed = ETIO series.

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.dominance change, aside from being related to thermal stress, this experiment did prove two things. First, thermal simulation studies with geologic metalloporphyrins can be run in a qualitative manner. Second, significant amounts of metalloporphyrins which are not extracted by 'routine' methods are present in sedimentary rocks. 429 The next thermal experiment involved the hydrous pyrolysis of deep sea sediment. In this case, an Upper Miocene calcareous claystone from 588.0 meters sub-bottom in the San Miguel Gap, California Borderlands (63-467-63-2: see Yeats, Haq et gl., 1981) was exhaustively (re-) extracted and submitted for hydrous pyrolysis at 350C over 48 hours (see Winterset gl., 1983). Previously, we had determined that the bitumen from this sample (63-467-63-2) contained only Ni-porphyrins (0/E = 3.37; 77% 0: Louda and Baker, 1981). The main thrust of this experiment was to artificially mature a sediment to the point of vanadyl porphyrin release (Table 48). That is, at site 467 in the San Miguel Gap, sediments buried about 450 meters deeper (+ 28C in situ) were found to contain VO-porphyrins (63-467-110-3), while all of those above did not (Louda and baker, 1981). Figure 123 is a comparison of the mass spectral distributions of the metalloporphyrins in the native bitumen (a) with those (b-e) found in the pyrolysate. First, vanadyl porphyrins were released and were therefore present in the bitumen-free rock, as were 'additional' nickel pigments. Second, both the Ni-and VO-porphyrins were found to be in low abundance, severely dealkylated and ETIO-series dominated. Though we shall return to this point, it is clear that thermal maturation as studied with the 'industry standard' parameters (350C, 2d: Winterset gl., 1983) is too severe to investigate tetrapyrrole catagenesis.

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Table 48. Yield and mass spectrometric data for the nickel and vanadyl porphyrins isolated from marine sediments and shales following hydrous pyrolysis. YIEL0(3 ) MASS SPECTROMETRIC CHARACTERIZATION(5 ) ug/g-sed.tdr.y wt. (4) VO:PHs NICKEL PORPHYRINS VANADYL PORPHYRINS SAMPLE(1 ) STAGE(2 ) -Ni:PHs VO:PHs Ni:PHS % DPEP A I. X % DPEP A I. X 63-467-63 -1 NATIVE 6.6 nd ---77.1 0 000 494. 4 63-46763-1 350/2d + ++ 0.54 < 1 0 .000 452.6 5 5 0.000 461.4 BAKKEN-6362 NATIVE 94.3 269.9 2.80 67.0 0.120 500.2 65.3 0.430 527.3 BAKKEN-6362 SOXHLET 0.6 7.6 13.03 74.4 0.110 497.3 75.7 0 .700 534. 5 BAKKEN-6362 300/3d 113.0 224. 0 1.95 67.1 0.066 439.3 67.2 0.067 510.6 BAKKEN-6362 350/2d 9.0 26.0 2.89 12.1 0.000 471.1 2.6 0.003 467.7 FOO'INOTES : (1)Samples are described in Tables 34 and 42 for the DSDP/IPOD sediment and the Bakken formation shale, respectively. (2) STAGE refers to treatment prior t o extraction of pigments.NATIVE= as collected,SOXHLET= additional extraction of ball-mill extracted sample.350/2d and 300/3d = temperature and time of exposure for hydr ous pyrolysis treatment of pre-extracted = 'new' bitumen).Hydrous pyrolysis through courtesy of Dr.Z.Sofer at Cities Service(Oxidental). (3)Calculated as given in Chapter 3 using millimolar extinction coefficients of 34.82 and 26.14 for nickel and vanadyl porphyrins,respectively. (4) Molar ratio. (5)Mass spectrometric analysis described in Chapter 3 and indices defined in Tabl e 23 +:-w 0

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a) b) I 24 c) 28 NICKEL 'native' NICKEL pyrolysate 32 VANADYL pyrolysate 20 24 28 32 36 40 CARBON NUMBER Figure 123. Mass spectral distributions of metallo?orph yrins isolated from DSDP/IPOD sample 63 -467-632 a)nickel porphyrins in native bitumen, b) nickel porphyrins in pyrolysate, and c) vanadyl porphyrins in pyrolystate.Hydrous pyrolysis performed on pre-extracted sediment at 350C for 48 hours.Solid = DPEP series,dashed = ETIO series. 431

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432 The last sample to be subjected to artificial thermal maturation trials was one of the Mississippian oil shales of the Bakken formation reported earlier in text (Tables 42-43, Figures 117-118). In this case, Bakken shale 6362, recovered with a R0 = 0.65 % was extracted, the nickel and vanadyl porphyrins separated and analyzed via mass spectrometry. The details of the MS results (Table 43) and the posi tioning of this sample in the evolution of the Bakken pigments (Figure 117) were given earlier as indicated. Following the normal exhaustive extraction of Bakken shale 6362 with ball-mill methods (see "Methods"), the sediment was re-extracted with soxhlet technique using a benzene methanol azeotrope for 304 days with 6 solvent changes. The last solvent change failed to extract any color or materials which fluor esced under ultra violet light. This re-extraction yielded an additional 2.7% of bitumen, considering both the original and soxhlet EOM to equal 100%. In both the Ni-and the VO-porphyrin arrays this reextraction was somewhat enriched in the DPEP-series (Table 48), but in other respects mimicked the first extract. Hydrous pyrolysis at 300C for three days resulted in the extraction/release of nickel and vanadyl porphyrins which mimicked the original extract, both in quantity and quality (Table 48, Figures 124-125). The only significant difference between the pigments in the pyrolysate and those in the original extract (viz. bitumen) is the absence of the higher alkyl members (>C34) in the vanadyl array of the former. To reiterate, hydrous pyrolysis was performed on extracted shale and the native bitumen, by standard usage, was absent. Therefore, these are additional pigments. Given the above, the total known metalloporphyrin concentration in Bakken shale is phenomenal. That is, on a shale weight basis, the

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BAKKE N-6362 (R. = 0.65) NATIVE BITUMEN (9 7 3 /o) SOXHLET BITUMEN Pre-HP ( 2 7"/o) HP 3ooc-3d HP 3soc 2d I I 24 26 / 28 CARBON \ 30 32 34 NUMBER Ni-PORPHYRINS 36 67.0"/oD 94 3,uQ!g-sed 5.01 mg/g-EOM 31.1!-Jg E / g-sed 74.4 Of. 0 0.6 0 .15 IJQE / g -sed 67. 1"/oD 1 1 3 0J.Jg/g-sed 5.97ma/ a EOM 37. 2 J.JQE/g-sed 12.1 "to D 9.0 JJg/g-sed 79).JgE/g-sed Figure 124. Low voltage(4. 5 8 .0eV) mass spectra of the nickel porphyrins isolated fro m Bakken shale number 6362 (R = 0 .65) prior to and following hydrous pyrolysis. i d = D PEP ( a l t CAP ) series dashed = E T I 0 series

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BAK KEN-6 362 C R. :0.55) N ATIVE BI TUMEN ( 9 7 3.,.) SOXHLET BITUMEN Pre-H P ( 2 70fo) HP Jooc d HP 35oc 2d VO PORP HYRINS 65. 3 t o D 269.9 )Jg / g -sed 14. 3mgtgEOM 95.6 )JQE / g -sed 75.70fo D 7.6 )JQ/ g-sed 1.6)JgE / g -sed 67. 2 Of. D 224. 01-'9 g-sed 11.6mg/ g-EOM 51. 1 )-19 E / g-5ed 2.6 Ofo D 26. 0 !Jg fg-sed 25. 3pgE /g-sed 22 24 26 26 30 32 34 36 36 40 42 44 CARBON NUMBER 434 Figure 125. Low voltage(4.5 eV) mass spectra of the vanadyl porphyrins isolated from Bakken shale number 6362( R = 0 .65) prior to and following hydrous pyrolysis. Solid =0 DPEP(alt. CAP") series, dashed= ETIO series.

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435 .extract plus 300C/3d-pyrolysate yield a total of 94.6 (NiPH, bitumens) plus 113.0 (NiPH, pyrolysate) plus 277.5 (VO PH, bitumens) p lus 224.0 (VO PH, pyrolysate) or 709.1 sediment, dry weight. As additional EOM was released during pyrolysis, the yields as normalized to EOM average rather than add. However, if we consider EOM to equal 'petroleum', then the yields of Ni-(ca. 5.5 mg/g-EOM) and VO-(ca. 13.1 mg/g-EOM) may also be viewed as tremendous. The second experiment with this shale was a conventional (HP) hydrous pyrolysis (350C, 2d: Winterset gJ., 1983). In this case (Figures 124-125; Table 48), results almost identical to those with the deep sea sediment (Figure 123) were obtained. That is, a low yield (Table 48) of mainly dealkylated (C23-C32) ETIO-series porphyrins maximizing at C27 in both the Ni(Figure 124) and VO-(Figure 125) arrays. However, this experiment yields significant alternate data. Specifically, since the two separate HP experiments were not sequential but parallel operations performed on identical sub-samples of pre extracted shale, then the pigments released in the 3d-300C run may be considered as evolving from those in the previous run. Therefore it is very clear that h ydrous pyrolysis at 350C, and certainly above, is too severe for the study of tetrapyrrole geochemistry. H owever, HP experiments with known amounts of synthetic pigments (viz. DPEP, ETIO) and selected catalysts at such high temperatures may aid in clarifying loss rates. It is becoming increasingly clear that industrial hydrous pyrolysis conditions (cf. Winterset gJ., 1983), aimed mainly at estimating a maximal bitumen yield for a rock, are too severe for the study of labile biomarkers.

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436 Rowland and co-workers (1986) explain the formation of unusual compounds in hydrous and anhydrous pyrolysates as being ... not simply a function of the type of organic matter or m i neralogy but rather of the high temperatures or fast heating rates." To date, the closest mimic in both the quantity and the quality of OM produced during in vitro thermal simulation appears to be from a 6 year study of a turbanite and a brown coal performed with slow i ncreases in temperature (Saxby et gj., 1986). The problem of mineral catalysis must also be addressed. The addition of carbonate or kaolinite to Kimmeridge kerogens has been reported to greatly assist the production of EOM and in generating known geochemical reactions (g .g. sterane and triterpane epimerizations) when compared to HP alone or with other minerals (Eglinton et gj., 1986). Therefore, e x periments using neat kerogen and reporting no bond breaking during porphyrin studies (g.g. Van Berkel et g]., 1989) should be re-evaluated. Alternately, it has been suggested that VO-etioporphyrins are released from kerogen and dilute the DPEP series during catagenesis (Sundararaman et gJ., 1988). However, as the data in that paper was gathered at 350-450C, one should question release and suspect degradation-dealkylation Clearly, the thermal simulation of metalloporphyrin catagenesis should continue but thought should be given to the use of neat pig ments, known mixtures, catalysis, longer times and lower temperatures. Further, it is also clear that the decrease in DPEP-to-ETIO ratios (% DPEP), the switch to ETIO dominance and the overall dealkylation of the metalloporphyrins (g.g. Figures 118, 120-121) stand as valuable markers for catagenetic progress and general overall thermal history.

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437 The above has done little to aid in the question of the source of the ETIO-series porphyrins. Certainly, some structures must be preformed entities stemming from the cytochromes of sulphate reducing bacteria, as isotopic evidence suggests (Bareham et gj., 1989, 1990), or from the early oxidative opening of the isocyclic ring(s) inherited from chlorophyll. However, the potential for the thermocatalytic conversion of a DPEP (CAP) to an ETIO skeleton still exists. It must be concluded here that most, if not all, of the current theories on the underlying mechanisms of the DPEP-to-ETIO dominance change are operative and operate to different degrees in different settings. Thus, for now, the switch to ETIO-dominance can be viewed as expressing a faster rate of DPEP destruction (cf. Barwise, 1987), the addition of ETIOdominated porphyrins from kerogen (cf. Sundararaman et gj., 1988) and a minor conversion of DPEP to ETIO structurally (cf. Baker and Palmer, 1978; Didyk et g}., 1975ab). It can also be noted that, when similar rocks are studied over a wide range of natural maturity (g.g. Monterey, Tables 40, 41, 45, 46: Bakken, Tables 42, 43), there always is a tremendous predominance of the DPEP types in the immature samples. So much is this excess that one could assume only a 2-4% survival for tetrapyrrole skeletons whic h thermally lose the isocyclic (cycloalkano) ring and still explain the quantity of etioporphyrins found in mature samples. The ETIO question, regarding maturation, remains enigmatic. The last facet of tetrapyrrole geochemistry to be covered herein concerns another set of enigmatic geoporphyrins, the benzoporphyrins. The benzoporphyrins were first considered rhodoporphyrins (see review in Baker and Louda, 1986a) due to the 'similarity' between the visible spectrum of these geologic pigments and the known rhodoporphyrins (g.g.

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438 rhodoporphyrin [LIX]: band order III>IV>II >I). In the true rhodoporphyrins, the rhodofying (viz., red absorber, green imparter) group is an electron withdrawer such as a carboxylic acid or formyl moiety (Stern and Wenderlein, 1936b). However, as detailed in Chapter 3 (see [CXVI], Figure 46f), the visible band order of the geologic free-base 'rhodoporphyrins' is III> I>IV>II, clearly distinct from the conventional rhodoporphyrins. Baker and co-workers {1967) suggested that the geologic pigments were benzoporphyrins with, what amounts to a butadiene moiety attached to adjacent B pyrrole positions. This structure was confirmed by the isolation of phthalamide, and the unexpected methyl phthalamide, following the chromic acid oxidative degradation of a georhodoporphyrin concentrate (Barwise and Whitehead, 1980). Since then, several individual benzoporphyrins have been isolated and structurally elucidated (Barwise and Roberts, 1984; Kaur et gl., 1986; Lash, 1993; Verne-Mismer et gl., 1987). The overwhelming consensus is that the benzo-moiety is on ring II (3,4 or 7,8-revised) with compounds bearing the conventional isocyclic ring inherited from chlorophylls Whether this unusual group arises early during aberrant biosynthesis (cf. Clezy and Mirza, 1982), via intramolecular cyclizations (Baker and Louda, 1986a; Lash, 1993) or via Diels Adler type diene/dienophile additions (Baker and Palmer, 1978; Quirke et gl., 1990) remains for the future. The current state of knowledge regarding structural variations and possible origins can be found in Lash's (1993) review In geologic matter, the benzoporphyrins have been found once as free-base pigments (Quirke et gj., 1990), a few times as nickel chelates (Baker and Louda, 1986b; Louda and Baker, 1981) and numerous

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439 times within vanadyl porphyrin arrays Baker and Louda, 1986a; Baker et gl., 1967; Kaur et gl. 1986; Lash, 1993; references in each). A large part of the present studies dealt with the improvement and standardization of analytical techniques for the study of tetrapyrrole geochemistry (Chapter 3). As part of those investigations the detection and quantitation of geologic benzoporphyrins was addressed with electronic (see Tables 12, 14: Figure 61) and mass (Tables 18-23, 30: Figures 80-81) spectrometric method. It was concluded that UV/VI S provided more reliable, accurate and repeatable results. Given here as Table 49 is the percentage benzoporphyrins, as determined via electronic absorption spectroscopy, in the majority of vanadyl porphyrin arrays reported herein. The e xact meaning of these data and any potential linkages to organic source (viz. precursor pigments) and/o r paleoenvironment will need to await the future. However, for now, the only immediately apparent trend is towards an increase in the relative amount of benzoporphyrins in the vanadyl pigments from more organically mature hosts As e x amples, consi der the two shale sequences and all of the oils by 0API gravity. In the shales from the WCA well, e x cluding the "S9" shale, due to a la rge change in organic facies and age between S8 and S9 (W. L. Orr, pers. commun.), there is a general increase from about 4 to about 8% benzoporphyrins with depth. A similar trend was found for the Bakken shales (11.0-16 4 % ) Considering all of the oils and using 0API-gravity as a proxy for general maturity, the trend is for increase percentages of the benzoporphyrins in more mature ( 0API>25) oils. As stated, these observations are preliminary, as this is the first data base dealing with this subject However, should these trends be real, then it is tempting to speculate as to why VO-

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Table 49. Percentages of benzoporphyrins in vanadyl porphyrin arrays isolated from shales and petroleum crudes of anoxic marine origins(!). ( R )(1) MS INDEX(2 ) (1) (1) MS INDEX(2 ) (1) 0 % BENz(3 ) % BENz(3 ) SAMPLE 0API % DPEP A. I. SAMPLE 0API % DPEP A. I. WCA-S5 (0 3) 84. 0 0.19 4 8 0-7072 16.8 75.8 1.02 3.9 WCA-S6 (0 .3) 91.3 0.50 7.9 0 -7377 16.8 78.0 0.35 4.2 WCA-S7 (0.35) 90. 3 0.51 10. 7 0-7073 17.1 70.8 0.86 5.3 WCA-S8 (0 4) 88.6 0 .72 9.0 0-8062 17.5 83. 0 0.54 4 0 WCA-S9 (0.4) 87.8 0.46+ 4.5 0 -3735 18.5 44.4 0.94 6.1 B -2650 (0.48) 82.2 0.15 13.0 0 -4936 20.5 70.2 0 .34 3.2 B -6362 (0.65) 65. 3 0.43 16.3 0 -7771 20. 8 66. 2 0.69 6 7 B -6182 (0. 77) 72.8 0 .46 17.2 0 -8515 21.4 76. 3 1.00 9 3 B -6486 (0. 97) 3.5 0.17 19. 4 0 -7077 21.5 66. 7 0 .65 4.2 0 -8061 9 6 84. 5 0.32 4 5 0 -3726 21.7 13.8 0.36 7.1 0-7768 9.6 42.5 1.00 4 7 0 -3729 23. 1 51.2 0 .66 4.4 0-7071 9.7 76.3 0.54 5.7 0-4938 23. 7 69.7 0.25 4.0 0 -7042 11.8 83.7 0.48 3.3 0-3915 26. 6 7.4 0 .11 6 4 0-7050 11.9 67. 6 0.13 1.4 0-8519 31.7 75. 7 0 .94 11.1 0-7044 14.4 74.8 0.55 2 1 0 -3922 31.9 4 8 0 .12 9 3 0-4907 14. 7 46. 5 0.11 4.2 0-3919(4) 33.2 44. 4 1.60 7 8 0-8067 16. 7 81.5 0.50 5.2 0-3145 34.0 7.4 0 .02 9 2 FCXITNOTFS : (1) Samples are more fully described in Tables 40 (WCA=wildcat"A"well), . 42 f B =Bakken)and 4 4 ("0"= oil,see FRL nos ) .Vitrinite reflec tance values of the Wildcat-"A well shales are very rough approximations(see Orr,1984). (2) Mass spectral index calculations are detailed in Table 23. (3) Percent benzoporphyrins(% BENZ) is calculated usin g electronic absorption spectroscopy as detailed in Chapter 4, Table 14 and Figures 6061. (4)0il # 0-3919(FRL# 3919)is from the Manderson field of the Big Horn Basin and has been shown to be a mature oil contaminated with immature biomarkers(cf.Baker al. ,1987) .p.. .p.. 0

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441 benzoporphyrins increase in shales bitumen and oil VO-porphyrin arrays with maturity. In the case of shales, we have shown that the first release ('early catagenesis') products are vanadyl porphyrins of a high %-DPEP and low A.I. character. Later the ETIO-series and more highly alkylated pigments of all series appear. In light of that scheme and the trend found here (Table 49), it is suggested that certain portions of the vanadyl benzoporphyrin population in sedimentary ('shale') OM are late release products Since there are VO-benzoporphyrins in immature bitumen as well, it is obvious that release occurs continually but a relative enrichment into the bitumen occurs during the latter stages of kerogen maturation. In the case of petroleum, which for the most part is divorced from its source via migrat i on, the enrichment of benzoporphyrins relative to the overall VO-porphyrin array appears to be due to enhanced thermal stability/survival. That is, c onsidering the three mature oils of 0API gravity greater than 25 (FRL-3915, -3922 and -3145), the deadline of porphyrin e x istence is being signaled by low c oncentrations 1 3-21 see Table 44), low% DPEP and low A.I. (Table 49). In these oils the percent benzoporphyrin appears elevated by survival. As noted in Table 49, oils FRL-8519 and -3919 are not as mature as 0API gravity alone indicates. That is, oil FRL-3919 (Manderson field, Big Horn Basin, Wyoming) was determined to be a mature oil which, during migration, 'extracted' immature vanadyl porphyrins from less mature shales (Baker et g]., 1987). The second oil not considered here even though its 0API gravity is over 25 is FRL-8519. Oil sampl es FRL-8519 and FRL-8515 were rec overed from the same well fiel d but at different depths.-In these cases the vanadyl porphyrin distributions are essentially identical (8515/8519: %DPEP =

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442 76.3/75.7; A.I. = 1.00/0.94; X= 542.2/541.3: Table 46). It has been concluded (W. L. Orr, pers. commun.) that oil FRL-8519 originated as FRL-8515 but, during migration and/or pooling with gas intrusions, became deasphalted with a resultant increase in API-gravity which is not linked directly to thermal maturity. Therefore, from the above, vanadyl benzoporphyrins appear to enriched relative to alkyl DPEP-and ETIO-porphyrins in the latter catagenetic products from kerogen and to survive a bit longer during the final stages of geoporphyrin maturation.

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443 CHAPTER 5 CONCLUSIONS The goals stated at the end of the Introduction were (1) to develop and/or refine techniques for the analyses of tetrapyrroles during all stages of organic maturation, and (2) to apply these methods to a wide range of samples and sample-suites in order to yield an overview of tetrapyrrole geochemistry, stressing the chlorophylls as precursors. This, it is felt, has been accomplished. Given the scope of both facets of this research, the "Results and Discussion" section was split into two individual chapters (4 and 5), each covering the results from the individual goals given above. Analytical Experimentation Since the primary result(s) of analytical experimentation is the development of methods by which to perform analyses, more attention will be given to the from the application of these techniques than to the techniques themselves. The analytical methods studied were electronic absorption spec troscopy, mass spectrometry and a variety of chromatographic methods. The goals and results of this section included: (1) the development of easy and rapid initial bitumen assay for the classes of tetrapyrroles (viz. 'chlorin,' free-baseand/or metalloporphyrins present; (2) development and refinement of 'soft' chromatography (CC; microcrystal line cellulose) for the separation of dihydroporphyrin arrays

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444 (phorbides, chlorins, purpurins) into three polarity classes (nonpolar/ esters and alkyl; mono-carboxylic acids; di-plus tri-carboxylic acids); (3) characterization of the behavior of metalloporphyrins during chromatographic purification steps by individual series (DPEP, ETIO, BENZ) and carbon number; (4) provision of a large UV/VIS data base, including previously unreported Soret bands and full spectra for many new synthetic pigments (see Appendix A); (5) establishing refined methods for the estimation of geologic tetrapyrroles either as individ ual types or in mixture and providing an estimate of accuracy by e xtensive testing with known pigments; (6) developing a chromophore identification scheme using the successive and parallel treatment of pigments with sodium borohydride and 63Cu2+ ; and (7) refining the methodology for gathering mass spectral data. Certain of the methods developed during these studies were presented during a symposium on porphyrin geochemistry (Baker and Louda, 1990; Louda and Baker, 1990). The concept of a method for 'chromophore identification' stems from a knowledge of the electronic spectra of tetrapyrrole pigments and the ways in which the spectra change due to alteration of the chromophore. There is nothing new or dramatic in this concept, however, it took the collection of a relatively large data base and the application of ce rtain simple reactions to prove its utility. The methods developed can be applied down to the nanogram level and with isolates which are not pure enough for continued analyses by other techniques. In essence, a geotetrapyrrole, usua l ly a free-base phorbide or chlorin, isolate is split into two aliquots. The first half (A: H2 ) is reacted first with Cu2+ to yield the metallo complex (= Cu:A), and then with sodium borohydride, to give an o x y-deoxo der ivative (= 00-Cu:A) if a

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445 conjugated carbonyl was present. Collection of the UV/VIS spectrum at each stage now gives either two (i.g. no carbonyl) or three (i.g. carbonyl present) spectral data sets. The second half of the native pigment {B:H2 ) is reacted directly with sodium borohydride and, if a conjugated carbonyl is present (OD-B:H2 ) provides a fourth spectral datum. Comparison of such UV/VIS data, before and after reaction with Cu2+ and/or BH4-, with similar data collected on authentic pigments {Tables 10, 11) has been shown to be a valuable method for chromophore identification and is especially useful when sample and isolate size is limited. The technique of EI-MS for the analysis of geoporphyrins is not new (Baker, 1966; Baker et gj., 1967). However, until the present investigations a specific methodology by which to obtain repeatable mass spectra with geoporphyrins was lacking in the literature. The resultant heating profile (Figure 82) and reproducibility (Figure 83) found allow here the first estimates of the precision attainable in such analyses. These precisions are found to be; % DPEP (%), weighted average mass (X, %), alkylation index (A.I., %) and percent benzoporphyrins (%Benz, %). As given earlier, the % Benz parameter should be reported from UV/VIS data (-2%). The net conclu sion from extensive mass spectral study i's that the low voltage (4-12eV) EI mass spectrum is sensitive and highly repeatable. Therefore, it is suggested that the averaged, tsotopically corrected and normal ized low voltage mass spectrum should be (re-)instated as the common fingerprinting method for geoporphyrins and that such a spectrum should be reported in histogram form, with an accompanying tabular account if wished, with all reports on geoporphyrin distribution and/or structural

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446 .identifications. That is, as attention in porphyrin geochemistry shifts to a more molecular emphasis, the overall geoporphyrin array from which individual structures are isolated is being dismissed. Further, as routine HPLC/RP-HPLC methods fail to detect geoporphyrins much above C33-C34, data on the extent of alkylation is lost. That is, the methods developed here for more precise determination of the porphyrin alkylation index (A.I.: Baker et gJ., 1977) allowed crossplots of %DPEP versus A.I. to be applied to the study of metallopor phyrin evolution (Figure 120). Once obtained, the average low voltage ElMS can be utilized as not only a fingerprint but also as a framework into which the relative abundances of each structural isomer may be placed for comparison to the whole. As structural studies on geopor phyrins began and as they continue there has been a noticeable lack of quantitation and/or relation of these structures to the entire array from which they were isolated. Review of the literature cited through out the present text reveals that only about 10% of the modern geopor phyrin structural studies even attempt quantitation. This, in the author's opinion, is a waste of data Consider the RP-HPLC chromatograms and the corresponding mass spectral histograms for the three vanadyl porphyrin isolates given as Figure 126. In this e xample only two isomers of the pigments with m/z values of 541 were e x amined. These are both C32 DPEP (Alt. CAP) structures. The RP-HPLC peak blackened is 'true' C32 VO-DPEP [LXXXVIII]. The a lternate C32 VO-DPEP ("CAP") is tentatively identified as a cyclopropano porphyrin (cf. Chicarelli et gJ., 1984; Wolff et gj. 1984: see Figure 13). Assuming for the moment that these are the correct assignments and that no other VO C32 DPEP series. porphyrins can be found via RP-HPLC, then the

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a) 400 500 mlz 600 22 24 26 28 30 32 34 36 38 4 0 42 CARBON NUMBER 700 II I 0 OH 20 40 TIME (minutes) 60 80 Figure 126. Comparisons of the mass spectral and RP-HPLC fingerprints of three vanadyl geoporphyrin isolates. (a) Estonian fire-shale ( A .Treibs, ca. 1934-5), (b) Bakken s h a le# 6362 (Tables 41-42), (c) Boscan petroleum asphaltene-(Tables 7-8,Fractions "4-9 pool"). RP-HPLC performed on a 4.6 x 250 mm C18(0DS) column(5 urn) using the conditions of Sundararaman(1985). MS DPEP(solid) and ETIO(dashed).In MS and RP-HPLC the solid colored peak is 'true DPEP[XXXVIII ] and the dashed peak is (tentatively) a C32 cyclopropane-porphyrin( C32, 541 m/z). .j::-.j::--.....J

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448 relative abundance of each type, based on Beer's Law calculations of the RP-HPLC peaks (i. g using either peak height or area after standardization), can be determined and that abundance can be placed into the conte x t of the entire array (Figure 126). Little by little, as more structures become identified, the entire picture of relative importance would unveil While it is true that, once all member s of a series at a carbon number are identified, simple percentage comparisons will show the relative importance at that mass (m/z) it is also true that without comparison to the whole, the real import is lost. Geochemical Investigations The current investigations were des igned to probe as fully as possible the entire continuum of tetrapyrrole geochemistry. The present studies resulted i n a des cription of the over a l l phenomenology of tetrapyrrole geochemistry and have provided a rather complete framework for the placement of molecular structural data Therefore, the reactions and reaction pathways suggested below stem not only from the work reported here but i ncorporate data from the literature. The d i vision of tetrapyrrole geochemistry into discrete stages (Baker and Louda, 1983, 1986a) was performed in a manner so as to both paral lel the overall evolution of sedimentar y OM ( cf. Hunt, 1979; Tissot and Welte, 1978) and to allow for d e s criptive c onvenience However, it should be remembered that these 'stages' or 'divisions' can and do overlap and that exceptions to the strict order given on paper are bound to occur in nature. The divisions of tetrapyrrole geochemistry considered herein are: Diagenesis, split into early, mid-and late-and correspond ing to defunctionalization, aromatization and

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449 nickel chelation, respectively; and, catagenesis--the maturation of nickel porphyrins, the release and maturation of vanadyl porphyrins and the destruction of pigment in later phases Currently, if discussion is limited to marine/brackish anoxic sedimentary environments, the evolution of tetrapyrrole pigments broadly follows this scheme: Immature bitumen, at the onset of petroleum generation, contains nickel and vanadyl porphyrins vastly dominated by the DPEP (a lt. 'CAP" ) series. As maturation (catagenesis) continues, nickel porphyrins eventually disappear with no overt trends in DPEP-to-ETIO ratios being expressed while, at the same time, the vanadyl porphyrins exhibit two distinct and rather straightforward trends. Initially, this DPEP dominance is maintained during a period in which higher carbon number members of both ('DPEP-', 'ETIO-') series appear. Later, both series are dealkylated and a shift to ETIO-series dominance occurs. The background for these changes was reviewed extensively in text. The questions at the heart of tetrapyrrole geochemistry then become, What are the sources of both the DPEP and the ETIO seri es?, and What processes lead to a series dominance change during maturation? Figure 1 was the pictorial representation of these queries. Direct 'Treibs' scheme (Treibs, 1936) geochemistry will yield a DPEP-compound (viz. DPEP [XXXVIII] per se from chlorophyll-a [I] and an ETIO-compound (viz. ETIO-III [LII]) from a heme-type (g.g. protopor phyrin-IX [LIII] precursor. However, the involvement of a 'crossover' from the DPEP-to-ETIO type via thermal (cf. Baker and Palmer, 1978; Didyk et 1975a-b; Morandi and Jensen, 1966) or o xidative (cf. Baker and Louda, 1980a, 1983a, 1986a; Corwin, 1960; Louda and Baker, 1986) means, has also been suggested The resu lts from studies

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450 reported herein support the conversion of chlorophylls to both OPEP and ETIO precursors. Further, these data reveal the potential for an 'ETIO-to-DPEP' (alt. CAP) crossover and the generation of rearranged DPEP-type compounds. Pigment yield studies revealed that the concentration of tetrapyrroles changed in predictable manners during the individual stages of organic evolution. Figure 127 is a representation of pigment yield profiles for oxic (dashed) and anoxic (solid) depositional conditions. In the case of oxic deposition and low TOC, tetrapyrrole pigments are found to be rapidly destroyed and any identifiable chlorophyll derivatives are usually dominated by chlorins and purpurins. In the case of anoxic deposition with substantial TOC 1%+), an initial period of rapid pigment concentration decreases, due to both loss and incorpora tion into 'geopolymers', is followed by a zone of relatively stable pigment yield. This profile, as shown in Figure 127, encompasses all of diagenesis and extends into strata with in situ temperatures of about 50-60C for sediments of about Miocene age. Younger or older deposits would, of course, require higher or lower present in situ temperatures to be of equivalent At the onset of catagenesis, pigment concentration increases rapidly and reaches very high levels (g.g. 10-15 mg/g-EOM). This e x treme ly rapid and significant increase is suggested as being due to the breakage of heteroatomic bands (g.g. C-S, C-0) at the very onset of petroleum generation. As catagenesis continues, pigment concentration decreases, first by direct dilution with additional non-porphyrin bitumen and later by the actual destruction of pigment.

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T ( C) 0-4 10 20 30 40 50 60 70+ -:;--->. -I... ::::J -Ctl E v c: Ctl Ol I... 0 0 let: 0..: L: w I-. w (9 > E -340 -t::. A.I ED Figure 127. Suggested overall tetrapyrrole yield trends for sediment s deposited in anoxic(solid) or oxic(dashed) conditions.Events and pigment array descriptions are covered in text. 451

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452 Diageneti c Studies The study of chlorophyll diagenesis involved the analyses of unialgal cultures, sediment traps, surface and sub-surface sed iments and sapropels The main conclus ion of these studies is that the geochemistry of chlorophyll is highly anastomotic However, out of that diversity, trends are emer ging which w ill greatly aid the interpretation of organ i c facies, paleoenvironment and thermal history. The period of early diagenesis encompasses the defunctionalization and structurally rearrangement of precursor pigments. As such, this period of geochemistry overlaps and is often difficult to separate from biological events occurring in pre-/syn-depositional conditions. Two main fating reactions are suggested as being involved i n determining the subsequent course(s) of chlorophyll der i vat ive evolu tion within sediments First, allomerization, the o xidative attack on the isocyclic ring (Hynninen, 1979), appears to initiate the cleavage of the isocyclic ring with purpur ins and chlorins being generated. Herein, purpurin-18 [XXVI] and chlorin-p6 [XXVIIa] were identified as key intermediates in this (o xidati ve) scheme. Thi s route, depicted here as Figure 128, also can lead to other chlor i n structures ( c f Fischer and Stern, 1940) and potentially to etioporphyrins at C31 or below. Second, the loss of the C-10 carbometho x y moiety, the so-ca l led 'pyro'-reaction, i s suggested as directing further diagenesis towards the DPEP [XXXVIII] type structure. An entire series of phorbides were tentatively identified as participating in the evolution of chlorophyl l into DPEP. These i nclude; bacteriopheophyt i n-a [XXI], pyro-pheophytin a [III], pheophytin-a [IIa], pheophorbide-a [VIa], pyro-pheophorbide-a [VIII], meso-pyropheophorbide-a [ X ] deox o pyropheophorb i de-a (cf.

PAGE 481

y M h 0 6s:-<;=o Ho bHOH l N HN CO 00H OH HOO PURPURIN -18 "UNSTABLE CHLORIN" +H 2 0 HOOC , CHLORIN-P 6 Figure 128. Structural comparison of the pigments involved in the oxidative rupture of the isocyclic ring in a chlorophyll derivative(pheophorbide-a), producing purpurin-18 and chlorinp6 +=' lJ1 w

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454 (IX]), and deoxomesopyropheophorbide-a (alt. 7,8-dihydro-DPE. [XII]). In the present studies, all of these pigments, except the phytyl esters (pheophytins), were isolated as non-acid structures. Thus, it is suggested that this group of phorbides are the decarboxylated (g.g. 7ethyl-7-despropio-) analogs of the structures However, both the carboxylated (Keely and Brereton, 1986; Keely et gj., 1988, 1990; Prowse and Maxwell, 1991) and the steryl ester (King and Repeta, 1991; Prowse and Maxwell, 1991) forms of several of the above are known. Given this, a scheme for the anoxic diagenesis of chlorophyll is forwarded here as Figure 129. It is reiterated here that this defunctionalization scheme can apply equally well to carboxylated (pheophorbides) decarboxylated, phytyl ester (pheophytins), steryl ester or bound pigments, and can include any of the chlorophylls as ultimate precursors. However, aside from the phenomenology of vanadyl porphyrin evolution in nature which strongly favors this, there is no data to support a kerogen-bound porphyrin precursor pool. The overall result of the early-diagenetic (defunctionalization) portion of this scheme (Figure 129) is the production of free-base phylloerythrin [XXXII], deoxophylloerythrin [XXXVII] and DPEP [XXXVIII] via aromatiz ation ("mid-diagenesis") of the corresponding defunctionalized phor bides. The chelation of nickel (II) during late diagenesis yields a very common array of metalloporphyrins (viz. NiPE [LXXVIII] NiDPE [LXXXVI] and NiDPEP [XV]). The production of the etioporphyrins, alternate DPEP-or CAPseries porphyrins and the benzoporphyrins has also been studied herein. The potential participation of the oxidative pathway (Figure 128) in the production of etioporphyrins was mentioned above. Here, the

PAGE 483

PIIEOPHYTIN-a ------l -COOCH 3 PIIEOPIIORBIDE-'!_ PYRO-PHEOP HYTIN-a -COOCH3 ..------phytyl PYRO-PHEOPHORBIDE-a +2H7 13, 17'-CYCLO-MESOPYROPHEOPIIORBIDE-a DEOXOPYROPHEOPIIORBIDE-a PHEOPIIORBIDE-a I -H 0 2 DEOXOMESOPYROPIIEOPIIORBIDE-a (DOMPPa-7,8-DiHydroDPE) } -H2 0 13' ,17'-CYCLO-NESOPIIEOPIIORBIDE-a ""' -co2 +2Hv/O -211 2 I 7, SiHHYDRO-DPEP + H -0 i +211 l-2H PE 2 H / O OPE CO DPEP + + -1 -2 (E-2 m/zseries) "lHDPEP (E-4 m/z series) -------t--------------(/} 1:'1 z 1:'1 "' (/} :>::1 () 1:'1 1:'1 C) z )> () >-l 1:'1 H 0 -z =-C) C) [r] )> >-l ::r: H )>["Tl C))> 1:'1::0 Zr' tTl.-< (/} H (/} 03: HH )>O C) I [r]' Zr' M)> (1}>--j Hl"'l (/} I Figure 129. Proposed routes(partial)for the anoxic/reductive diagenesis of chlorophyll derivatives ending in the production of DPEP and 'DiDPEP'porphyrins. _p.. Ln Ln

PAGE 484

456 relationship between the chlorins found and three nickel porphyrin acids reported from Messel shale (Ocampo et gl., 1985a) is made. That is, Ni pyrroporphyrin (cf. [LX]) is easily derived from purpurin-18 [XXVI], with free-base or Ni rhodoporphyrin (cf. [LIX]) as an intermediate. Similarly, Ni phylloporphyrin (cf. [LVI II b ] ) can easily derive from chlorins such as -e6 [XXIII] and -e4 Following decarbo x ylation, the above Ni porphyrin acids could yield C28-C30 Ni alkyletioporphyrins. Primarily from literature study, the main sources of C32 ETIO-porphyrins (viz. etioporph yrin-III [LII]) appears to be heme-type compounds (see g.g. Bonnett et gl., 1987; Bareham et gl., 1989, 1990; Quirke and Maxwell, 1980). The production of alternate DPEP-type porphyrins, the so-called (CAP), has attained considerable interest in the past several years (see Callot et gl., 1990). During the present work intermediates potentially leading to two of these alternate DPEP forms were partially identifi ed. First, c y clop y ropheop horbide-a e nol and its meso-analog were found to e xist in deep sea sediments. This phorbide appears to form (Figure 130) via a dehydration reaction, possibly involving clay catalysis, and can theoretically (cf. Callot et gl., 1990; Fookes, 1983b) lead to a cyclobutano substituted porphyrin, if rupture of the isocyclic ring occurs. As far as can be told, the present study is the first report of the meso-analog of the cyclop heo phorbide-a enol. The above alternate DPEP-series or CAP structure does ultimatel y derive from a chlorophyll precursor However, the fo rmation of a DPEPser i es (viz. M-2 in M S., see Baker et gl., 1967) mimic with a cyclo propano moiety (Chicarelli et gl., 1984; Wolff et gl., 1984) or a

PAGE 485

-H20 '-.!:2 H "-H20 ""' e:-O H '0 PYROPHEOPHORBIDE-a +2 H J--r..l N I Cf= 'bH' HEO PHORBIDE-a "D i DPEP"( E-4 ) ;'( Figure 130. The cyclization pyropheophorbide-a and mesopyropheophorbidea and the participati on of the cyclopheophorbidea enols in the production of DiDPEP compounds. Asterisks known geoporphyrphyrins(see Chicarelli et al.,1987; Kee l y and Maxwell,1990). V1 -......1

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458 methyl-substituted cyclopropano moiety (Wolff et 1984; cf. Lash et gl., 1990) in geologic settings is indicated by the respective identifications of the alkyl porphyrins. In the present study. compounds with electronic spectra matching the known rhodins ([XLI], [XLVI]) were isolated. Figure 131 is a scheme showing the cyclization of mesoporphyrin-IX [LV] to yield mesorhodin-IX [XLI}, a long known in vitro reaction (Fischer and Orth, 1937). The decarbo xyl ated analog of mesorhodin-IX [XLI] was tentatively identified from deep sea sediments during these studies. The conversion of the 'ethyldespropio' mesorhodin-IX analog to the known geologic porphyrin is a straightforward reduction-dehydration-reduction process. It i s known that the cyclization of mesoporphyrin-IX yields both possible isomers (Fischer and Orth, 1937) and it is therefore not surprising that both of the possible (Figure 131) isomers were indicated during the characterization of these "DPEP-6" compounds from Gilsonite (see Wolff et gl., 1984). Given as Figure 132 is an overall, albeit abbreviated, scheme for the diagenesis of chlorophyll and heme-derived precursors. Note that the products shown are metallo-porphyrins. the products derived after mid-(aromatization) and late-(chelation) diagenesis. The overwhelming fate of tetrapyrrole pigments in the geosphere is to become true DPEP structures. That is, not only from the studies reported here but from literature review, the geologically dominant metalloporphyrins are C30-C33 DPEP, with DPEP [XXXVIII] per seas the absolute most abundant. Further, it is becoming increasing well documented that the major C32 ETIO-porphyrin is etioporphyrin-III. Therefore, these end member precursor-product pairs, chlorophyll/DPEP and heme/etio-III,

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OOH'COOH MESOPORPHYRI N COOH -H20 DEHYDRATION/CYCLIZATION COOH MESORHODIN(S) ( 2 isomers) +2H -C02 .. C33 CYCLOPROPANOPORPHYRIN 5(2 isomers) Figure 131. Formation of cyclopropane-porphyrins from mesoporphyrin-IX[LV]. p. V1 \.!)

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-----CHLOROPHYLL HEME ......_ -........ C H Le6 M-DPEP(true) M-PSEUDO -0(7) M -PSEUD0-0(6) M -ET lOs \ \ \ t t Figure 132. Suggested overall geochemical transformations of chlorophyll and heme structures. Abbreviations, PUR= purpurin, CHL= chlorin, M= metallo D= DPEP(alt. CAP), R= alkyl or H. (j'l 0

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461 fulfill the Treibs' hypothesis (Treibs, 1936) perfectly. Modifications to that scheme are suggested within Figure 132. The oxidative scission of the isocyclic ring of pheophorbide-a [VIa] can lead to ETIOporphyrins through a series of purpurins (g.g. [XXVI]) and chlorins (see [XXIII], [XXVIIa]). Cyclization of the propionic acid moiety of heme derivatives such as mesoporphyrin-IX [LV] can lead to pseudo-OPEP-6 compounds (cyclopropane-series) through mesorhodins (see [XLI]). Potentially, the cyclization of the propionic and acetic acid moieties in chlorin-e6 [XXIII] could lead to the pseudo-DPEP-7(cyclobutano) compounds. However, since a cyclopheophorbide-a enol route to these compounds is better known (Figure 130), the cyclization of chlorin-e6 [XXIII] remains entirely speculative. The bottom-most route in Figure 132 infers the potential thermal or thermocata lytic conversion of the DPEP-(alt. CAP-) series into ETIO-structures. This potential conver sion has never been proven, nor has it been disproved. In order to cover precursor-product relationships above, the majority of mid-(aromatization) and late-(nickel chelation) diagenesis has been covered. In brief, mid-diagenesis covers that period during which defunctionalized phorbides are aromatized to DPEP-series porphyrins. The direct generation of the ETIO-series has never been observed, outside of the peat-coal realm (cf. Bonnett et gl., 1983, 1984, 1987). Late-diagenesis, for marine-aquatic sediments, involves the chelation of nickel by porphyrins in the bitumen. Catagenesis includes the maturation/destruction of the nickel porphyrins as well as the release of vanadyl porphyrins from a bound (kerogen ?) state. The vanadyl and nickel porphyrins mature together, though the nickel species never exhibit any trends and disappear

PAGE 490

462 completely well in advance of the vanadium pigments. The release and maturation of the vanadyl geoporphyrins have been shown here to follow well defined, nearly predictable, trend lines. At the onset of catagenesis, vanadyl porphyrins of restricted carbon number ranges (C28-C33) and very high %-DPEP values (80-95 % ) are released i nto the bitumen (viz. juvenile petroleum) in often enormous quantities 1-20 mg/gEOM). Subsequently, the production of additional bitumen from k erogen/ asphaltene main phase oil generation) dilutes the originally highly concentrated vanadyl pigments and, via additional macromolecular d isruption. adds higher carbon number species C34-C40+) to both the DPEP-and ETIO-series. At this point in catagenesis, the vaoadyl porphyrin array maintains a high percentage of the DPEP-series 75-85% DPEP) and has an elevated alkylation inde x A.I. = 0.7-1.1). It is during this portion of catagenesis that the cross-plot of %DPEP versus A.I. (Figure 121) becomes e x treme ly useful in the evalua tion of organic maturity and especially so when comparing con-generic samples. As catagenesis continues, dealkylation of both main series (D, E) and a shift to ETIO-dominance occurs. Towards the end of catagenesis, as far as the tetrapyrroles are concerned, low concentra tions 1-2 of severely dealkylated (C22-C32, C26-27 max.) etioporphyrins (g.g. 2-5% DPEP) signal the deadline for th ese pigments. Certain portions of the overall geochemist ry of tetrapyrroles have now been outlined. The future must hold the answers for several important queries regarding these pigments As examples: Does a thermal-thermocatalytic conversion of DPEP-(CAP-) to ETIO-series porphyrins occ ur?; Are tetrapyrroles bound to bulk OM (kerogen)?; If

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463 so, what type bonds (C-S, C-0, C-C) are involved?; What are the precursors and reactions involved in the geochemical production of the benzoporphyrins? and What is the pre-/syn-depositional fate of the chlorophylls-c?

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465 Bacon M. F and Holden M. (1967) Changes in chlorophylls resulting from various chemical and physical treatments of leaves and leaf extracts. Phytochem. 193-210. Baker E. W. (1966) Mass spectrometric characterization of petropor phyrins. J. Am. Chern. Soc. 88, 2311-2315. Baker E. W. (1969) Porphyrins. In Organic Geochemistry (Edited by Eglinton G. and Murphy M. T. J.), pp. 464-497. Springer, Berlin. Baker E. W. (1970) Tetrapyrrole pigments. In Initial Reports of the Deep Sea Drilling Project (Edited by Bader R. G. et gj.) Vol. 4, pp. 431-438. U.S. Govt Printing Office, Washington, DC. Baker E. W. (1971) Porphyrins in deep ocean sources; Petroporphyrins from Challenger knoll oil bearing cores. Chern. Geol. z, 45-49. Baker E. W. and Corwin A. H. (1966) Mesoporphyrin-IX. In Biochemical Preparations (Edited by Maehly A. C.), Vol. 2, pp. 76-79. JohnWiley and Sons, New York. Baker B. L. and Hodgson G. W. (1961) Rate of formation of the nickel complex es of pheophytin-a. J. Phys. Chern 65, 1078-1079. Baker E. W. and Louda J. W. (1980a) Products of chlorophyll diagenesis in Japan trench sediments, Deep Sea Drilling Project sites 438, 439 and 440. In Initial Reports of the Deep Sea Drilling Project (Edited by Langseth M., Okada H. et gJ.), Vol. 5657, pp. 13971408. U.S. Govt Printing Office, Washington, DC. Baker E. W. and Louda J W (1980b) Geochemistry of tetrapyrrole pigments in sediments of the north Philippine Sea, Deep Sea Drilling Project Leg 58. In Initial Reports of the Deep Sea Drilling Project (Edited by deVries-Klein G., Kobayashi K. et gl.), Vol. 58, pp. 737-739. U.S. Govt Printing Office, Washington, DC. Baker E. W. and Louda J. W. (1980c) Organic geochemistry: Highlights in the Deep Sea Drilling Project. In Advances in Organic Geochemistry 1979 (Edited by Douglas A. G. and Max well J. R.), pp. 295319. Pergamon, O x ford. Baker E. W. and Louda J. W. (1981a) Chlorophyll derivatives in sediments of the south Philippine Sea. In Initial Reports of the Deep Sea Drilling Project (Edited by Hussong D Uyeda S. et gj.), Vol. 60, pp. 497-500. U.S. Govt Printing Office, Washington, DC. Baker E. W and Louda J W (1981b) Geochemistry of chlorophyll derivatives: DSDP/IPOD Leg 61, Site 462, northern Nauru basin. In Initial Reports of the Deep Sea Drilling Project (Edited by Larson R. L Schlanger S. et gj.), Vol. 61, pp. 619-620 U.S. Govt Printing Office, Washi ngton, DC.

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466 Baker E. W. and Louda J. W. (1982) Geochemistry of tetrapyrrole, tetraterpenoid and perylene pigments in sediments from the Gulf of California: Deep Sea Drilling Project Leg 64; Sites 474, 477, 479 and 481 and Scripps Institution of Oceanography Guaymas Basin survey cruise Leg 3, Sites lOG and 18G. In Initial Reports of the Deep Sea Drilling Project (Edited by Curray J. R., Moore D. G. et gl.), Vol. 64, pp. 789-814. U.S. Govt Printing Office, Washington, DC. Baker E. W. and Louda J. W. (1983) Thermal aspects in chlorophyll geochemistry In Advances in Organic Geochemistry 1981 (Edited by Bjoroy M. et gl.), pp. 401-421. Wiley, Chichester. Baker E. W. and Louda J. W (1984) Highly dealkylated copper and nickel etioporphyrins in marine sediments. In Advances in Organic Geochemistry-1983 (Edited By Schenck P. A., deLeeuw J W and Lijmbach M.). Org. Geochem. 183-192. Baker E. W. and Louda J. W (1986a) Porphyrins in the Geologic Record, in Biological Markers in the Sedimentary Record. Methods in Geochemistry and Geophysics, Vol. 24 (Edited by Johns R. B.), pp. 125-225. Elsevier, Amsterdam. Baker E. W. and Louda J W. (1986b) Porphyrin geochemistry of Atlantic Jurassic-Cretaceous black shales. In Advances in Organic Geochemisty 1985 (Edited by Leythaeuser D. and Rullkotter J.), Org. Geochem. 10, 905-914. Baker E. W. and Louda J. W. (1987) Porphyrins as Biomarkers: A Current View. 194th National A.C.S. Meeting, New Orleans, August 3DSeptember 4, 1987. Abs. Geoc. #48. Baker E. W. and Louda J W. (1990) Qualitative and quantitative geopor phyrin analysis-!!. Mass spectrometeric characterization, indices and organic maturity trends 199th Natl. A.C.S. Meeting, Division of Geochemistry, Boston, April 22-27, 1990. Abs. Geoc. #77. Baker E. W. and Palmer S E. (1978) Geochemistry of Porphyrins, in The Porphyrins (Edited by Dolphin D.), Vol. 1, pp. 486-552. Academic, New York. Baker E. W. and PalmerS. E. (1979a) Chlorophyll diagenesis in !POD Leg 47A, Site 397 core samples. In Initial Reports of the Deep Sea Drilling Project (Edited by vonRad U., Ryan W. B. F. et gl.), Vol. 47 -Part 1, pp. 547-551. U.S. Govt Printing Office, Washington, DC. Baker E. W. and Palmer S. E. (1979b) Tetrapyrrole pigments in cretaceous sediments from the Bay of Biscay, !POD Leg 48, Hole 402A. In Initial Reports of the Deep Sea Drilling Project (Edited by Montadert L., Roberts D. G. et gl.), Vol. 48, pp. 931-933. U.S. Govt Printing Office, Washington, DC.

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467 Baker E. W. and Smith G. D. (1973) Chlorophyll derivatives in sedi ments, Site 147. In Initial Reports of the Deep Sea Drilling Project (Edited by Heezen B. C., MacGregor I. D. et gl.), Vol. 20, pp. 943-946. U.S. Govt Printing Office, Washington, DC. Baker E. W. and Smith G. D. (1975a) Chlorophyll derivatives in DSDP Leg 31 sediments. In Initial Reports of the Deep Sea Drilling Project (Edited by Karig D. E., Ingle Jr. J. C. et gl.), Vol. 31, pp. 629632. U.S. Govt Printing Office, Washington, DC. Baker E. W. and Smith A. D. (1975b) Chlorophyl l derivatives in DSDP Leg 14, 20, 26, 27 and 29 sediments. In Initial Reports of the Deep Sea Drilling Project (Edited by Karig D. C., Ingle Jr. J.C. et gl.), Vol. 31, pp. 905-909. U S Govt Printing Office, Washington, DC. Baker E. W., Ruccia M. and Corwin A. H (1964) The preparation of mesoporphyrin-IX and etioporphyrin-III Anal. Biochem. a, 512. Baker E. W., Yen T F., Dickie J. P., Rhodes R. E. and Clark L. F. (1967) Mass spectrometry of porphyrins. II. Character ization of petroporphyrins. J. Amer. Chern. Soc. 89, 3631-3639. Baker E. W., Corwin A. H., Klesper E. and Wei P. E. (1968) D eoxophyllo erythroetioporphyrin J. Org. Chern. 33, 3144-3148. Baker E. W PalmerS. E. and Parrish K. L. (1976) Tetrapyrole pigments in DSDP Leg 38 sediments. In Initial Reports of the Deep Sea Drilling Project (Edited by Talwani M., Udinstov G. et gl.), Vol. 38, pp. 785-789. U.S. Govt Printing Office, Washinton, DC. Baker E. W., PalmerS. E. and Huang W. Y. (1977) Intermediate and late diagenetic tetrapyrrole pigments, Leg 41: Cape Verde Rise and Basin. In Initial Reports of the Deep Sea Drilling Project (Edited by Lancelot Y., Seibold E. et gJ.), Vol. 41, pp. 825-837. U.S. Govt Printing Office, Washington, DC. Baker E. W., PalmerS. E: and Huang W Y. (1978a) Chlorin and porphyrin geochemistry of DSDP Leg 40 sediments. In Initial Reports of the Deep Sea Drilling Project (Edited by Bolli H. M., Ryan W. B. F. et gl.), Vol. 40, pp. 639-647. U.S. Govt Printing Office, Washington, DC. Baker E. W., PalmerS. E. and Huang W Y. (1978b) Early and intermediate chlorophyll diagenesis of Black Sea sediments: Sites 379, 380 and 381. In Initial Reports of the Deep Sea Drilling Project (Edited by Ross D. A., Neprochnov Y. P. et gJ.), Vol. 42-Part 2, pp. 707-715. U.S. Govt Printing Office, Washington, DC.

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512 Willstater R. und Stoll A. (1913) Untersuchungen uber Chlorophyll, 385 pp. Spring, Berlin. Note: English translation published as; Willstater R and Stoll A., as translated by Schertz F. M. and Merz A R (1928) Investigation on Chlorophyll, 385 pp. Science Printing, Lancaster, Pennsylvania. Wilschke A (1914) Uber die fluoreszenz der chlorophyllkomponenten. wissensch. Mikr. 31, 338-361. Wilson D. F and Erecinska M (1979) Cytochrome Oxidase, in The Popphyrins (Edited by Dolphin D.), Vol. V II, pp. 1-70. Academic, New York. Winters J. C., Williams J. A and Lewman M. D. (1983) A laboratory study of petroleum generation by h y drouspyro ly sis. In Advances in Organic Geochemistry 1981 (Edited by Bjoro y M. et a l.), pp. 524533. Wiley, Chichester. Wolfbauer C. A. (1973) Criteria for recognizing paleoenvironments in a playa-lake complex: The Green River formation of Wyoming. 26th Field Conference, Wyoming Geol. Assoc. Guidebook, pp. 87-91. Wolff G. A., Murray M., Maxwell J. R., Henter B. K. and Sanders, J. K. M. (1983) 15,17-Butano-3,8-diethyl-2,7,12,18-tetramethylporphyrin:A novel naturally occurring tetrapyrrole. J Chern. Soc. Chern. Commun. 922-924. Wolff G. A., Chicarelli M. I., Shaw G. J., Evershed R. P., Quirke J. M E. and Maxwell J. R. (1984) Structure analysis of naturally occurring alkyl porphyrins by hydrogen chemical ionisation mass spec trometry Tetrahedron 40, 3777-3786. Yeats R. S., Haq B. V. et (1981) Introduction and Site Reports. In Init. Repts. DSDP (Edited by Yeats R. S., Haq B. V. et Vol. 63, pp. 5-349. U. S. Govt. Printing Office, Washington. Yen T. F., Boucher L J., Dickie J. P., Tynan E. C. and Vaughan G. B (1969) Vanadium complexes and porphyrins in asphaltenes. J. Inst. Petrol. 55, 87-99 Yentsch C. S. (1965) Distribution of chlorophyll and pheophytin in the open ocean. Deep-Sea Res. 12, 653-666. Yentsch C. S. (1983) A note on the fluorescence characteristics of particles that pass through glass-fiber filters. Limnol. Ocean. 28, 597-599 Yentsch C S. and Menzel D. W. (1963) A method for the determina t ion of phytoplankton chlorophyll and pheophytin by fluorescence. DeepSea Res. 10, 221-231.

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513 Yentsch C. S. and Ryther J. H. (1959) Absorption curves of acetone extracts of deep water particulate matter. Deep-Sea Res. 7273. Zelmer P. P. and Man E. H. (1983) Analysis of chlorophyll diagenesis-!. Kinetics of the phorbide to porphyrin transition. Orq. Geochem. 2. 43-49. Zelmer P. P. and Man E. H. (1984) Analysis of chlorophyll diagenesis!!. Solvent effects on metal invorporation into chlorophyll diagenetic products. Orq. Geochem. I. 223-229. Zscheile F. P. and Harris D. G. (1943) Fluorescence of chlorophy lleffects of concentration, temperature and solvent. J. Phys. Chern. 47, 623-637.

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APPENDIX A Preparation. Purification. Derivat i zation and Physicochemical Characterization of Authentic Standard Tetrapyrrole Pigments 515 During the course of these studies, it soon became apparent to the author that a great many authentic standards were needed. This need derived from many fronts. First, while the classic and monumental studies of the school of Hans Fischer (see Fischer and Orth, 1937; Fischer and Stern, 1940; and references therein) have provided the world with the greatest body of tetrapyrrole chemistry ever assembled, the technical limitations of electroni c absorption spectroscopy, by today s standards, leave much to be desired. Specifically, the lack of obtaining spectra much below the blue E.A. = 454; "ende absorbierung" [end or limit of absorption] = 454 nm: cf. Fischer and Stern, 1940) precluded information as to the position of the Soret or y-band (cf. Gouterman, 1978) and its relationship ratio) to absorption maxima in the visible. Some information on the position of the Soret band for several tetrapyrrole pigments can be found in the works of Stern and co-workers (Pruckner and Stern, 1936) but the list is far from complete. Further, while the order of absorbancies of visible bands has been known for years purpurin -18 ME: I>III >II>IV>V: Fischer and Stern, 1940) and the extinction coefficients given Purpurin -18 ME; = 58.25 at 698 nm in dio xane solvent: Stern and Wenderlein, 1936b), the

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516 numerical ratio of all bands (g.g. Purpur i n -18 ME; Soret = 2 4, V = 0 06, IV = 0.12, III = 0 44, II = 0 16, I = 1 .00; setting highest absorption maximum in the visible [g.g. 400-700 nm] equal to unity: Louda, this study) is often hidden in the literature or totally nonexistent. The choice of solvent or solvent-systems employed during electronic spectroscopy was of concern as well. That is, ''pyridine ether" was usually given as the solvent system, but not the ratio of the two components (see Fischer and Orth, 1937; F i scher and Stern, 1940; Stern et gl., 1937-1937b). Given that changes in solvent composition, often minute, can lead to either noticeable or drastic changes in both the position and intensity of absorption bands (see g.g. Rao, 1961; Williams and Fleming, 1966), I concluded that 'simple' easily standardized solvents should be used. Thus, in the present stud i es all spectra are recorded either in benzene or pero xide-free ethyl ether. Pyridine, capable of acting as a base or nucleophile, is further found objectionable since its effects on the spectra of geoporphyrins, often with open a -positions (Antipenko et gl., 1979; Chicarelli and Maxwell, 1984; Fookes, 1983a+b; Hein et gl., 1985; Quirke, 1983; Quirke et gl., 1979), unique exocyclic ring structures (Barwise and Roberts, 1984; Chicarelli et gJ. 1984; Ekstom et gl., 1983; Fookes et gJ., 1983b; Ocampo et gl., 1984; Qui rke et gl., 1980, 1982; Wolff et gl., 1983) or e x tended a-alkylation patterns (cf. Baker, 1966; Baker and Palmer, 1978; Baker et gl., 1967; Quirke et gl., 1980) are totally unknown. The author does not presume to 'rewrite the book' on tetrapyrrole pigment electronic absorption spectra, as a great many e xcellent chapters on the theoretical and empirical nature of such spectra (g.g. Fischer and Orth, 1937; Fischer and Stern, 1940; 1952, 1973,

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517 1978; Longuet-Higgins et gj., 1950; Stern et gj., 1934-1937b; Treibs, 1973) exist. Rather, aside from becoming totally competent/familiar with the technique, I wished to obtain the empirical data on compounds known or suspected as being related to natural tetrapyrrole pigments in various stages of diagenesis and catagenesis. That is, to provide an empirical data base of electronic absorption spectra, resulting from known chromophoric systems, which can then be employed as a 'tool' for the preliminary identification of geopigment isolates. Second, while the mass spectra of numerous tetrapyrrole pigments are known (g.g. Baker et gj., 1967; Budzikiewicz, 1978; Budzikiewicz and v.d. Haar, 1968; Hood et gj., 1960; Jackson et gj., 1965; Mead and Wilde, 1961; Meot-Ner et gj., 1973; Shaw et gj., 1978; Sundararam et gj., 1984), it is imperative to characterize known compounds with the individual instrument utilized in any study This stems from the inherent peculiarities of mass spectrometers due to differences in geometries, ionization mode and/or voltages, accelerator potential, method of detection, operator and other variables ad infinitum (g.g. Biemann, 1962; Budzikiewicz et gj., 1964; Mclafferty, 1962; Silverstein et gj. 1974; and lead references in each). Next, the techniques of separation science require knowns for the development, refinement and standardization of chromatographic methods, be they "classical" column (cc) or the newer high-performance liquidchromatography (hplc), or liquid/liquid (g.g. organic/aqueous acid or base; organic/aqueous alcohols). Additional testing with known compounds, utilized during the present studies, includes derivatizations as esters (g.g. methyl, d h h 1 ) 11 h 1 t ( N I nat L I saN 60N C I nat L I 63C ) 1 yrop yty meta o-c e a 10n l.g. 1 1, 1, u u

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518 and reduction of carbonyl functions (i.g. ketone, aldehyde) with sodium borohydride. Lastly, all of the above 'justification' for a rather extensive program of authentic compound (viz. 'standards') formulation becomes secondary to possible wasted time and effort spent isolating and tentatively identifying pigments from often expensive and/or unique geologic samples on the basis of what may be only theoretical reality. Thus, I can only conclude from the above arguments, among others, that much more than passing notice should be, and is herein, given to authentic standard tetrapyrrole pigments during biogeochemical investigations. More than two-thirds {69%) of the compounds given subsequently were formed by the author from easily verified chlorophyll{-a,-b,-c), or hemin (viz. equine) derivatives. Numerous compounds (30%) were 'on hand' in the laboratory of Professor Earl W. Baker (g.g. DPEP, cf. Baker et 1968) as remnants of tetrapyrrole studies carried out at the Mellon Institute of the Carnegie-Mellon University in Pittsburg, Pennsylvania (g.g. E. W. Baker, J. G. Erdman, T. F. Yen, and others) during the late 1950's early 1960's. These latter compounds, source identified as "EWB" or "JGE" (i.g. Earl W. Baker or J. Gordon Erdman) herein were either utilized as received (after verification), were purified (g.g. deuteroetioporphyrin: "JGE"), and/or were derivatized further. Aside from the above 'on-hand' compounds, the use of which was generously provided by Professor Baker, the outright gift of 3 tetrapyrroles to the author must be noted. The vanadyl and nickel chelates of synthetic benzoetioporphyrin' (viz. 13,17-diethyl7,8,12,18-tetramethylbenzo[2,3]-21H,23H-porphine: Clezy and Mirza,

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519 1982; Clezy et gl., 1977) were graciously provided by Professor Peter S. Clezy. Mesopyropeophorbide-a M.E. was a most welcome, and timely (cf. Baker and Louda, 1982; Louda and Baker, 1986), gift of Dr. Pamela P. Zelmer (Zelmer and Man, 1983). A few compounds (g.g. XLI, XLIII, XLV, LIII) were obtained from the Master's research of Mr. Gustavo Guteirrez (Florida Atlantic University, 1986). These were further derivatized, purified and all data presented was collected by the present author. Given that the field of tetrapyrrole chemistry; including the subspecialties of biochemistry, biological/chemical oceanography and geochemistry; is presently fraught with nomenclatural difficulties (cf. Bonnett, 1978) due to the extensive/extended use of 'common names', and in light of recent attempts to rectify this situation (g.g. Bonnett, 1978; IUPAC-IUB, 1978), it becomes necessary to provide comment. Throughout this treatise a modified 'Fischer' ("F") nomenclature (Fischer and Orth, 1937; Fischer and Stern, 1940) wil l be maintained for both the tetrapyrrole pigments (cf. Bonnett, 1978) and the chloro phylls (cf. Seely, 1966) per se. Thus, in this section only, except for discussion of unambiguously assigned structures, will the recently proposed IUPAC-IUB (1978: viz. 'revised' "R"*) nomenclature be crossreferenced. Detailed discussion of nomenclature can be found in a recent publication of this author (Baker and Louda, 1986a). *while the author has made every attempt to provide a correct revised nomenclature identification, the present confused state, including both systematic and semisystematic revisions, begs error. Identification should thus be placed with the Fischer nomenclature and corresponding trivial name.

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520 Each compound, employed as a standard, is cross-referenced by Roman enumeration and are ordered into classes or types according to macrocyclic nuclei, shown in Figure Al, which form the basic skeletons of these pigments Peripheral substituents, which lead to the indi-vidual character of each pigment within the separate classes, are given in Table A -I. Phorbide Nucleus, Free-base and Mg Chelates All known chlorophylls, including the various bacterial forms, contain, in essence, the phorbide nucleus (Figure Ala). It was the presence of the 'extra' ring structure, the so-called 'isocyclic ring' (asterik in Figure Ala and Ale}, in geologic pigments that first allowed linkage of a constituent of 'fossil fuels (g.g. shales, petroleum, coal) to biotic sources. In this case, DPEP type (Figure Ale) geoporphyrins to parental phorbides, namely the chlorophylls (Treibs, 1936). As a large part of the author's studies deals with the pre/syn-depositional and early diagenetic continuum of tetrapyrrole geochemistry, much emphasis was given to the isolation of parental chlorophylls and in vitro derivatives which may or may not be correla-tive to geochemical isolates. (I). Chlorophyll-a: Magnesium -1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9-oxo-10-carbomethoxyphorbin 7-propionic phytyl ester. ("F''); ( pheophyt ina to _g.) magnes i urn (II) ( "R") ; C55H7205N4Mg, 892 amu. Chlorophyll-a was extracted from (a) garden variety spinach (Spinacea oleracea), (b) the fresh-water green alga (Chlorophyta) Closterium sp., and (c) the marine red alga (Rhodophyta) Acrochaetium sp. Spinach was obtained locally while the uni-algal cultures

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521 ("concentrated quarts") were purchased from Carolina Biological Supply Company, Burlington, North Carolina, U.S.A. The details of extraction and chromatographic techniques are given in the main body of the text. Chlorophyll-a was isolated andre-purified by chromatography over microcrystalline cellulose. Elution with 6% acetone in petroleum during the first recycle over microcrystalline cellulose provided a pure (viz. free of chlorophyll-b via 2nd derivative absorption spectra-scopy) isolate (cf. Strain, 1958: CC, sugar). Chlorophyll-a (I): UV/VIS. (ethyl ether) 380 (infl), 410, S = 429, 537, 578, 615, 661.5 nm = I. Band order; I>II>III >IV, S/I = 1.3, I/IV = 23; M.S. (EID) sporadic 892 m/z (M+: 0-15%), 278 m/z (phytadiene: 100%), ion cluster at 610-620 m/z (624 m/z = M-phytadiene: 5-10%). Note, poor quality (i. g. low S/N) mass spectra, probably related to thermal lability, and expected peak at 833 m/z (M-59, M-COOCH3 "pyro" reaction) is totally absent. (II). Pheophytin-a: 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9-oxo10-carbomethoxy-phorbin-7-propionic acid phytyl ester. ("F"); pheophytin-a ("R"); (Trivial) = pheophorbide-a-(IIa) phytyl ester; C55H7405N4 870 a.m.u.: -(lib) dihydropheophytin-a, or pheophorbide-a dihydrophytyl ester); C55H7605N4 872 a.m.u. Pheophytin-a (IIa) was either prepared from native chlorophyll-a (I) via mild acidic demetallation or purified chromatographically from commercially available preparations of "pheophytins-a and-b." In the former case, to chlorophyll-a (I) in acetone (g g 5 ml) was added (0.1 ml) 10% aqueous HCl (w/v) under a blanket of nitrogen. The flask was stoppered, swirled a few times to affect total admixture and set in the dark at room temperature for ca. 5-10 minutes. Magnesium removal from chlorophyll-a (I), in solution, was easily noted by a color change from bright grass-green to a dull olive-brown. The acidified acetone solution of, now pheophytin-a was added to ethyl ether (ca. 5X Vol) in a separatory funnel and extracted with copious

PAGE 550

522 (g.g. lOX vol.t) amounts of distilled water, adding strong artifical brine (NaCl) in small amounts if necessary to form a sharp biphasic aqueous/ethereal system. Preliminary neutralization of the acidified acetone solutions with saturated aqueous sodium bicarbonate (NaHC03 ) was also employed but appeared to offer no distinct advantage to resultant liquid:liquid separations, in this case. Water washing of the ethereal epiphase was continued until pH reaction showed 7.0 for at least three times running. Pheophytin-a (IIa) was recovered by evaporation of the ethereal solution in vacuo. Electronic spectro scopic and chromatographic checks usually revealed 100% conversion (I to I I). The alternate method utilized to obtain pure pheophytin-a, and the method of choice herein, was to affect the total separation of commercially available (Carl Roth KG, Karlsruhe, F. R. G.) "pheophytins-a + b" through complexation (Figure A2) of the 3-formyl moiety of pheo phytin-b (XV) with Girard's reagent "T'' (carboxymethyltrimethyl ammonium chloride hydrazide) according to the method of Wetherell and Hendrickson (1959) as refined by Kenner and co-workers (1973). Girard's reagent "T", as received (A.C.S. reagent grade), was noted to be randomly discolored (i.g. amber to brown discoloration on white crystals) and possessed a nondescript strong odor. Thus, fearing unwanted 'side reaction,' the "T'' reagent used was freshly twice recrystallized from absolute ethanol (cf. Merck #4257 in Windholz et li. 19 7 6 ) Separation of pheophytin-a (Ila) from the commercial "a plus b" mixture was, essentially a scaled-down mimic of Kenner et _gJ. (1973), as follows: 2.5 g of "pheophytins-a plus-b" was dissolved in a mixture

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523 of chloroform (70 ml) to which was added Girards "T" (1.04 g) in methanol (87 ml) plus glacial acetic acid (12 ml); the solution was flushed with nitrogen for 5 minutes and brought to reflux for 1 hour; after cooling, under a gentle flow of nitrogen, the solution was evaporated in vacuo and residual acetic acid removed by azeotrope (acetic acid/toluene: 28/72:v/v) with toluene; dissolution of the residue with methylene chloride (CH2Cl2 ) allowed direct chromatographic loading and elution with the same solvent from alumina (Al203 neutral, Grade III, 6% H20); following evaporation in vacuo a black semi-solid (pheophytin-a, IIa) was obtained. Pheophytin-a (IIa), obtained as above, was found, via TLC over silica gel or by LPHPLC with silica gel or alumina, to be pure and thus did not require recrystallization (g.g. CH2Cl2/MeOH; Kenner et 1973). During the development of a reversed phase low-pressure high performance liquid chromatographic (RP-LPHPLC; C18 silica, 13-24 pm; see text), for the standardization of pheophytin-a (IIa) from dihydro pheophytin-a (lib) separations/ca-LC identifications, 3 minor 'contaminants' were noted. Shown as Figure A3 is the resultant chromatogram of one of many semi-preparative (g.g. 75-100 pg) RP-LPHPLC determinations. Essentially there are 3 components eluting prior to, and therefore less alkyl in character than, true pheophytin-a (IIa). Albiet, the 'separation' of these four alleged components is not what one would desire, given the potential of true hplc (cf. Schoch et 1977, 1978). However, it was my desire to develop a system which could handle large (g.g. > 100 pg -1 mg) geochemical injections and provide a semi-pure isolate, of at least a single chromophore, for

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524 further fractionation, when available, by "state-of-the-art" technique. Thus, the reader's imagination is requested during the interpretation of this chromatogram by the author. During the investigations of Schoch et g]. (1977, 1978) on chloro phyll biosynthesis they formulated ... "a sequence in which Chlid is first esterified with GG and then hydrogenated via ChloHGG and ChlrHGG to ChlP." (Schoch et g], 1978)* In the study mentioned, Schoch et g]. used a C18 column eluting with methanol:acetone (9:1, v/v) for the separation of standards prepared by the esterification of pheophorbide-a free-acid (VIa) with geranylgeraniol (GG) and derivatives in successive stages of saturation (DHGG, THGG, p*). I employed the same chromatographic environment but of lesser resolving power (i.g. fewr plates; 13-24 versus 5 etc. cf. Schoach et g], 1978). Thus, I can only conclude that the 3 "peaks" eluting prior to 'true' pheophytin-a (II, i.g. phytl ester of VIa) are the corresponding GG, DHGG and THGG esters (peaks 1-3, Figure A3), respectively. Chromatographically, these assignments are reason able as, in reverse order (THGG, DHGG, GG), each 'pheophytin' would be expected to express successively less alkyl character relative to the hexahydro-geranylgeranyl (viz. phytyl) species. If one assumes that each of these 'pheophytins-a' exhibit identical milli-molar extinction coefficients, as they most likely do considering absolutely identical chromophoric character, then a reasonable estimate of the relative amounts of each component can be arrived at. Thus, using recorder *chlid = chlorophyllide-a; GG = geranylgerniol; DHGG = dihydro geranylgeraniol; THGG = tetrahydrogeranylgeraniol; p =phytol (i.g. hexahydrogeranylgeraniol; Chl =chlorophyll-a (I).

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525 response at A = 410 nm as a proxy for spectrophotometric quantitation, I estimate that only 1-2% of the pheophytin-a used in the present studies consisted of these less saturated ester forms. A scaling factor of 4x for the true pheophytin-a {Ila) peak in Figure A3 is to be noted. Pheophytin-a {Ila) utilized for 'normal-phase' (i.g. adsorption) chromatographic and electronic spectral standardizations and further derivatizations was the 'mixed' form with the 1-2% 'alternate' (i.g. GG, DHGG, THGG) esters. Pheophytin-a {Ila) used in reverse-phase chromatography was further purified, as exhibited in Figure A3. As each 'alternate' pheophytin-a made up less than 1% of total pigment, detection via solid-probe mass spectrometry was not achieved. Pheophytin-a {IIa): UV/VIS (ethyl ether) S = 409.5, 475, 505.0, 534.5, 561, 609, 666.5 nm = I. Band order, I>IV>III>II>IVa>IIa, S/I = 1.9, I/IV = 4.8; M.S. (F.D.) M+ = 870 m/z (E.I.: see text). Dihydropheophytin-a (Ilb) was prepared, in low yield, through the Lewis acid (viz. BF3 ) catalyzed esterification of pheophorbide-a free acid {VIa) with dihyrophytol. This compound was deemed of geochemical interest on two fronts. First, during the earlier work of Baker and Smith (Baker, 1970; Baker and Smith, 1973, 1974, 1975; Smith and Baker, 1974) presumed molecular ions were reported at 872 m/z. As some but, it must be noted, not all of the isolates yielding the 872 peak also exhibited a pheophytin-a-like electronic spectrum, thus precluding vinyl reduction as accounting for the 'extra' 2 mass units, a dihyrophytyl nature was proposed for the esterifying moiety (Baker, 1970). Second, phytol, or phytyl esters, and dihydrophytol, or dihydrophytyl esters, have been proposed

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526 as geochemical sources for phytadienes and phytenes, respectively (see Blumer and Thomas, 1965; Didyk et gl., 1978; Van DeMeent et gl., 1980; references in Tissot and Welte, 1978), although more recent studies place the majority of acyclic diterpenoids as deriving from archaebacterial lipids (g.g. Risatti et gl., 1984, and references given). Dihydrophytol a.m.u.) was either purchased* (Analabs, North Haven,. Conn., U.S.A.) or received as gifts of the alcohol (J. W. deleeuw) and the acetate ester (B. R. T. Simoneit). The first attempt to estrify pheophorbide-a (PPa, VIa) free-acid with dihyrdophytol (DHP) went as follows; 100 mg DHP (0.35 m moles) and 120 mg. PPa (0.21 mmoles) were mixed into 20 ml peroxide-free ether ether and, under nitrogen with stirring, 1.5 ml fumming boron trifluoride-etherate (45% BF3 : Eastman #4272: ca. 12 m moles) was added. The mixture was stoppered and left overnight in the dark at room temperature. The reaction was stopped through the addition of water (20 val) and, after adding fresh ether (ca. 5 val), pigments transferred to the epiphase. Following 3 additional water washings of the ethereal pigment solution product was recovered through evaporation in vacuo. Chromatography on columns (CC, sugar or cellulose) or thinlayers (TLC, silica) failed to reveal any non-polar (i.g. esterified) pigment, only unreacted starting material (VIa) and nondescript alteration products could be found. This reaction probably failed through the author's over conservatism on the part of dihydrophytol. That is, blanket statements such as "The use of BF3-etherate is simple and gives high yields." (March, 1968) need to be modified by addition of a mass*Production now discontinued.

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527 action (re alcohol) requirement (g.g. Marshall et gl., 1970 and references in Hickinbottom, 1957). Thus, the author's faux pas here was attempting a nearly equi-molar reaction between alcohol (i.g. OHP) and acid (i.g. PPa, VIa). Subsequent reaction between DHP and PP-a {VIa) went as follows: 400 mg DHP (1.34 m mole) and 9.44 mg PP-a {VIa, 15.95 p mole) were mixed in 30 ml anhydrous peroxide-free ether under nitrogen and 5 ml fuming BF3/etherate (ca. 40 m moles as BF3 ) added. The reaction mixture was then stoppered, under nitrogen, and placed in the dark at room temperature for 12-14 hours. In this case the reaction mixture was not washed with water but, rather, evaporated in vacuo directly, with a water trap to deactivate gaseous BF3 and chromatographed on cellulose. In this manner unreacted dihydrophytol was recovered. Contrary to my normal procedure during cellulose liquid (viz. column) chromatography (see text), the reaction residue was dissolved with acetone (ACE) and loaded into a 'head' of petroleum ether (PE) in such a way as to have the loading solvent be the equivalent of 3.5-5% ACE/PE, thus precipitating unreacted pheophorbide-a free acid and allowing development with 5% ACE/PE to elute a first fraction contain ing both DHP and PP-a-DHP ester (i.g. dihydropheophytin-a, IIb). PP-a free acid (VIa) was recovered by development of the column with 15-25% ACE/PE, as per usual. The net result of this reaction, with all yields given as mole% of original PP-a free acid (VIa), was; 524.96 pg (0.602 p mole; 3.8%) PP-a-DHP (i.g. dihydropheophytin-a: IIb), 8.17 mg (13:8 p mole; 86.8%) unreacted PP-a (VIa) and, by difference, 9.4% unaccounted/unidentified 'side-products'. The approximate molar ratios for this (viz. 2nd) reaction were taken from the work of Baker et gl.,

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528 1970 in which no indications of proximate yields or reaction conditons were given. Going on the previous 2 reactions; the first a dismal failure, ostensibly due to the lack of mass-action (re. alcohol) and not the activation of reactants, as BF3 is a very efficient carbonium-ion initiator/promoter (f.g. RCOOH+BF Reo + BF30H-=March, 1968); I concluded that there were only two conditions of reaction remaining which could be enhanced. That is, according to rea ction kinetics and rate theory (ref. Morrison and Boyd, 1966; Sienko and Plane, 1966; f.g. general t e xts and references therein), I could next alter only the concentration, a proxy for entropy and/or collision rate, or temperature, equating to enthalpy and/or kinetic energy Given that dihydro porphyrins, as most organic pigments, are prone to thermal alteration (Holden, 1976; Liaaen-Jensen, 1971; Svec, 1978) it was decided to alter concentration and re-attempt 'room-temperature' (ca. 27C, S. Florida) reaction. The third, last and most 'successful' reaction proceeded as follows: to the recovered unreacted PP-a (VIa) of the previous reaction (8.17 mg, 13.85 mole) and about 600 mg. DHP (ca. 2.02 m mole) in 7.5 ml ethyl ether was added 5 ml BF3-etherate, with reaction conditions and product recovery proceeding as given above. Recovered was 1.849 mg DHP-PP-a (IIa: 2 .12 15.3%), 5.71 mg PP-a (VIa: 9.68 69.9%) and, by difference, 14.8% (2.05 unaccountedfor 'side' or 'alteration' products During this run, aside from increasing the concentration of all reactants (viz. molarities), I also altered their ratios, inadvertently Thus, the ratios of D HP:BF3:PPa, in for the previous reaction and the present were; 77:2:1 and

PAGE 557

529 146:3:1, respectively. The net result of the third reaction scenario is that, it is not conclusive whether the increase in absolute concen-trations or the ratio of DHP/PP-a elicited the approximate four-fold increase in yield of product (i.g. PP-a-DHP, lib). However, achieving greater than 10% yield and a usable amount of product, for my present requirements militated cessation of these experiments. Dihydropheophytin-a (lib: pheophorbide-a-phytyl ester: UV/VIS (ethyl ether) S = 409.5, 475, 505.0, 534.5, 561, 609, 666.5 nm = I. Band order, I>IV>III>II>IVa>IIa, S/I = 1.9, I/IV = 4.8 (see IIa); M.S. (E.I., 70 eV) 874 (M+2), 872 (M), 814 (base, M-58), 460/461 m/z. (III) Pyropheophytin-a: 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9oxo-phorbin-7-propionic acid phytyl ester. ("F"); 132-decarbomethoxy pheophytin-a. ("R"); C53H7203B4 812 a.m.u. Pheophytin-a (IIa), derived via the Girard's reagent "T"/chroma tographic method given earlier, was dissolved (ca.1 mg) in 5-10 ml pyridine, cooled, evacuated (25 in. Hg) and sealed. The sealed tube and contents were heated at 100C for 48-50 hours, according to the method of Pennington et g]. (1964), cooled and the solvent evaporated in vacuo. Purification was via a preliminary chromatography over microcrystalline cellulose eluting with 5% acetone in petroleum ether followed by LPHPLC over silica (see text) thus allowing separation of the less polar pyre-derivative (III) from unreacted, and/or oxidized, starting material (IIa). Pyropheophytin-a (III): UV/VIS (ethyl ether) S = 409.5, 475, 505.0, 543.5, 561, 609, 666.5 = I. Band order, I>IV>III>II>IVa>IIa. S/I = 2.0, I/IV = 4.7. M.S., not determined. CIV). 10-0xy-pheophytin-a: 1,3,5,8-tetramethyl-4-ethyl-2-vinyl10-hydroxy-9-oxo-19-carbomethoxy-phorbin-7-propionic acid phytyl esters. ( "F"); 13 -hydroxy-pheophyt in-a. ( "R"); pheophyt 1 n-a -a llomer. ("Trivial"); C53H7406N4 886 a.m.u.

PAGE 558

530 Allomerized pheophytin-a (IV) was not prepared as such but, rather, found as an oxidation product formed during the demetallation of chlorophyll-a (I). That is, due to time constraints while visiting a FD-MS facility (see Acknowledgements), additional pheophytin-a was required and I hastily demetallated chlorophyll-a without the precautions of nitrogen blanketing and yellow light or dark. It must be noted that I am not certain as to whether the chlorophyll-a (I) was allomerized, thus yielding both native and allomerized pheophytin derivatives, or if allomerization occurred during the demetallationneutralization-isolation procedure. The result, of which duplication may be next to impossible, was an oxidized (viz. a preparation of pheophytin-a (IIa) in which the 'allomer' amounted to ca. 33%, by FD-MS peak intensities [Figure A4: 870 (100.0), 886 (47.1)m/z]. "Allomerized" pheophytin-a (IV) was not isolated as such, due to the very small amount of this mixture ( <20 but the preparation was later used for hplc standardization tests. 10-0xy-pheophytin-a (IV): UV/VIS: not determined. M.S. (FD) 886 m/z. (V). 9-0xydeoxo-pheophytin-a: 1,3,5,8-tetramethyl-4-ethyl-2vinyl-9-h{droxy-10-carbomethoxy-phorbin 7-propionic acid phytyl ester. ("F"); 13 -hydroxy-131-deoxo-pheophytin-a. ("R"); 9-00-pheophytin-a. ("Trivial"; cf. Louda and Baker, 1986); C55H7605N4 872 a.m.u. Pheophytin-a (IIa) was reduced with NaBH4 in ethanolic-ethyl ether, as given under "Procedures" in the text, and the product (V) purified via cellulose chromatography. Upon electronic absorption spectroscopy, hypsochromic shifts in the position of all maxima and a concurrent loss of fine structure were taken as indicating the removal

PAGE 559

of the auxochrome effect of the 9-keto moiety via reduction to hydroxyl. Attempts at mass spectrometry with this compound have, to date, failed. 531 9-0xydeoxo-pheophytin-a (V): UV/VIS (acetone) S = 396. 5, 500.0, 597, 651.5 nm =I. Band order; I>IV>II, S/I = 3.4, I/IV = 3.0. M .S., not determined. (VI). Pheophorbide-a; VIa free-acid, VIb methyl ester: 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9-oxo-10-carbomethoxy-porbin propionic acid. ("F "); pheophorbide-a ("R"); VIa, C35H3605N4 592 a.m.u.; VIb, C36H3805N4 606 a.m.u Pheophorbide-a free acid (VIa) can be derived from pheophytin-a (IIa) either through the use of chlorophyllase (Hill, 1963; Holden, 1976; Willstater and Stoll, 1913) or cold concentrated aqueous HCl (Seely, 1966). More typically, pheophorbide formation from the parent chlorophyll or pheophytin is performed via acid catalysed transestrification in one of the lower (C1 C2 ) alcohols and the product obtained is the corresponding methyl (g.g. VIb) or ethyl ester. As examples, cold methanolic HCl or 5% H2S04 in methanol are commonly used (see Fuhrhop and Smith, 1975; Seely, 1966; Svec, 1978). Large quantities (g.g. >20 g) of pheophorbide-a free acid (VIa) were 'on-hand' in the laboratory of Professor E. W. Baker and aliquotes of this supply were utilized, following cellulose chromatography, essentially as such. Pheophorbide-a methyl ester (VIb) was prepared by the author in a variety of ways, as a means of checking reaction conditions, yields and artifact formation before applying these to the much more valuable geochemical isolates. The details of each reaction is given elsewhere (see text) as the results apply to more than the present compound and problems exist with each method. Pheophorbide-a ME (VIb) was most

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532 commonly formed from the free acid (VIa) with diazomethane (CH2N2 ) in peroxide-free ethyl ether which had been washed (H20) free of base (yields X= 90+% molar). Alternately, pheophorbide-a ME (VIb) was separated from pheophorbide-b ME (XVII), with the Girard's reagent "T" method described earlier, following 'methanolysis (5% H2S04 in methanol) of commercial pheophytins-a+b. Purification of pheophorbide-a free acid (VIa) was by chromatography over microcrystalline cellulose eluting with 15% acetone in petroleum ether. The corresponding methyl ester (VIb) was purified over grade III (5-6% H20) neutral alumina developed with dichloro methane. Pheophorbide-a (VI): UV/VIS (VIa-b: ethyl ether) S = 409.5, 468 (IVa), 505.0, 534.5, 558 (IIa), 608, 666.5 nm = I. Band order; I>IV>III>II>IVa>IIa, S/I = 1.8, I/IV = 4.4: M.S., (FD) 606 m/z (M): (EI) 606 (M), 591, 574, 547/8, 531, 519, 459 m/z (see text). (VII). 9-0xydeoxo-pheophorbide-a ME: 1,3,5,8-tetramethyl-4ethyl-2-vinyl-9-hydroxy-10-carbomethoxy-phorbin-7-propionic acid methyl ester. ("F"); 13'-hydroxy-13'-deoxo-pheophorbide-a methyl ester. ("R"); 9-00-pheophorbide-a ME "Trivial" (cf. Louda and Baker, 1986); C36H4005N4 608 a.m.u. The 9-keto moiety of pheophorbide-a ME (VIa) was selectively reduced to the hydroxyl through reaction with NaBH4 in ethanolic ethyl ether as given for compound V and in the text. Hypsochromic shifts in the postion of all absorption maxima (UV/VIS} and the 'expected' mass spectrum (M = 608, 590 m/z = M -H20) confirmed the success of the procedure. Purification, when required, was over cellulose, developed with 15% acetone in petroleum ether. 9-0xydeoxo-pheophorbide-a ME (VII): UV/VIS (acetone) S = 396.5, 500.0, 597, 651.5 nm =I. Band order, I>IV>II, S/I = 3.4, I/IV = 3.0;

PAGE 561

533 (VIII). Pyropheophorbide-a ME: 1,3,5,8-tetramethyl-4-ethyl-2vinyl-9-oxo-phorbin-7-propionic acid methtl ester. {"F"); 3-vinyl-3desethyl-phytochlorin methyl ester. {"R" ); C34H3603N4 548 a.m.u. Pyropheophorbide-a ME is often prepared via reflux in collidine {Fuhrhop and Smith, 1975; Kenner et gj., 1973) or pyridine {Fischer and Stern, 1940; Pennington et gJ., 1964). Alternately, pheophytin-a {IIa) in ether (1 val) can be added to concentrated HCl (2.5 val) and heated to boiling, driving off the ether, for 1 hour (Baker et gJ., 1 968). The latter method requires esterification of the free acid to arrive at the desired product {VIII). Pyropheophorbide-a ME {VIII) was 'on-hand' as product from the previously mentioned Carnegie-Mellon investigations ("EWB" cf. Baker et _g.J_. 1968) Pyropheophorbide-a ME (VIII): UV/VIS (ethyl ether) S = 409.5, 468 (IVa), 505.0, 534.5, 558 (IIa), 608, 665.5 nm = I. Band order; I>IV >III> II>IVa >IIa, Sj,I = 1.8, I/IV = 4.4: M.S. (EI/0, EI) 548 {M+), 533, 461, 433, 237 (M+) m/z (see text). (IX). 9-0xydeoxopyropheophorbide-a ME: 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-9-hydroxy-phorbin-7-propionic acid methyl ester. ("F"); 3-vinyl-3-desethyl-9-hydroxy-9-deoxo-phytochlorin methyl ester. {"R"); C34H3803N4 550 a.m.u. Pyropheophorbide a ME (VIII) was subjected to NaBH4 reduction in ethanolic ether as given earlier (compound V) and in the text. Hypsochromic shifts in the p osition of all absorption maxima and a loss *As the revised nomenclature (Bonnett, 1978; IUPAC-IUB, 1978) makes no mention of 'pyro' 10-decarbomethoxy in the Fis c her terminology) and strongly states that includes this moiety, I can but assume that the 'pyropheophorb1des are to be named according to the new "phytochlorin" skeletal designation.

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534 of fine structure revealed the success of the reaction. Purification was via chromatography over cellulose (TLC, 15% ACE/PE). 9-0 xydeoxopyropheophor bide-a ME (IX ): UV/VIS (ethyl ether) S = 396.5, 500.0, 597, 651.5 nm =I. Band order, I >IV>III, S/I = 3.4, I/IV = 3.0: M.S., not determined (X). Mesopyropheophorbide-a ME: 1,3,5,8-tetramethyl-2,4-diethyl-9-o x o-phorbin-7-propionic acid methyl ester. ("F" ): phy tochlorin methyl ester. ("R") ; C34H3803N4 550 a .m.u. Mesopyropheophorbide-a ME (X) was not prepared by the author but, rather, was the gift of Drs. Pamela Zelmer and Gene Man of the Univer-sity of Miami (Florida : cf. Zelmer and Man, 1983). My part i cipation in that project entailed verific ation of purity and identity via lphplc and electronic absorption and mass spectrometry, respectively. This compound, the chromophore of which I believe to e xist in sediments (cf. Baker and Louda, 1984), was thus added to the standards available and subject to further derivatizations (i.g. compounds XI, XXXII, XXXIII, LXIII, LXIV, LXXVIII-LXXXI). Mesopyropheophorbide-a (X) is commonly prepared by catalytic reduction of the vinyl moiety of pheophorbide-a ME (VIb) with an atmosphere of H2 in pyridine : a c etone (1:40 v/v) over 10% Pd-charcoal, followed by reflux of the primary product (vi z. meso-pheophorbide-a ME) in pyridine (Kenner et gj. 1973) or collidine (Pennington et gl., 1973) to complete the removal of the 10-carbometho x y moiet y (i.g. the 'pyro' reaction: cf. Fische r and Stern, 1940; Fuhrhop and Smith, 1975). Mesopyropheophorbide-a ME (X). UV/VIS (ethyl ether) S = 405.2, 475-8, 501.0, 530.5 558, 600, 656.0 nm = I. Band order, I >IV>III> II>IIa = IVa, S/I = 2.4, I/IV = 4.8; UV/VIS (benzene) S = 410.5, 496, 504.0, 533.5, 558, 603, 658.5 = I. S/I = 2.4, I/IV = 4 6. M.S. (EI) 550, 548, 463, 275, 238. 0 m/z.

PAGE 563

535 (XI). 9-0xydeoxomesopyropheophorbide-a ME: 1,3,5,8-tetramethyl2 ethyl-9-1hydroxy-phorbin-7-propionic acid methyl ester. ( "F"): 13 -hydroxy-13 -deoxo-phytochlorin-methyl ester. ("R"); C34H4003N4 552 a.m.u. Mesopyropheophorbide-a ME (X), above, was subjected to reduction with NaBH4 in ethanolic ethyl ether as given elsewhere (see text). As with other "oxy-deoxo" derivatives (g.g. V, VII, IX) in the 9-keto phorbide series, positive reaction results (i.g 9-keto to 9-hydroxy) were immediately apparent both by a visually detectable color change of the solution and hypsochromic shifts in the position of all absorption maxima, as noted by electronic spectroscopy. The resultant electronic spectrum of 9-oxydeoxo-mesopyropheophorbide-a-ME (XI) was found to be essentially identical to deoxomesopyropheophorbide-a ME (XII) and the 7-propyl-despropio derivative (XIII) of the latter. This is not unexpected, as the ?-propionic acid moiety (i.g. versus ethyl or propyl) or hydroxyl (i.g. versus hydrogen) substitution has little effect upon the position or extinction of the absorption bands of phorbides (this study cf. Holt, 1959; Treibs, 1973). 9-00-Mesopyropheophorbide-a M.E. (XI). UV/VIS (benzene) S = 394.8, 497, (524), 586, 642.0 nm =I. Band order= I > IV>II>(III), S/I = 4.4, !/IV= 2.6; M.S., not determined. (XII). Deoxomesopyropheophorbide-a ME (DOMPP-a M.E.): 1,3,5,8-tetramethyl-2,4-diethyl-phorbin-7-propionic acid methyl ester. ("F"): 13'-deoxo-phytochlorin methyl ester. ("R"): 7,8-dihydro deoxophylloerythrin methyl ester. (Trivial) C34H4002N4 536 a.m.u. Deoxomesopyropheophorbide-a M.E. (XII) was available in good supply to the author as an 'on-hand' compound in the laboratory of Professor E. w. Baker. DOMPP-a M.E. (XII) was prepared by Baker et (1968) via "mild" Wolff-Kishner reaction with pyropheophorbides-a (VIII) and -b as starting materials.

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536 DOMPP-a M.E. (XII), in the author's hands, was purified further via LPHPLC over silica (see text}, in order to remove the decomposition products (viz. o x idation) attributable to age, utilized as a chromatographic standard, characterized via electronic and mass spectrometric technique and derivatized (i. g compound LXV}. Deoxomesopyropheophorbide-a M .E. (XII) UV/VIS (ethyl ether) S = 390. 0, (488}, 497. 0, 523.8, (540}, 585. 2, (612}, 639.6 nm =I. Band order= S/I = 3.4, I/IV = 3.3; M .S. (F. D ) 536 m/z. (E.I./0.} 536, 507 m/z. (E.I., 70 eV) 536, 521 (M-15}, 507 (M+2-31, and M-29}, 463 (M-73) m/z. (XIII) 7-Propyl-7-despropio-deo x omesopyropheophorbide-a : 1,3,5,8-tetramethyl-2,4-diethyl-7-propyl-phorbin ( "F"). 4,9,13,18tetramethyl-9,14-diethyl 3-propyl-phorbine, or 17-propyl-17-depropio-13' -deo x o-phytochlorin ( "R"): 7P7DP-DOMPP-a (Trivial ; Louda, this study). C33H40N4 492 a.m.u. Geochemically, this compound (XIII) and its 7,8-didehydro-derivative (XXXVI}, the ''expected" C33 DPEP product of chlorophyll-a (I) diagenesis (cf. Table 4.9 in Baker and Louda, 1986; Louda and Baker, 1983}, are extremely important knowns. That is, as an alternate reaction to the a-decarboxylation of the ?-propionic acid moiety of phorbide or 7,8-didehydro-phorbide free acids, apparently the major route (cf. Baker and Louda, 1982, 1986a}, reduction to a 7-propyl moiety is quite possible (cf. Louda and Baker, 1983). 7P7DP-DOMPP-a (XIII) was obtained as a remnant of the aforementioned Mellon-Institute studies and "is believed" to have been formed by the LiAlH4 reduction ("JGE") of DOMPP-a (XII, "EWB" ) in the free acid state (E. W Baker, pers. commun. 1986). The author's role with this compound (XIII) was that of purification (viz. purity verification) via LPHPLC over silica and alumina (see

PAGE 565

text), characterization via spectroscopic techniques and various derivatizations (i.g. compounds XXXIX, LXVI, LXXXIX, XCI). 537 7P7DP-DOMMP-a (XIII) can also be thought of as a c33 dihydro-DPEP (see XXXIX). Thus, it becomes important as a model for potential redox equilibria thought to exist in highly reducing sediments between DPEPand 7,8-dihydro-DPEP-series porphyrins (Baker and Louda, 1986b; cf. Blumer and Omen, 1961). The electronic spectra of 7P7DP-DOMPP-a (XIII: Figure AS) and that reported (Blumer and Omen, 1961) for "deoxomesoetiopyropheophor bide-a" (viz. 7,8-dihydro-DPEP: see XII), allowing for the difference in data presentation (i.g. absorptivity versus scales, respectively), are identical. The importance of this compound and its derivatives, mentioned earlier, cannot be overemphasized and, as far as I can ascertain, resultant physicochemical data are presented here for the first time. 7-Propyl-7-despropio-deoxomesopyropheophorbide-a (XIII). UV/VIS (ethyl ether) S = 389.5, (491), 498.0, 524.5, (542), 586, (616), 640.0 nm = I, S/I = 3.0, I/IV = 4.0. UV/VIS (benzene) S = 396.5, (493), 499.6, 527.0, (545), 587, (618), 641.0 nm =I, S/I = 3.3, I/IV = 3.7 (Figure AS). Band order= M.S. (E.I.) 492 (M+), 449 m/z (M-43, a-cleavage of n-propyl: see text). (XIV). Chlorophyll-b: Magnesium-1,5,8-trimethyl-4-ethyl-2-vinyl3-formyl-9-oxo-10-carbomethoxy-phorbin-7-propionic acid phytyl ester. ("F"); chlorophyll-b. or (pheophytinato .Q.) magnesium (II). ("R"); 3Formyl-3-desmethyl-ch lorophyll-a. (Trivia 1); C55H7006N4Mg, 906 a.m. u. Chlorophyll-b (XIV) was isolated from both spinach oleracea) and the chlorophyte Closterium sp., as given earlier for chlorophyll-a (I). Chlorophyll-b (XIV), free from the a-series pigment (I) as shown via 2nd derivative absorption technique, eluted during the first

PAGE 566

recycle over microcrystalline cellulose (see text) with 7. 5-10.0 % acetone in petroleum ether. 538 Chlorophyll-b (XIV): UV/VIS (ethyl ether) 430 (infl), S = 442.5, (550), (568}, (595}, 642. 0 nm = I. Band order= I>II>III>IV, S / I = 2.7, 1/IV = 8.3: M.S., not determined (viz. unrecognizable pyrolytic breakdown). (XV). Pheophytin-b: 1,5,8-trimethyl-4-ethyl-2-vinyl-3-formyl-9oxo-10-carbomethoxy-phorbin-7-propionic acid phytyl ester. ("F"); Pheophytin-b ("R"}; Pheophorbide-b-phytyl ester. (Trivial}; C55H7206N4 884 a.m.u. Pheophytin-b (XV} was obtained ether via the mild acidic demetallation of chlorophyll-b (XIV) or, more commonly, via the chromato graphic separation of commercial "pheophytins-a and -b, as described previously for pheophytin-a (IIa} except for the omission of formyl complexation with Girard's reagent "T." Final purification of pheophytin-b (XV}, in either case, was with LPHPLC over methanol deactivated silica (see text) ran isocratically with 3.5% acetone in petroleum ether. Pheophytin-b (XV}: UV/VIS (ethyl ether) 372, 412, S = 434.5, 523, (552}, 600, 654.8 nm = I. S/I = 4.7l !/IV= 2.9. Band order= M.S. (F.O.} 884 m/z. (XVI). 3-Methanol 3 -d esformyl 9-oxy-9-deoxo-pheophytin-b: 1,5,8-trimethyl-4-ethyl-2-vinyl-3-methanol-9-hydroxy-10-carbomethoxy-phorbin ?-propionic acid phytyl ester. ("F"); 7-Demethyl-7-(methanol}-13' deoxo-13'-(hydroxy}pheophytin .Q. (semisystemic, "R") or: 4,8,18trimethyl-14-ethyl-13-methanol-20-hydroxy-phorbine-3-propionic acid phytyl ester (systematic, "R"}; 3 MDF-900-pheophytin-b. (Trivial}; C55H7606N4 888 a.m.u. 3MDF-900-Pheophytin-b (XVI} was formed through the reduction of both carbonyl moieties of pheophytin-b (XV} with sodium borohydride in *Field desorption mass spectrum determined on mixture of pheophytins-a plus -b, from which XV was isolated, but not on XV per

PAGE 567

539 ethanolic ether. A large (i.g. 34 nm) hypsochromic shift in the position of Soret (S) absorption (i.g. XV, S = 434.5; XVI, S = 402.0) attests to the removal of 2 carbonyl moieties from ring conjugation (see text). Given that the presence or absence of a propionic ester moiety, be it an isopenoid or Cl/C2 alcohols, has little, if any, effect upon the chromophore of pheophorbide nuclei (Fischer and Orth, 1940; Gouterman, 1978; Stern and co-workers, 1934-1937b; Treibs, 1973; cf. Table 10, this study), then one can reasonably expect, as shown elsewhere (see text: Table 10), similar, if not identical, results with identical chromophores (see compound XVIII): In this light, the electronic spectrum reported for the methyl ester of 3MDF-90D-pheophorbide-b (XVIII, herein) reported by Holt (1959) should essentially match that obtained here for 3MDF-90D-pheophytin-b (XVI) and, subsequently, XVIII, specifically. In the main, close agreement was found, with one notable exception. That is, in the original study of Holt (1959), he reports a maximum at 622 nm. This peak I did not observe in the spectra of either XVI or XVIII. However, using the data base presented in the present work, this spurious peak is immediately recognizab l e. The maximum which Holt (1959) reported at 622 nm for both the ''oxy-deoxo" derivatives of pheophorbide-a ME (i.g. VII) and pheophorbide-b ME (i.g. XVIII), and, by arguments given above, expected in the present case (XVI), I attribute to contamination with the corresponding copper chelates. Specifically, the intense low-energy bands of Cu-900 pheophorbide-a ME (LXII) and Cu-3MDF-90D-pheophytin-b (LXVIII) are located in the 620-630 nm region (cf. Table 10; in text).

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540 It is reported that "mild" borohydride reduction of pheophytin-b (XV) or pheophorbide-b ME (XVII) will yield only the corresponding 3methanol-3-desformyl derivatives (Hynninen, 1979). In the present study such a 'stepwise' reduction was not attempted. Rather, I opted for the use of excess reductant in all cases, in order to obtain results more directly applicable to geochemical isolates which are often, if not usually, present in small amounts and accompanied by uncharacterized, potentially ketonic co-chromatographic), contaminants. 3-Methanol-3-desformyl-9-oxy-9-deoxo-pheophytin-b (XVI): UV/VIS (ethyl ether) S = 402.5, 503.0, (528), (547), 595, 650.0 nm = I. S/I = 3.6, I/IV = 2.8. Band order= I>IV>II>(IIIa)>(III); M.S., not determined (pyrolytic). (XVII). Pheophorbide-b ME: 1,5,8-trimethyl-4-ethyl-2-vinyl-3formyl-9-oxo-10-carbomethoxy-phorbin-7-propionic acid methyl ester. ("F"); Pheophorbide-b methyl ester ("R"); 3-Formyl-3-desmethyl pheophorbide-a ME (Trivial); C36H3606N4 620 a.m.u. As given for pheophorbide-a ME (VIb), pheophorbide-b ME (XVII) can be obtained from the parent chlorophyll -b, XIV) or pheophytin (XV) via the acid catalyzed demetallation/transesterification, respectively, with mineral acids HCl, H2S04 ) in methanol (Fuhrhop and Smith, 1975; Seely, 1966; Svec, 1978). Similar acidic methanol 5% H2S04 in MeOH, dry) treatment of pheophorbide-b free acid will also yield the methyl ester. In the present study pheophorbide-b ME (XVII) was prepared in 2 ways. These being the decomposition of the Girard's reagent "T" ('hydrazone') complex (see compound II, Figure A2) and essentially the same, with the products of the chromatographic separation of the methyl

PAGE 569

pheophorbides-a {VIb) and -b (XVII) formed via the acidic meth anol transesterification of commercial 'pheoph ytins-a and b.' 541 In the former case, the pheophy tin-b/Gira rd's "T" comple x elu ted from alumina (neutral, Grade II-III, 3 5 % H20) with CHCl3/MeOH, 2 0 :1 (v/v) and, following evaporation of the eluting solvent, was disso lved in methanol (200 ml)/ acetone (10 ml)/H2S04 (6.5 ml) and sti r r ed, at room temperature, overn i ght (ca 14 hrs.) under nitrogen i n t h e dark The reaction mixture was diluted with an equal volume (c a. 215 ml) o f chloroform, washed several times with water, dilute aqueous NaHC03 and water, in that order. The organic hypophase fro m the last wash was evaporated in vacuo, dissolved with minimal dichlo romethane (CH2C l2 ) and chromatographed over alumina (Grade II-III, neutral). Elut ion with CH2Cl2 afforded pheophorbide-b ME (XVII), which developed as a deep brownish-grey band. The above procedure is a modification of that given by others (Fuhrhop and Smith, 1975; K enner et . 1973) Alternately, commercial 'pheophytins-a and -b' were conver ted to the corresponding pheophorbides via 5 % H2S04 in MeOH treatment (phytyl methyl transesterification) and separated as f ollows; 2.5 g com mercia l {Roth, F. R. G ) 'pheophytins-a and -b' were stirred with 2 0 0 ml 5 % H2S04/95% MeOH (vjv) for 18 hrs with the passage o f a s low (1-2 ml min-1 ) stream of nitrogen. Reaction was stopped by dilution of the above mixture with 200 ml CHC13 and washed w i th equal v olumes (i.g. 400 ml) of water 5 times. The chloroform hypophasic solu t ion was dehydrated over activated Na2S04 and evaporated in v acuo. Subsequent reaction of the 'pheophorbides-a and -b methyl esters' with Girard's reagent "T'', chromatographic separation and the regene r ation of p heo-

PAGE 570

phorbide-b ME (XVII) followed that given earlier (compound IIa) and above, respectively. 542 Pheophorbide-b ME (XVII): UV/VIS (ethyl ether) 372, 412, S = 434.5, 552, 600, 654.8, nm = I. S/I = 4.6, I/IV = 2.9 Band order= M.S. (E.I./0.) 622, 620 (M+), 607 (M+2-15), 606 (M, free ac1d?), 578 (606-28), 563 (M-58+1), 548 (M-73+1; and/or 606-58), 533 (M+2-87), 473 (M-147), 471 (M-2-147 and/or M-145) m/z (see text). (XVIII). 3-Methanol-3-desformy l -9-o x y-9-deo xo-pheophor bide-b methyl ester: 1,5,8-trimethyl-4-ethyl-2-vinyl-3-methanol 9-hydro x y-10carbometho x y-phorbin-7 propionic acid methyl ester. ("F"); 7-methanol7-deformyl-13'-hydro x y-13'-deo x o-pheophorbide-b methyl ester, -or4,8,18-trimethy l -14-ethyl-9 vinyl-20-hydro x y-13-methanol-phorbine-3propionic acid methyl ester. ("R"); 3-MOF-9-00-pheophorbide-a ME (Trivial); C36H4006N4 624 a.m.u As with other 'oxy-deoxo' derivatives, pheophorbide-b ME (XVII) was reduced to 3-MOF-9-00-pheophorbide-b ME (XVIII) with sodium borohydride in ethanolic ethyl-ether. Conversion of the chromophore, as detected by electronic absorption spectroscopy, to essentially that of a 'vinyl-phorbide' (see text) revealed the success of the reaction. No attempt was made to prepare the partially reduced product (i.g. 3-methanol-3-desformyl-pheophorbide-b ME), according to the arguments given above for compound XVI (cf. Hynninen, 1979). An absorption maximum at 622 nm, reported for XVIII by Holt (1959), was not observed. As with the 'dioxy-dideo xo' form of pheo phytin-b (i.g. XVI), I attribute the previous report (Holt, 1959) of said absorption to contamination with small amounts of the corres ponding copper chelate (cf. compound LXVII), which is lacking in the present case. 3-Methanol-3-desformyl-9-o x y-9-deo x o-pheophorbide-b ME (XVIII): UV/VIS (ethyl ether) S = 402.5, 503.0, (528), (547), 595, 650. 0 nm = I. S/I = 3.2, !/IV= 2.6. Band order= I > IV>II>(IIIa) >(III); M.S. (E.I./0.) pyrolytic (see text).

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543 (XIX). "Chlorophylls-c": Chlorofucine (Sorby 1873) chloro phyllin-y 1906); chlorophyll-y 1913); chlorophyll-c (W1lschke, 1914); Chlorophyll-c =magnesium complex of POfphip-7-acrylic acid 2 ("F"); Chlorophyll-c1 ("R", trivial); 3\3, 17 ,17 -tetradehydro-13 -(methoxycarbonyl)phytoporphinatomagnesium (II). ("R"); Chlorophyll-c/ =magnesium complex of 1 3 58-tetraacid. <;F"); Chlorophyll-C2 ("R", trivial); 3\32,8,8 ,17\172-hexade hydro-13 -(methoxycarbonyl)phytoporphinatomagnesium (II). ("R"): C35H3005N4Mg, 610 a.m.u.; C35H2805N4Mg, 608 a.m.u. Aside from structural differences between 'chlorophylls-c' (XIX) and the 'regular" chlorophylls-a (I) or -b (XIV), the history, albiet even the existence (cf. Willstater and Stoll, 1913), of chlorophyll-c is quite worthy of note. The existence of chlorophyll-c (viz. 'the third component') was, perhaps, first mentioned by Sorby (1873). This was followed by Tswett's (1906) studies of the adsorption of chlorophylls, and caroten-aids, on an extremely wide range of solids (i.g. development of chroma-tography) in which a "chlorophyllin-y" was suggested. As an outcome of the first truely detailed phyisico-chemical studies on the chloro-** phylls, Willstater and Stoll (1913) concluded: "The assumed third component is not a natural pigment." The following year, A. Wilschke (1914), using fluorescence technique, concluded 'chlorophyll-c' (= 'chlorophyllin-y' of Tswett, 1906; as modified to 'chlorophyll-y' by Willstater and Stoll, 1913) to be a natural product and suggested the *The Fischer ("F'') nomenclature given is this author's attempt to predict the systematic names that the Fischer school 'would' have given to chlorophyll-c1 and -c2 (c1/cz designation from Jeffrey, 1969; structures from Dougherty et gl., 1966, 1970). Phaeoporphyrin-a5 was used as the nomenclatural model Fischer and Stern, 1940). **ouring which, it must be noted, the majority of terms/ reactions/descriptive parameters (g.g. 'allomerization,' number, HCl number, chlorophyllase activity, visible order, ac1d versus alkali degradative routes, et cetera) were def1ned.

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544 moniker we use today. The monumental work of the Fischer school (Fischer and Stern, 1940) paid little more than 'lip-service' to this 'third component,' perhaps placing more emphasis on the results of Willstater and Stoll (1913) than those of Wilschke (1914). Thus, the 'phaeoporphyrin nature of chlorophyll-c' might not have gone unproven for nearly 2 decades, as eventually brought out by S. Granick (1949). That is, in this author's opinion, had any of the Fischer school given even cursory exam to chlorophyll-c, its phaeoporphyrin nucleus would have immediately been recognized. This, of course, rests on the fact that a minimum of 51, including the "magnesium complex", compounds with the 'phaeoporphyrin-a5 nucleus (alt. chromophore) were reported (cf. Fischer and Stern, 1940). The importance of chlorophyll c in the global photosynthetic budget, as well as its biosynthetic/structural relationships (g.g. 'allomerization,' fluorescence, etc.), was, however, first brought to scientific fact in the study of H Strain and W. Manning (1942). So steadfast in their convictions on the role of chlorophyll-c in the biosphere they went on to state: Between 455 and 490 the amount of light absorbed by chloro fucine was much greater than that absorbed by chlorophyll a .. One is forced to the conclusion that chlorofucine may be an important pigment in the carbon economy of nature (Strain and Manning, 1942). The concept that 'chlorophyll' is in direct relationship to "phyto plankton standing crop" is definitely not new (cf. Harvey, 1934; Kreps and Verbinskaya, 1930). However until the true importance of chloro-. phyll-c in the aquatic (incl. marine) realm was accepted did 'routine' estimation of its abundance enter standard procedure (Creitz and Richards, 1952; Jeffrey, 1974, 1980; Parsons and Strickland, 1963;

PAGE 573

545 Richards and Thompson, 1952; UNESCO, 1966). As a near 'aside,' the range of light attenuation for which chlorophyll-c exceeds chlorophyll a (i. g. 455-490 nm), as noted (viz stressed) by Strain and Manning (1942) is, (i.g. natural selection; cf. Darwin, 1858) in no small manner linked to the natural transm i ttance of light in water (i.g. max. = 475 2nm: see g.g. Riley and Chester, 1971: cf. Richards 1952). Thus, together with certain, albiet adaptive, carotenoid (i. g tetraterpenoid) chromophores (g.g fucoxanthin, peridinin, etc., Jeffrey, 1980; Jensen and Sakshaug, 1973; Ramus et gJ. 1976a-b; Yentsch, 1983), chlorophyll-c allows photoautotrophism to extend to depths not allowed by the strict quantum mechanisms of 'chlorophyll-a plus -b' photo systems (cf. Jeffrey, 1980; Parsons et gJ. 1977; Richards, 1952). Least the reader consider the above mere ramblings, let me state, with much hindsight, that while I (Louda, this study) and others (Bidigare et gl., 1985; Jeffrey, 1974, 1980; Richards and Thompson, 1952) have been able to identify 'chlorophylls-c' in water column detritus, its 'apparent' absence in sediments deposited/collected beneath the photic zone (cf. Gillan and Johns, 1980) presents an enigma. That is, is chlorophyll-c (i.g. C1 and C2 : XIX) preferentially destroyed or, rather, is it in some manner complexed or taken into early geopolymers and thus 'hidden' as an identifiable species as of yet? Going on the above, it therefore was necessary to become analytically familiar with 'chlorophylls-c' (XIX) and certain of its breakdown products. 'Chlorophylls-c' (XIX) were extracted both from the pelagic brown alga Sargassum sp. (Phaeophyceae), collected locally as fresh specimens within the adjacent coastal waters of South Florida, and the freshwater

PAGE 574

diatom Synedra sp. (Bacillariophyceae) purchased from Carolina Biological Supply Company (Burlington, North Carolina, U.S.A.). 546 Initial 'experimental' extractions revealed the necessity for pretreatment of algal material in a manner which would, apparently (cf. Holden, 1972; Jeffrey, 1969), inactivate cellular enzymes and/or acids. That is, the 'chlorophylls-c' (XIX) fraction (i.g. cellulose fraction 3 : see "Materials and Methods" in text) obtained from the extracts of fresh viable Phaeophyta always contained unidentified 'polar chlorins' as well (Figure A6a). However, if brown algae were first frozen (cf. Jeffrey, 1969) these artifacts failed to form (Figure A6b). Similar polar chlorins' are found by the author in e xtracts of, pre-frozen, aged diatom cultures and were observed to increase with depth in the marine water column (see text), as is detailed elsewhere Having therefore observed that the extraction of fresh viable material is artifactual, all subsequent work was performed on samples which were initially frozen for 6-24 hours. Based on the above preliminary studies, the extraction-isolation of 'chlorophylls-c' (XIX), primarily from Sargassum sp., was as follows. Frozen Sargassum sp., previously freed of epiphytic growths and the heavier ribs and pneumatophores, was ground with acetone: methanol (1:1, vjv) in an all glass 'hand-homogenizer.' A trace of MgC03 commonly used to prevent 'pheophytinization' (see Hill, 1963; Holden, 1972; Smith and Benitez, 1955; Strain and Svec, 1966) due to 'cellular-acids,' was included only in the first 2 extractions. The cursory use of base is due to more modern evidences that neutralization of such cellular acid activity is neither fast nor complete (Svec, 1978) and that tetrapyrrole carboxylic acids (g.g. pheophorbides,

PAGE 575

547 'chlorophylls-c') can be essentially irreversibly bound to excess carbonate (Holden, 1972). Following 5 extractions with acetone/ methanol, as given, further extraction was with acetone/petroleum ether (1:1, vjv) until additional extracts were colorless and lacked fluor-escence. Pooled extracts were reduced in volume in vacuo {T<40C), the pigments driven into ether by liquid/liquid technique and washed free of salts and residual methanol with water. The crude e xtract, in ether, and the aqueous layer were checked spectrophotometrically for the disposition of pigment. This check is necessary as it is possible to attain an alcoholic aqueous phase which leads to the unwanted disposal of chlorophyll-c {cf. Smith and Benitez, 1955). The ethereal pigment extract was next evaporated in vacuo and chromatographed over microcrystalline cellulose, as given elsewhere (see text). The fraction containing 'chlorophyl ls-c' (XIX) eluted from cellulose with acetone/methanol (9:1, v/v). This fraction was found to be either contaminated with artifactual 'polar chlorins' (i.g. probable di-and tri-carboxylic acids), as detailed above (Figure A6a) or to be essentially pure, relative to other p i gments, 'chloroph ylls-c' (XIX, Figure A6b). According to the electronic spectral data given by Jeffrey (1969) on separated chlorophylls-c1 and -c2 the reported here (Figure A6b) appears to contain appro x imately equal amounts of the 2 forms. Further fractionation of 'chlorophyl ls-c was not attempted in the present case 'Chlorophylls-c' (XIX): UV/VIS (ethyl ether) S = 447, B = 580, a = 629 nm. S/a 11-12, a/B 1.1. (Figure A6b: cf. 1 a/B = 1.5: 2 a/B = 0.9; Jeffrey, 1969*). M .S., not determ i ned.

PAGE 576

548 (XX): Pheophytin-c ("F," trivial), pheophorblde-c1 and -c2 ( R, trivial). Systematics in "F" or "R" systems, refer to compounds XIX and deleate magnesium chelation: 1 C35H3205N4 588 a.m.u.; 2 C35H3005N4 586 a.m.u. According to standard definition (see Bonnett, 1978; Seely, 1966), affecting the removal of Mg from a chlorophyll by mild acid treatment yields the corresponding pheophvtin. Implicit in the same definition is, however, retention of the esterifying isoprenoid alcohol phytol, geranylgeraniol, etc.). Thus, upon the structural elucidation of chlorophylls-c (Dougherty et QJ., 1966, 1970) it became evident that simple Mg-loss from 'chlorophylls-c' (XIX) can but yield the corres ponding 'pheophorbides-c' (XX). Recent nomenclatural revisions "R") now reflect these facts (cf. Holt, 1966). 'Pheophorbides-c' (XX) were generated from Sargassum sp. 'chlorophylls-c' (XIX) by the treatment of the later in acetone with cold 10% aqueous HCl. The solution was neutralized with saturated aqueous NaHC03 diluted with water and pigment transfered to ethyl ether. 'Pheophorbides-c' (XX) were recovered by evaporation of the ethereal epiphase, after water washing several times. 'Pheophorbides-c' (XX): UV/VIS (ethyl ether) S = 430, 495, 574, 595, 625 =I. S/I = 25, I/IV = 0.6. Band order= M.S., not determined. (XXI). Bacteriopheophytin-a: 1,3,5,8-tetramethyl-4-ethyl-2acetyl-9-oxo-10-carbomethoxy-(3,4)-dihydro-phorbin-7-propionic acid phytyl ester ("F"); ("R", trivial) or 2,7,12,18-tetra methyl-8-ethyl-3-acetyl-13 -oxo-13 -(methoxycarbonyl) cyclopenta[at] bacteriochlorin-17-propionic acid phytyl ester ("R", systematic); 2acetyl-2-desvinyl-3,4-dihydro-pheophytin-a (trivial): C55H7606N4 888 a.m.u. The source of bacteriopheophytin-a (XXI), used as a 'standard' herein, was an intertidal (littoral) bacterial 'mat' collected by the author on the south side of Gandy Boulevard (St. Petersburg, Florida)

PAGE 577

549 in Tampa Bay (27 52.3'N x 82 35.7'W). This bacteria l bloom was noted in the mid-littoral only upon disruption of the surface sediments footprints). Collection of the 'purple zone' (ca 1 em th ick) was via 'spade and bucket' and included much sand, as well as psammon diatoms and other probable interstitial unicellular algae of mixed classes, as noted below. Highly reducing conditions color change to grey/ black with sulfides and smell of H2S) was not encountered until depths of an additional 5-6 em below the 'purple zone' were excavated. Thus, it is likely, but certainly not definitive, that the population sampled was Athiorhodaceae versus Thiorhodoceae). Further classification was not attempted n or pertinent to this study, as only the presence of large amounts of bacteriochlorophyll-a (Figure A7) was required and the presence of absorption at 772 or 757 nm is highly specific for bacteriochlorophyll-a or bacteriopheophytin-a (XXI), respectively (cf. Palmer et gl., 1982; Pratt and Gorham, 1970; Sanger and Gorham. 1973; Smith and Benitez, 1955; Strain and Svec, 1966). Since the source of the bacteriopheophytin-a (XXI) 'standard' was not a pure culture, although mono-specific 'blooms; are not atypical in nature (cf. Kormondy, 1969; Stanier et g] 1970), prudence dictates more than passing note on the isolation and purification of this compound (XXI). Further, experience during this phase was employed during the study of more strictly geochemical samples "Mangrove Lake" and "Pond Apple Peat," see text). The bacterial 'mat,' containing more sand than biomass, was transported in a sea-water wet condition from the coliection site to Florida Atlantic University (Boca Raton, Florida) and extractio n was begun about 6-8 hours later. Extraction of this sample was via ball-

PAGE 578

550 milling (see text) with the following solvent systems, used for periods of 5-15 minutes each: acetone (ACE)/methanol (MeOH), 1:1 (1x); ACE/MeOH, 9:1 {4x); and benzene/MeOH, 1:1 (Bx). The acetone used, in this case, was 1/10th saturated with respect to MgC03 1 val. MgC03-saturated ACE plus 9 vols. pure ACE). E xtracts were filtered, sequentially pooled via evaporation in vacuo, dissolved in deperoxided ethyl ether and washed several times with distilled water to remove both sea salts and MgC03 At this point, the electronic abso rption spectrum of the 'crude extract' {Figure A7) revealed the presence of bacteriochlorophyll-a (A1 = 772 nm; cf Smith and Benitez, 1955) and chlorophyll-a (A1 = 662; compound I). 'Chl?rophylls-c' (XIX) absorption is masked by the presence of large quantities of carotenoids and the non-band I absorptions of chlorophyll-a (I) and bacteriochlorophyll-a. 'Chlorophylls-c' (XIX) were noted as being present during subsequent chromatographic fractionation but were not thoroughly typed in this case. The crude e xtract, in ethyl ether, was washed twice with cold 5 % HCl, aqueous (w/v), in order to affect magnesium removal, and rinsed with water until neutral to pH reaction 'pheophytinized' mixture from above was fractionated over microcrystalline cellulose with the 'multi-fraction' technique (see text) using increasing percentages of acetone in petroleum ether. As per usual, the extract was "loaded'' in acetone and 'locked' onto the adsorbant with copious petroleum ether relative to acetone volume) such that the initial eluate was ca. 0.5 % in acetone. Follow ing the less sorbed carotenes and carotenols, bacteriopheophytin-a (XXI) and pheophytin-a {IIa) eluted in 3.5-5.0 % a cetone/petroleum ether, in that order. Development and elution resulted in the 2

PAGE 579

551 pheophytins emerging as partly overlapping Gaussian distributions, skewed relative to emergence from the column) positively and negatively, as found by elution analysis (Table AI!), in the cases of bacteriopheophytin-a (XXI) and pheophytin-a (IIa), respectively. Fraction 4 (Table AI!) was found to contain a near co-dominance of carotone-diols and bacteriopheophytin-a (Figure AS). Thus, a common carotenoid fractionation technique, referred to as hexane/aqueous methanol partition (cf. Foppen, 1971; Krinsky, 1963; Louda, 1978; Petracek and Zechmeister, 1956), was applied in order to affect a rapid partial purification. In this case, fraction 4 from above was 'wet' with a few drops of CH2Cl2 taken into n-hexane (pre-saturated with 90% aqueous methanol) and thrice extracted with 90% aqueous methanol (pre saturated with n-hexane) via conventional liquid:liquid technique. The electronic spectrum of the methanolic hypophase (Figure A9 'dashed') is characteristic of only carotenoid (viz. carotenol/carotene-diol) chromophores, while that of then-hexane epiphase (Figure A9 'solid') revealed effective diminution of same with respect to bacteriopheo phytin-a (XXI). Carotenoids remaining in the epiphase, obviously lacking hydroxyl moieties and exhibiting low energy maxima 460, 485, 523 nm), relative to carotenoid systems with eleven double bonds in conjugation (cf. Davies, 1976; Foppen, 1971), suggest themselves as being aromatic carotenoids (g.g. renierapurpurin, renieratene, etc.: see Davies, 1976; Liaaen-Jensen, 1978), thus lacking a propensity to partition into aqueous methanol. The carotenoid 'contaminants' of the bacteriopheophytin-a (XXI) fraction were not typed further. Then-hexane epiphase from above, and containing all of the bacteriopheophytin-a (XXI) complement of fraction 4 described

PAGE 580

552 was next subjected to reversed phase LPHPLC (see text). In this case, the residue obtained upon evaporation was dissolved in minimal acetone and injected onto a 8 x 570 mm column of C-18 bonded ODS) silica gel (13-24 Whatman #LRP-1) and developed isocratically with methanol/acetone 95:5 (v/v) at 150 p.s.i.g. (2.3 ml-min-1). The resultant chromatogram (Figure AlO}, and subsequent electronic spectral analysis, revealed the complete separation* of bacteriopheophytin-a (XXI; fraction D, Figure AlO} from carotenoid (fractions A-C, Figure Al9) and pheophytin-a (IIa; fraction E, Figure AlO} contamination. The 'heart-cut' of the RP-LPHPLC fraction "D" from above, conformed with published data on more highly confirmed isolates Smith and Benitez, 1955; Strain and Svec, 1966). The electronic spectrum of the resultant isolate (Figure All} also matches pure bacteriopheophytin-a (XXI}. Bacteriopheophytin-a (XXI): UV/VIS (ethyl ether) 52**= 362.0, Sl = 388, (465), (500), 530, 635, 688, 757.0 nm = I. Band order, I>IV>II>IVa>IVb>III, S2/Sl = 1.7, S2/I = 1.4, I/IV = 3.0 {Figure All); M.S., not determined. (XXII). 2-a-hydroxy-ethyl-2-desacetyl-9-oxy-9-deoxo-bacteriopheo phytin-a: 1,3,5,8-tetramethyl-4-ethyl-2-a-hydroxy-ethyl-9-oxy-10carbomethoxy-{3,4}-dihydro-phorbin-7-propionic acid phytyl ester {"F"). 2,7,12,18-tetramethyl-3,8-diethyl-3',13'-dioxy-132-(methoxycarbonyl) cyclopenta[at]-bacteriochlorin-17-propionic acid phytyl ester {"R''). 3,9-dioxy-3,9-dideoxo-qacteriopheophytin-a (trivial). C55H8006N4 892 a.m.u. *The 'shoulder' of fraction "0" (Figure AlO} is not a separate pigment but, rather, a particle size distribution phenomenon (see text) peculiar to this packing. **As compound XXI exhibits a bifurcated Soret band, I have arbitrarily designated the high and low energy maxima as S2 and Sl, respectively.

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553 Bacteriopheophytin-a (XXI) was treated with NaBH4 in ethanolic ether as given elsewhere (see text). Chromophore alteration, via reduction of 2 conjugated carbonyl moieties was immediately obvious upon electronic spectrometric analysis of the product (XXII. Figure A12}. In this case, the bifurcated Soret band(s) switched extinction order (i.g. S1>S2} and were hypsochromically shifted ca. 13 nm. Major visible absorption maxima, the so-called bands I and IV, were also found to have hypsochromically shifted 41 and 33 nm, respectively. The entire 'appearance' of the product's (XXII) electronic spectrum also has taken on a sharper fine structure. That is, major absorptions have narrower half-band widths resulting from the removal of carbonyl conjugation which tends to extend the energy range of each allowed transition (see text; cf. Gouterman, 1978}. As far as I can tell, this product is reported herein for the first time, a somewhat amazing statement given the simplicity of the techniques involved. An attempt was made to prepare the copper chelate of 2,9-dioxy2,9-dioxo-bacteriopheophytin-a (XXI). The resultant spectrum, excepting absorption at ca. 360-362 and 728, is all too reminescent of Cu-9-oxy-9-deoxo-pheophorbide-a ME (LXII) and Cu-9-oxy-9-deoxo mesopyropheophorbide-a (LXIV) to claim success. That is, given the reported facile nature of 'bacteriochlorin' oxidation to 3,4-didehydrobacteriochlorin nuclei (see Fischer and Stern, 1940; Inhoffen, 1968; Scheer, 1978; Scheer and Inhoffen, 1978}, it appears that the attempt at room temperature metallation with cupric sulfate in methanolic CH2Cl2 even with nitrogen flush/blanket and the absence of non-yellow (1 = 575-585 nm) light, either the presence of cupric ion or an enhanced reducing nature of the copper product (Cu XXII) lead to the

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554 oxidative formation, ostensibly, of Cu-2-a-hydroxy-ethyl-2-desvinyl-9oxy-9-deoxo-pheophytin-a. Investigation of the above was halted due to time limitations and the small scale (<10 of the reaction. 2-a-hydroxy-ethyl-2-desacetyl-9-oxy-9-deoxo-bacteriopheophytin-a (XXII): UV/VIS (ethyl ether) S2 = 350.6, S1 = 374.0, 441, 468, 497.2 (IV), (602-8), (660), 682, 715.8 nm = I. Band order, I>IV>IVa = II>III>IVb>IIIa (Note: "Fischer" order would be; I>V>VI > II>III>VII>IV) Sl/I = 1.6, I/IV = 3.0 (Figure A12). M.S., not determined. Chlorin-Nucleus. Including Purpurins As Professor Alsop Corwin, no doubt inferring or assuming knowledge of the Fischer 'data base' (cf. Fischer and Orth, 1937; Fischer and Stern, 1940) yet with geochemical inflection, so succinctly stated: "Acid degrades chlorophyll in one direction and alkali in a different direction. Once the original acid or alkaline degradation has been initiated, a whole series of thermal degradation products is possible, differing in the acid and alkaline sequences." (Corwin, 1960). Excepting the benchmark linkage of fossil tetrapyrroles, and therein petroleum/coal et cetera to ab initio origins in the biosphere by the late Professor Alfred Treibs (1934a,b; 1935a,b; 1936), no single statement on the chemistry/geochemistry of tetrapyrrole pigments has affected the author's views on the subject more. That is, given the present and past quandries on the geochemical origins of ETIO-series porphyrins, as detailed elsewhere (see text), the potential for ETIOseries generation via early oxidative scission of the isocyclic ring structure, inherited from chlorophyll, dictates more than passing familiarity with compounds in the 'alkaline' (i.g. oxidative, cf. Corwin, 1960; Fischer and Stern, 1940) sequence.

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555 Initial studies (g.g. Baker and Louda, 1980; Louda et . 1980) lead me to conclude that, of the myriad of chlorins and purpurins possible with in vitro techniques (cf. Fischer and Stern, 1940), a few geochemical chlorins/purpurins begged recognition. These compounds (i.g. XXIII-XXVII), plus others (i.g. XXVIII-XXXI) for alternate reasons, were therefore investigated with physicochemical characterization/familiarization in mind. (XXIII). Chlorin-e6 : {XXIIIa) tri-methyl ester: {XXIIIb) freeacid: 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-chlorin-6-carboxylic acid-yacetic acid-7-propionic acid = (XXIIIb). Trimethyl ester= (XXIIIa). 31,32-Didehydrorhodochlorin-15-acetic acid-13,15,17-trimethyl ester, or (25, 3S)-20-(carboxymethyl)-18-carb9xy-13-ethyl-3,7,12,17tetramethyl-8-vinylchlorin-2-propionic acid-2 ,181,202-trimethylester. C37H4206N4 638 a.m.u. Chlorin-e6-TME (XXIIIa) was not synthesized by the author but, rather, was purified from a remnant stock of the Mellow Institute studies As received, chlorin-e6-TME (XXIIIa) was found to contain appreciable chlorin-e6-DME as well as unrecognizable breakdown (oxidation ?) products. Purification of the desired known (XXIIIa) was via microcrystalline cellulose chromatography (see text) eluting with 5% acetone in petroleum ether Purity checks were made via chromatography over alumina eluting with CH2Cl2 and TLC on silica in a variety of solvents (g.g. 20% acetone/petroleum ether). Chlorin-e6 free-acid (XXIIIb), one of the 2 tricarboxylic acid -chlorins (i.g. XXIIIb, XXVIIa) required for chromatographic standardization (see text), was generated from the trimethyl ester parent (XXIIa) via strong acidic hydrolysis (30% HCl, w/v, aqueous, cold). Following neutralization of the acid solution with NaC03 pigment was transferred to ethyl ether and back-extracted into 2.5-3 .0%

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556 .HCl (w/v). The last step allowed preliminary separation of the majority of the tricarboxylic acid (XXIIIb) from remaining ester forms (cf. Smith, 1975, pp. 14-15). At this stage it was evident that the total de-esterification of chlorin-e6-TME (XXIIIa) was not as facile as I had wished, yields were poor (i.g. <10%). Final purification was via cellulose chromatography eluting with 35-50% acetone in petroleum ether. Chlorin-e6-TME (XXIIIa) was found to easily chelate Cu*(LXIX) and was totally unreactive to borohydride, Chlorin-eq-TME (XXIIIa): UV/VIS (ethyl ether) S = 400.5, 500.0, 529.5, (561), 610.5, 665.0 nm = I S/I = 2.1, !/IV= 3.8. Band order = M.S. (F.D.) 638 m/z, (E.I.) 638 (M+), 606, 579, 565, 551, 479 m/z. {XXIV). Purpurin-7-TME: 1,3,5,8-tetramethyl-4-ethyl-2-vinylchlorin-6-carboxylic acid-y-glyoxylic acid-7-propionic acid-trimethyl ester. ("F"); 18-Methoxycarbonyl-13-ethyl-3,7,12,17-tetramethyl-20[(methoxycarbonyl)formyl]-8-vinylchlorin-2-[2-(metho x ycarbonyl)ethyl]. ( R ) C37H 4007N 4 6 52 a m u Purpurin-7-TME (XXIV) was prepared by a modification of the method of Kenner et (1973: as reported by Fuhrhop and Smith, 1975). In the present case; 1.5 g pheophytin-a (Ila) was dissolved in 20 ml warm pyridine, diluted with 630 ml ethyl ether, and air was bubbled through the solution while 10 g KOH in 35 ml n-propanol was added. The reaction mixture was stirred for an additional 1/2 hour with continued aeration. The 'organic-alkali' reaction mixture was next extracted twice with 250 ml volumes of water and the ethereal epiphase discarded. The basic aqueous combined extracts contained "unstable chlorin" (cf. Fischer and Stern, 1940) was next neutralized with aqueous sulfuric acid (ca. 20 ml H2S04 50 ml H20) and e xtracted twice with equal volumes of CH2Cl2 discarding the aqueous solution remaining. The

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557 pooled CH2Cl2 solution of 'unstable chlorin' was washed 3x with H20 and split into 2 portions. One-half of the above preparation was used to prepare purpurin-18-ME (XXVI) while the remaining half was immediately treated with excess ethereal diazomethane and placed in the dark at room temperature for 10 minutes. Evaporation of the CH2N2 treated aliquote from above yielded the crude purpurin-7-TME preparation. Chromatography over alumina (Grade II-III, 3.5% H20) in CH2Cl2 afforded 2 main fractions. The first eluting band was pale-olive on the column, yielded intense red-fluorescence to ultraviolet light (A = 366nm) and exhibited a chlorin-like chromophore (S = 403, I = 673 nm; CH2Cl2 ) upon elec-tronic spectrometric exam. This fraction was discarded. The second and major band was dark brownish-grey to the eye, lacked noticable fluorescence and had the proper electronic spectrum for purpurin-7-TME (XXIV: S = 404, I = 682 nm; CH2Cl2 ) except for a noticable band width broadening to the low energy side of band I. Second derivative electronic spectroscopy revealed a minor band at ca. 695 nm, thus indicating the possible presence of purpurin-18 ME (XXXVI). LPHPLC of this major fraction from alumina chromatography over methanol-deactivated silica (see text) developed isocratically with 5% acetone in petroleum ether cleanly separated the less polar purpurin-18 ME (XXVI: S = 406.5, I = 696.2; in eluant) from the main and desired product, purpurin-7-TME (XXIV). Purpurin-7-TME (XXIV): UV/VIS (ethyl ether) S = 403.0, (475), 502, 540, (630-640), 678. 0 nm = I. S/I = 3.6, I/IV = 3.7. Band order = I >III>IV> II>IVa; M.S. (E.I./0.) 652 (M+), 593, 565 (base), 491 m/z. (XXV). 'Oxy-deoxo'-purpurin-7-TME: 1,3,5,8-tetramethyl 4 -ethyl-2-vinyl-chlorin-6-carboxylic acid-y-glycolylic acid-7 propionic acid-

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558 trimethyl ester. ("F"). 18-Methoxycarbonyl-13-ethyl-3,7,12,17-tetra methyl-20-[(methoxycarbonyl)methanol]-8-vinylchlorin-2-[2(methoxycarbonyl)ethyl]. ("R"). C37H4207N4 654 a.m.u. 'Oxy-deoxo' (i.g. '00,' trivial)-purpurin-7-TME (XXV) was prepared from the parent compound (i.g. XXIV) via sodium borohydride reduction in ethanolic ether, as detailed elsewhere (see text). Purification of the product (XXV) was via conventional column chromatography over microcrystalline cellulose developed with 3.5-5.0 % acetone in petroleum ether. 'Oxy-deoxo'-purpurin-7-TME (XXV): UV/VIS (ethyl ether) S = 401.0, 501, 530, 611, 665.0 nm =I. S/I = 2.7, I/IV = 4.1. Band order= I>IV>III>II; M.S. (E.I./0.) 654 (M+), 622/623 (M-CH30H/CH30), 577 m/z (base, M-18-59). (XXVI). Purpurin-18 (XXVIa, free acid: XXVIb, methyl ester): 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-chlorin-6,y-dicarboxyl i c acid anhydride-7-propionic acid. ("F"). 3,7,12,17-tetramethyl-13-ethyl-8vinylchlorin-18,20-dicarboxy-2-propionic acid o-lactone. ("R"): C33H3205N4 (XXVIb), 564 a.m. u. One-half of the CH2Cl2 solut ion of 'unstab l e chlorin,' described earlier (compound XXIV), was subjected to repeated evaporationredissolution (CH2Cl2/benzene, 1:1, v/v) until no further increase in absorption at 695 nm was noted (cf Kenner et gl., 1973; as given in Fuhrhop and Smith, 1975). Initial purification was via column chromatography over (see text) eluting purpurin-18 free acid (XXVI a) with 121 5 % a c etone in petroleum ether. At this point, about half of the product was retained as the free-acid (XXVIa) while the other portion was converted t o the methyl ester (XXVIb) with ethereal diazomethane. Purpurin-18-ME (XXVIb) was reacted with NaBH4 and yielded a positive reaction via electronic spectroscopy. That is, the purpurin type spectrum was altered to that of a more classic chlorin via

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559 hypsochromic shifts in the postion of all maxima (Product: S = 405, 502, 534, 613, 668 nm = I S/I = 3.9, !/IV = 3.8. Band order = I >IV>III>II). Mass spectrometric analyses (F.O. E.I./0. ) were inconclusive. Close electronic spectral agreement between the product obtained via borohydride reaction with purpurin-18 ME (XXVIb) and chlorin-p6 (XXVII), together with poor mass spectral data, militates omission of this compound as a 'standard.' It appears that NaBH4 together with the high atmospheric humidity of South Florida, catalyzed the hydrolysis of the diketo-lactone rather than actually reducing the carbonyl moieties. Purpurin-18, as the free-acid (XXVIa) or methyl ester (XXVIb), consistently reacted to borohydride, in ethanolic ether (see text), in the manner given above. Thus, similar products obtained from geochemical isolates were considered valid, as to the nature of the in vitro test. Purpurin-18-ME (XXVIb). UV/VIS (ethyl ether) 359, S = 407. 0, 478, 505.5 (IV), 541. 5, (588), 639, 695. 5 nm = I. S/I = 1.6+ !/IV = 7 0. Band order = I>III>II > IV>IVa>IIa; M.S. (E.I./0.) 578 (M ), 491/493 447 m/z. (XXVII). Chlorin-06 (XXVI!a, free-ac id: XXVIIb, trimethyl ester): 1,3,5,8-tetramethyl-4-ethyl-2-vinyl-chlorin-6,y-dicarboxylic acid-7-propionic acid ("F"). 3,7,12,17-tetramethyl-13-ethyl-8vinylchlorin-18,20-dicarboxy-2-propionic acid. ( R"): C36H4006N4 (XXVIIb), 624 a.m.u Chlorin-p6-free acid (XXVIIa) was prepared via the alkaline hydrolysis of purpurin-18 free acid (XXVIa) using a modification of the method of Fischer and Stern (1940), as follows: 20 mg purpurin-18 free acid was dissolved with minimal pyridine, diluted with 30 ml ethyl ether and 1.7 ml KOH/MeOH (1: 3 w/v) was added with vigorous mix ing, which was continued for 10 minutes (Note: upon addition of methanolic

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560 alkali an immediate color change from purple-brown to brilliant kellygreen was apparent); 40 ml H20 was added, the solution transferred to a separatory funnel and the alkali hypophase collected, discarding the ethereal epiphase; Fresh ethyl ether was added and chlorin-p6 free acid (XXVIIa) driven into the epiphase by neutralization of the hypophase with dilute (ca. 1.5 N) HCl; the ethereal solution of crude chlorin-p6 free-acid (XXVIIa) was washed several times with water and evaporated in vacuo. The crude preparation of chlorin-p6 free-acid (XXVIIa) was initially purified via chromatography over microcrystalline cellulose with elution of the desired product occurring at about 35-50% acetone in petroleum ether (see text). At this point the product (XXVIIa) was split into halves, one being retained as such as a tricarboxylic acid known (XXVIIa) and the other half transformed to the trimethyl ester via esterification with ethereal diazomethane (see text). Chlorin-p6 -TME (XXVIIb) was further purified via chromatography over silica gel and subsequently over alumina, in order to assure the complete removal of partially esterified chlorin-p6-DME) products. Reported data, herein, on the electronic and mass spectral behavior of chlorin-p6 are for_ the free-acid (XXVIIa) and trimethyl ester (XXVIIb), respectively. (XXVII). UV/VIS (ethyl ether: XXVIIa) S = 399.9, 498.6, 531, 615, 670.8 nm = I. S/I = 2.8, I/IV = 4.0. Band order= I>IV>III>II; M.S. (E.I./0.) 624 (M+), 593 (M-31), 565 (M-59), 537 (M-87), 505 (M-119) m/z. (XXVIII). Rhodin-g7-trimethyl ester: 1,5,8-Trimethyl-4-ethyl-2vinyl-3-formyl-chlorin-6-carboxylic acid-y-acetic acid-7-propionic acid-trimethyl ester. ("F"). 7-Formyl-7-demethyl-31,32-didehydro rhodochlorin-15-acetic acid-13,15,17-trimethyl ester, or (2S,3S)-20(carboxymethyl)-18-carboxy-7-formyl-13-ethyl-3,12,17-trimethyl-8-vi nylch lorin-2-propioni c aci d-23 181 ,202-trimethyl ester. ( "R"): C37H4007N4 652 a.m.u.

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561 Rhodin-g7-TME (XXVIII) was an 'on-hand' compound in the laboratory of Professor E. W. Baker. This compound {XXVIII), following chromatography over alumina to remove possible oxidation products accumulating with age, was utilized as such. Rhodin-g7-TME (XXVIII). UV/VIS {ethyl ether) S = 426, 521, 552-4, 600, 654 nm =I. S/I = 7.1, !/IV= 2.0. Band order= I>IV>III>II; M.S. {E.I./0.) 652 {M+), 620 {M-32), 593/579/565 {M-59, -73, -8 7). (XXIX). 3-Methanol-3-desformyl-rhodin-07-trimethyl ester: 1,5,8-Trimethyl-4-ethyl-2-vinyl-3-methanol -c hlorin-6-carbo xylic acid-y-acetic acid-7-propionic acid-trimethyl ester. ("F"). 7-Methanol-7-demethyl31,32-didehydrorhodochlorin-15-acetic acid-13,15,17-trimethyl ester. {"R"). C37H420 7 N4 654 a.m.u. Rhodin-g7-TME (XXVIII) was treated with sodium borohydride in ethanolic ethyl ether (see text). That the desired product {XXIX) was indeed generated was evidenced upon electronic spectral exam. In this case, the position of the Soret maximal absorption was hypsochromically shi fted ca. 22 nm {XXVIII, S = 426 nm: XXIX, S = 403.5 nm). Band I absorption was found to have undergone a 7 nm bathochromic shift {XXVIII, I = 654 nm; XXIX, I = 661.5 nm), akin to removal of the auxochrome effect of 3-formyl conjugation in other 'b-series' chlorophyll derivatives XVI, XVIII). The product 3-MDF-rhoding7-TME* (XXIX), might also be trivially named as 3-methanol-3desmethyl-chlorin-e6-TME (see XXIIIb). In that light, the electroni c spectra of 3-MDF-rhodin-g7-TME (XXIX) and chlorin-e 6-TME (XXIIIb) might well be expected to closely resemble each other, given the usual minor effects of alcohol versus alkyl commutation upon a given chromophore Platt, 1956; Williams and Fleming, 1966; Treibs, 1973). Comparison of these 2 compounds (XXIIIb, XXIX) on the basis of chromophore, *MDF =methanol desformyl.

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562 one finds them exceedingly similar in fine structure and differing only by about 3 nm in the postion of corresponding maxima. 3-Methanol-3-desformyl-rhodin-g7-TME (XXIX). UV/VIS (ethyl ether) S = 403.5, 501.0, 530.0, 558, 605, 661.5 nm = I. S/I = 3.0, I/IV = 3.2. Band order= I>IV>III>II >IIa; M.S., not determined (XXX). 7-0xo-octaethylchlorin: 1,2,3,4,5,6,8,8-0ctaethyl-7-oxochlorin. ("F"). 3,3,7,8,12,13,17,18-2-oxochlorin. ("R"). 7-oxoOEC (trivial). C36H46N40, 550 a.m.u. 7-0xo-OEC (XXX) was of interest to the author as a possible model chromophore for 'chlorins' reported in the literature (see g.g. lead references in Baker and Palmer, 1978; cf. Baker et 1978a,b) and found personally (Baker and Louda, 1980, 1981, 1982; Louda and Baker, 1981) which exhibited band I absorption at or near 640 nm. The preparation of 7-oxo-OEC (XXX) was, in essence, a 1/10 scaled version of that reported by Bonnett et (1969; cf. Fuhrhop and Smith, 1975). In the present case: about 15 mg. octaethylporphyrin (OEP: L) in 7.5 ml concentrated H2S04 was treated with 1 ml 20% aqueous hydrogen peroxide for 6 minutes while being stirred at ice-bath temperature; The mixture was then added to 60 ml iced H20, neutralized with saturated aqueous NaHC03 and the pigments driven into ethyl ether; Following water washing of the ethereal product and evaporation in vacuo the residue was subjected to chromatography over alumina and silica gel, in that order; Final purification was via rechromatography over alumina with acetone (5-10%) in petroleum ether. This preparation was found to contain, besides 7-oxo-OEC (XXX: A1 = 642 nm), unreacted OEP (L) and a chlorin-like compound with band I absorption at ca. 686 nm. The latter was possibly a dioxo-chlorin but further study was not attempted.

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563 7-0xo-OEC (XXX) was found to exhibit an extremely narrow half-band width at the postion of band I absorption (I = 642 nm; 7 nm), thus being 'spike-like' in appearance and did not resemble the geochemical isolates mentioned above. Further study with this compound was deemed unnecessary. 7-0xo-octaethylchlorin (XXX). UV/VIS (Chloroform) S = 408, 512, 550, 586-"8, 615, 642 nm = I. S/I = 5, I/IV = 3. Band order = I>III>IV>IIa>II; M.S., not determined. (XXXI). Mesopyrrochlorin-methylester*: 1,3,5,8-tetramethyl-2,4-di!thyl-chlorin-7-propionic acid methyl ester. ("F"). Pyrrochlorin-17 -methyl ester, or 2,7,12,18-tetramethyl-3,8-diethyl-17-[(173 -methoxycarbonyl)ethyl]-chlorin. ("R"). C32H3802N4 510 a.m.u. Mesopyrrochlorin-ME (XXXI) was not one of the author's planned standards but, rather, was isolated from an ostensibly pure preparation of pyrroporphyrin (LX) purchased from Carl Roth (F.R.G., Catalog #7134). As mesopyrrochlorin-like absorption (i.g. A1 = 648 nm, benzene) was present in the pyrroporphyrin free-acid (see LX) prepara-tion, as received, generation of chlorin structure by the author is ruled out. Mesopyrrochlorin-ME (XXXI) was isolated from crude pyrroporphyrin (see LX) preparation following esterification of the latter in methanolic H2S04 (5-7%, v/v: see text). Mesopyrrochlorin ME(XXXI: alt. 7,8-didehydropyrroporphyrinME; trivial, "F") was separated from its porphyrin analog (LX) as an early running fraction during chroma-*The suggested revised nomenclature (UPAC-IUB, 1978) allows the new trivial system to include 'pyrrochlorin.' However, this trivial moniker includes reduction of the 2-vinyl moiety ("F") to 2-ethyl. This differs from the time honored designation of pyrrochlorin in the Fischer system (cf. Fischer and Stern, 1940), which includes the 2vinyl function, and, aside from the desired (IUPAC-IUB, 1978) inference of substituent similarity between 'pyrrochlorin' ("R") and pyrropor phyrin ("R" and "F"), I find this suggestion ludicrous.

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tography over alumina in CH2Cl2 Purification of this 'impurity' (XXXI) was achieved via rechromatography over Al203 (II-III) eluting with 2 .5% acetone in petroleum ether. Subsequent TLC and CC tests failed to show this as anything but a single compound (viz. XXXI). Mesopyrrochlorin-ME (XXXI). UV/VIS (ethyl ether) S = 395.5, 497.8, 532, (578), 588, 646.6 nm =I. S/I = 6.0, I/IV = 2.0. Band order = M.S. not determined (see LXXVII). DPEP-Nucleus DPEP-series porphyrins are so-named in reference to the 564 archtypical compound deoxophylloerythroetioporphyrin (viz. DPEP, XXXIII: see Fischer and Stern, 1940). The distinguishing feature of the DPEP-series is the presence of an isocyclic ring (i.g. 6,ycycloethano) directly inheritable from the chlorophylls (g.g. I, XIV, XIX, XXI; see Figure Ale) either via in vitro (Fischer and Stern, 1940; Fuhrhop and Smith, 1975; Seely, 1966) or geochemical (Baker and Louda, 1983; Baker and Palmer, 1978; Hodgson, 1973; Louda and Baker, 1986a; Treibs, 1936) reactions. The generation of 'true' DPEP-series porphyrins via the diagenesis of chlorophyll drivatives (viz phorbides) is the main area of the author's studies and, as such, total familiarization with these compounds (XXXII-XL) was of paramount importance The distinction made above regarding 'true' DPEP series (i.g. cycloethano exocyclic ring) stems from recent findings of the existence of DPEP-series (i. g. E-2 m/z, where E = ETIO-series porphyrins in mass spectral terminology: see text: cf. Baker, 1966; Baker et 1967; Yen et 1969) mimics which contain either 6-(viz. cyclopropano: Chicarrelli et 1984; Quirke et 1982) or 7-(viz. cyclobutano: Barwise and

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565 Roberts, 1984; Ekstrom et . 1983; Wolff et 1983) membered exocyclic structures. These points are more fully covered elsewhere in text. DPEP, per se, has been reported to have been prepared via a variety of routes (Baker et 1968; Fischer and Stern, 1940; Flaugh and Rappoport, 1968; Smith et 1984) including both total syntheses from pyrrylmethenes and derivatization from natural chlorophylls (i.g. pheophytin-a). Given known in v itro methods and the total lack of commercial DPEP knowns (i. g standards) it is somewhat surprising to the author that more work along these lines is apparently not underway. That is, as I proposed via 'theoretical diagenesis' (Louda and 1983; cf. Baker and Louda, 1986a) a minimum of twelve DPEP-series compounds, extending from C29 to C33, are possible beginning only with chlorophylls-a (I) and -b (XIV). The reactions involved include reduction, vinyl scission, devinylation, deformylation and decarboxylation, all of which have been successful with tetrapyrrole pigments (see, g y Baker et 1968; Fischer and Orth 1937; Fischer and Stern, 1940; Fuhrhop and Smith, 1975; Seely, 1966; and lead references in each). New, decarbo xylation reactions are now known (g.y. Barton et . 1983) and afford great promise (J. M. E Quirke, pers. commun., 1985) in formulating many more alkyl (viz. decarboxylated) tetrapyrrole standards The point of the above 'ed itorial' is that soon after beginning my studies on the geochemistry of tetrapyrrole pigments it became blatantly apparent that the vast majority of investigations in this field paid little or no attention to standards This fact makes tetrapyrrole geochemistry, unfortunately, unique.

PAGE 594

566 In order to standardize the separation and spectrometric techniques utilized in the present study, unfortunately not including true hplc (see Barwise and Park, 1983; Barwise and Roberts, 1984; Hajibrahim et gl., 1978, 1981; Quirke et gl., 1979, 1980a-b, 1982; Sundararaman, 1985), only a few structures (viz. DPEP-nuclei) were required. Thus, the following 9 compounds (XXXII-XL) were relied upon as chromatographic and spectrometric standards. (XXXII). Phylloerythrin-methyl ester: 1,3,5,8-tetramethyl-2,4-acid methyl ester. ("F"). Phytoporphyrin-17 -methyl ester. ("R"). PEME (Abbreviation, trivial). C34H3603N4 548 a .m.u. Phylloerythin, as the free acid, is reported to be formed via a variety of syntheses. These include; the HBr (Fischer and Stern, 1940) or HI (Hodgson and Baker, 1967) treatment of pheophorbide-a (VIa), the oxidation of deoxophylloerythrin (XXXVIIa) in oleum (Fischer and Stern, 1940), the cyclization of chloroporphyrin-e4-DME with alcohol-free ethylate (metal = ?) in xylene at 150C (Fischer and Stern, 1940), the reduction of pyropheophorbide-a (VIII) to a leuco product (?) and reoxidation to PE in air for 48 hours (Fischer and Stern, 1940), the hydrolysis/reduction/oxidation of pheophorbide-a (VIa) in refluxing 20% (w/v, aqueous) HCl (Fischer and Stern, 1940; cf. Seely, 1966*) and the oxidation of mesopyropheophorbide-a ME (X) with DDQ (i.g. 2,3-dichloro-5,6-dicyano-benzoquinone) in refluxing acetone:benzene (1:1, v/v. Kenner et gl. 1973; cf. Fuhrhop and Smith, 1975). *Note: The reference of Wickliff and Aronoff, 1963 given in Seely, 1966 does not contain any mention as to the preparation of phylloerythrin.

PAGE 595

567 Interest in the phylloerythrin-type chromophore (viz. NiPE, see LXXVIII) stems from the initial uncertainty I found with geologic isolates resembling, but not exactly, the nickel complex of phylloerythrin (cf. Baker and Louda, 1980). More solid evidence, specifically the transformation of a NiPE-like (see LXXVIII) spectrum to a Ni DPEP-like (see, LXXXVI and XC) spectrum upon borohydride treatment (Louda and Baker, 1981), militated that I become more familiar with the physicochemical manifestations of this compound (XXXII). The apparent ease of one of the reported methodologies for the formation of phylloerythrin, as the free acid, was intriguing and attempted first. In this case, pheophorbide-a free acid (VIa) was treated with 20% (w/v) HCl under reflux (cf. Fischer and Stern, 1940; Seely, 1966). At first, the production of a product with an oxo-rhodo type visible spectrum (i.g. III>II>IV>I: see Baker and Palmer, 1978; Marks, 1969; Stern and Wenderlein, 1936b) hinted of success in this reaction. However, further study showed almost total conversion to 2oxo-phylloerythrin (XXXV), which is detailed later. Having failed at this first attempt in the formation of phylloerythrin (XXXII), even with a nitrogen blanket until reflux began, a more precise single step reaction was decided upon. The second, and more successful, react1on was the aromatization of meopyropheophorbide-a ME (X) with DDQ in warm benzene (40-45C) for 5-8 minutes (Clezy et gj., 1977; cf. Kenner et gJ., 1973. In the present case, reaction was with ca. 1,000 mesopyropheophorbide-a ME (X) and all other conditions mimic the literature (Clezy et gj., 1977). Yields were still low (
PAGE 596

568 Phylloerythrin ME (XXXII). UV/VIS (benzene) S = 420.0, 521, 562.5, 586, 639.0 nm = I. S/I = 200, I/IV = 0.21. Band order = III>II>IV >I; M .S. (E.I., 12 eV) 548 (M+) m/z. (E.I., 70 eV) 548 (M+), 533 (M-15), 475 (M-73), 274 (M +), 237.5 ((M-73)++) m/z. (XXXIII). 9-0xy-9-deoxo-phylloerythrin-methyl ester: 1,3,5,8-tetramethyl-2,4-diethyl-6,!-ethano(9)-porphin-7-propionic acid methyl ester. ("F"). 13'-0xy-13 -deoxophytoporphyrin-173-methyl ester. ("R"). 9-00-PE-ME (Abbreviation, trivial). C34H3803N4 550 a.m.u. Phylloerythrin-ME (XXXII), ca. 20 was treated with a few crystals of NaBH4 in ethanolic-ethyl ether (see text). Alteration of the chromophore from oxo-rhodo (XXXII) to phyllo-modification of a DPEP-type (i.g. IV> I > II >III) attested to removal of the conjugated carbonyl (i.g. 9-oxo to 9-oxy) auxochrome. (XXXII). UV/VIS (benzene) S = 406.0, 504.0, 537. 5, 567.8, 621.2 nm =I. S/I = 90, I/IV = 0.3. Band order= M.S., not determined (pyrolytic). (XXXIV). 7.8-Dioxy-deoxophylloerythrin-ME: 1,3,5,8-tetramethyl-2 ,4-diethyl-7 ,8-dioxy-phorbin-7-propionic acid methyl ester. ( "F"). 17,18-Dioxy-13'-deoxophytoporphyrin-173-methyl ester. ("R"). DioxyDPE-ME (Abbreviation, trivial). C34H4004N4 568 a.m.u. 7,8-Dioxy-DPE-ME (XXXIV) was on-hand in the stocks of Professor E W. Baker as a compound formed by Dr. G. D. Smith (see Baker and Smith, 1974, 1975). This compound (XXXIV), actually a modified phorbide rather than a porphyrin, was utilized, as such, by the author only as an electronic absorption comparator 7,8-Dioxy-DPE-ME (XXXIV). UV/VIS (ethyl ether) S = 402.6, 506, 544, 585, 641.0 nm = I. S/1 = 6.9, 1/IV = 2.2. Band order = I > IV>III>II; M.S., not determined. (XXXV). 2-0xo-phylloerythrin-methylester: 1,3,5,8-tetramethyl-4-ethyl-2-acetyl-6 y-ethanone(9)-porphin-7-propionic acid methyl ester. ("F"). ester. ("R"). 2-Acetyl-2-desethyl-phylloerythrin-ME (trivial). C34H3404N4 562 a.m.u.

PAGE 597

569 As mentioned earlier (compound XXXII), 2-oxo-PE-ME (XXXV) was formed via the "one-pot" hydrolysis/reduction/o x idation of pheophorbide-a (VIa) in reflux ing aqueous 20% (w/v) HCl (Fischer and Stern, 1940; cf. Seely, 1966). Apparently, the e x clusion of molecular oxygen was not successful and the 2-vinyl moiety of pheophorbide a (VIa) was o x idized to acetyl r ather than being reduced to ethyl, as reported (Fi scher and Stern, 1940). However, the formation of 2-o x oPEME (viz. following esterification: Section liB) was, in retrospect, fortuitous to the present study That is, I have suggested the intervention of vinyl o x idation as a potential route to 2-methyl-2-desethyl 'homologs' of DPEP (Louda and Baker, 1983, 1986) such as "Abelsonite" (XL: cf. Storm et 1984). Thus, compounds such as 2-acetyl-9-k etophorbides, tentatively identified i n marine sediments (Baker and Louda, 1982), could yield 2-o x o-phylloerythrins following aromatization. Going on the above, 2-oxo-PE-ME (XXXV) was pur ified via chroma tography over alumina, eluting with benzene/methylene dichloride (1: 3, v/v) or acetone/petroleum ether (1:9, v/v) during rechromatography, verified spectrometrically and retained as a standard 2-0 x o-phylloerythrin-ME (XXXV). UV/VIS (benzene) S = 422.8, 528.5, 572.5, 598. 0, 650.5 nm = I S/1 63+ 1/IV = 0.5, III/I = 3.8. Band order= III>II>IV>I; M.S. (E.I.) 562 (M ), 547 (M-15), 489 m/z (M-73). (XXXVI). 2 (a-Hydro x y-ethyl)-2-desethyl 9 o x v-9-deo x o phylloerythrin -ME: 1,3,5 8-Tetramethyl-4-ethyl 2-(a-hydro xyethyl)-6,yethanol(9)-porphine-7-propionic acid methyl ester. ( F "). 3 '-Hydrox y13'-oxy-13'-deo x ophytoporphyrin-173-meth y l ester. ("R"). Dioxydideo x o-2-oxo-PE ME (trivial}. C 3 4 H380 4 N 4 566 a.m. u The dioxy-dideo x o derivative (XXXVI} of 2-o xo-PEME (XXXV) was prepared v i a treatment of the latter with NaBH4 in ethanolic ethyl ether. Removal of the auxochrome effect of both conjugated carbonyl

PAGE 598

570 functions lead to a change in the electronic spectral 'type' from the parental (i.g. XXXV) oxo-rhodo to that of a phyllo-like compound (cf. LVIII). Enhanced band I absorption (viz. DPEP-like modification of phyllo-type) yielded a band order of IV>II>I>III rather than IV>II>III>I or IV>I>II>III characteristic of true phyllo or DPEP type spectra, respectively. 2-(a-Hydroxy-ethyl)-2-desethyl-9-oxy-9-deoxophylloerythrin-ME (XXXVI). UV/VIS (ethyl ether) S = 400.8, 500.5, 536, 567.5, 620.8 nm = I. S/I 48, I/IV = 0.3. Band order= IV>II>I>III. M.S., not determined. {XXXVII). Deoxophylloerythrin (a) free acid. {b) methyl ester: 1,3,5,8-Tetramethyl-2,4-diethyl-6,y-ethano-porphine-7-propionic acid. ("F"). 13'-deoxophytoporphyrin. ("R"). OPE, OPE-ME (abbreviations of trivial for XXXVII-a and -b, respectively). 534 a.m.u. (XXXV lib). Deoxophylloerythrin (XXXVIIa) was on-hand in the laboratory of Dr. E. W. Baker (cf. Baker et gl., 1968) and was used as received, after checking purity via various chromatographies. OPE-ME (XXXVIIb) was formed from the free-acid (XXXVIIa) by treatment with methanolic sulfuric acid (7%, v/v) followed by purifica-tion over alumina in CH2Cl2 Deoxophylloerythrin-methyl ester (XXXVIIb). UV/VIS (benzene) S = 402.0, 499.8, 534, 566.5, 619.5 nm = I. S/I 38, !/IV= 0.4. Band order= IV> I>II>III. M.S. (E.I.) 534 (M+), 461 m/z (M-73). (XXXVIII). Deoxophylloethroetioporphyrin: 1,3,5,8-Tetramethyl2,4,7-triethyl-6,y-ethano-porphine. ("F"). 172-Decarboxy-13'-deoxo phytoporphyrin. ("R"). DPEP (trivial, abbreviation). C32H36N4 476 a.m.u. DPEP (XXXVIII), derivatized from pheophytins-a and -b (see II, XV) by E. W. Baker while at the Carnegie-Mellon Institute (Baker et gl., 1968) was 'on-hand' as the nickel (XC) and vanadyl (LXXXVIII) chelates. The former (XC) was demetallated with methanesulfonic acid (MSA),

PAGE 599

571 _purified by HCl/ethyl ether portion and chromatography, as indicated for free-base alkyl porphyrins (see text; cf. Baker and Palmer, 1978). The electronic spectrum of DPEP (XXXVIII) was found to be essentially identical to OPE-ME (XXXVIIb) and is not repeated here. Oeoxophylloerythroetjoporphyrin (XXXVIII). UV/VIS (cf. XXXVIIb); M.S. (E.I., 70 eV) 476 (M ), 461 m/z (M-15); M.S. (E.I., 4.5-6.0 eV) 476 m/z. (XXXIX). 7-Propvl-7-desethyl-deoxophylloerythroetioporphyrin: 1,3,5,8-Tetramethyl-2,4-diethyl-7-propyl-6,y-ethane-porphyrin. ("F"). 17-Propyl-17-depropio-13'-deoxophytoporphyrin. ("R"). 7-PDE-DPEP (trivial, abbreviation). C33H38N4 490 a.m.u. 7-Propyl-7-desethyl-DPEP (XXXIX) was prepared by the MSA catalyzed demetallation of the corresponding Ni chelate (XCI; cf. XIII) and purified as given earlier for DPEP (XXXVIII) per se. 7-Propyl-7-desethyl-DPEP (XXXIX). UV/VIS (benzene) S = 402.0, 500. 0, 534, 566, 619.5 nm =I. S/I = 37, I/IV = 0.4 (Figure 13). Band order= IV>I>II>III; M.S. (E.I.) 490 (M+), 475 (M-15), 461 (M-29) m/z. (XL). 2-Methyl-2-desethyl-deoxophylloerythroetioporphyrin: 1,2,3,5,8-Pentamethyl-4,7-diethyl-6,y-ethane-porphyrin. ("F"). 3Methyl-3-desethyl-172-decarboxy-13'-deoxophytoporphyrin. ("R"). 2Methyl-2-desethyl-DPEP (trivial). C31H34N4 462 a.m.u. 2-Methyl-2-desethyl-DPEP (XL) was prepared by the MSA demetallation of the organic "mineral" Abelsonite (XCII). As the sample used was a portion of that for which unequivocable identification has been made (Storm et gl., 1984) it is reasonable to assume identical structure herein. 2-Methyl-2-desethyl-DPEP (XL). UV/VIS (benzene) S = 402.0, 500.0, 534, 566, 619.5 nm = I. S/I = 32, I/IV = 0.4 (cf. Figure 13). Band order= IV>I>II>III; M.S. (E.I.) 462 (M+), 447 m/z (M-15).

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572 Rhodin Nucleus The term 'rhodin,' based upon visible absorption spectral similarities to rhodin-g7 (XXVIII), was given by the Fischer school (Fischer and Orth, 1937) to a variety of porphyrins formed via acid (i.g. oleum) catalyzed cyclizations (viz. dehydrations) between propionic acid moieties and methine bridges of the parental compounds (see Fischer and Orth, 1937; Fuhrhop, 1975, 1978; Treibs, 1973). The resultant structures (Figure A1d) all contain a 6-membered (viz cyclopropano) exocyclic ring with a carbonyl function (i.g. ketone) in conjugation with the macrocyclic aromatic nucleus. The 'Fischer' mode of nomenclature for the 'rhodin' class employed only the description 'the rhodin of' ... (parental compound) or 'rhodin'(roman numeral). In the later case the roman numeral made reference by inference to the parental compound. For example, meso-rhodin-IX (see XLI), actually 2 structural isomers (Fuhrhop, 1978), was the rhodin(s) formed from mesoporphyrin-IX (see LV). In the present work, I shall retain the Fischer common names but will add a 'Fischer-style' semi systematic nomenclature using phylloerythrin (XXXII) as a model. Aside from the classic rhodin formations mentioned above, other routes of forming 6-membered isocyclic ring porphyrins, of recent geochemical interest (Chicarelli et gJ., 1984; Wolff et gJ., 1984) which may hold synthetic possibilities include the anhydrobonellin (Agius et g]., 1979; Pelter et gJ., 1978) and neopurpurin (Seely, 1966) reactions. In the present study, the parental "rhodin-nucleus" compounds (i.g. XLI, XLVI) were formed by the stated oleum catalyzed reaction of the appropriate precursors (viz. LV and LX, respectively), but not by

PAGE 601

573 this author. The original syntheses of these compounds XLI, XLVI) were performed by the late Professor Alfred Treibs while part of the Hans Fischer group. Under the guidance of the late Or. Wolfgang Seifert, the geochemists of Chevron (Richmond, California) were becoming interested in the porphyrins and r equested samples from Professor Treibs Compounds XLI and XLVI were sent to Dr. Seifert' s laboratory, which was only in the initial stages of set-up for porphyrin analyses (Seifert, pers. commun. 1982), who forwarded them to Dr. Earl Baker and myself for confirmation via mass spectrometry. Thus, meso-rhodin-IX ME (XLI) and pyrrorhodin-ME (XLVI) were added to the present battery of standards and several derivatives XLII, XLVII, XCIII-XCVI) formed by this author During the course of the present studies given both the ease of reducing the carbonyl moiety of parental rhodins (see XLII, XLVII) and recent reports of geoporphyrins with an e x ocyclic cyclopropane ring (Barwise and Park, 1983; Chicarelli et gl. 1984; Wolff et gl., 1984), a graduate student (Mr. Gustavo Guteirrez, M S 1986) assigned to Professor Earl Baker at Florida Atlantic University (Boca Raton, Florida) was given the task of repeating, on a larger (viz. gram quantity) scale, my preparations of XLI and XLVI and further derivatizing same until the purely alkyl porphyrins with unsubstituted 6-membered (viz. cyclopropane) e x ocyclic rings were obtained. Although the ultimate goal of this master's project was not attained by Mr. Guteirrez, 3 additional compounds, intermediates in various derivatization steps XLIII-XLV), were made available to me as spectroscopic standards All data on compounds XLI-XLVII, presented herein, were collected by the present author.

PAGE 602

574 {XLI). Mesorhodin-IX M.E.: 1,3,5,8-Tetramethyl-2,4-diethy l-7,ypropanone (73)-porphin-6-propionic acid methyl ester. ("F"*). 8,13-0iethyl-2' ,22-dihydro-3,7,12,17-tetramethyl-23-oxocyclohexa [at] porphyrin-18-propionic acid methyl ester. ("R"). C35H3803N4 562 a.m.u. Mesorhodin-IX M.E. (XLI), obtained as described above, was purified via lphplc over silica (see text) and characterized via electronic and mass spectral techniques. Meso-rhodin-IX M.E. (XLI): UV/VIS (benzene) S = 411.0, 511.5, 548.5, 583, 637.0 nm =I. S/I = 15, I/IV = 0.8. Band order= IV>I>III>II. (ethyl ether) S = 405.5, 508.5, 545.0, 583.5, 635.5 nm = I. S/I = 15, I/IV = 0.8. Band order= IV>I>III>II (Figure A14, solid trace); M.S. (E.I., 70eV) 562 (M), 560 (M-2), 530 (M-32), 500 (M-4-58) 489/487 (M-73/M-2-73), 281 (M++), 244 ( (M-73)++), 239 ( (M-73-2-28)++) m/z (see text). (XLII). ME: i,3,5,8-Tetramethyl-2,"4diethyl-7,y-propanol (7 )-porphin-6-propionic ester. ("F"). 8,13-0iethyl-2' ,2'-dihydro-3,7,12,17-tetramethyl-2 -oxycyclohexa[at] porphyrin-18-propionic acid methyl ester. ("R"). 00-mesorhodin-IX ME (00 = oxydeoxo, see text: trivial). C35H4003N4 564 a.m.u. In order to remove the auxochrome effect of the carbonyl moiety of meso-rhodin-IX ME (XLI) reaction with sodium borohydride in ethanolic ethyl ether (see text) was employed. The product, oxydeoxomeso-rhodinM.E. (XLII), was found to have been obtained both by electronic absorp-tion and mass spectral evidences. -In the former case, band I absorption was diminished to the point that an ETIO-type spectrum (IV>III>II>I) resulted (Fig. A14). Upon mass spectral examination, the expected parent or molecular ion at 564 m/z was found but was subordinate to both 560 m/z (M-4) and 546 m/z (M-18). Thus, it is suggested (see text) that this porphyrin-alcohol (XLII) yields essentially 2 base peaks through competing reactions. *Alternate isomer: 1,3,5,8-Tetramethyl-2,4-diethyl-6,y-propanone (63)-porphin-7-propionic acid.

PAGE 603

575 The first being M-4 production via reo x idation of the alcohol to a ketone concurrent with further asomatization of the e x ocyclic ring to yield, in essence, the meso-verdin-IX ME (XLIII) structure. Secondly, dehydration, which effectively blocks further e x ocycle aromatization in this case {cf. Gutei rrez, 1986), yields the 'ex pected' M-18 ion. the loss of ketene (-42, H2C=C=O) was also found as resulting from the M-4 process (i.g. 518 m/z = M-4-42). These points are more fully covered elsewhere (see text). Oihydro-mesorhodin-IX M.E. (XLII) UV/VIS (ethyl ether) S = 400.0, 500.5, 532.0, 571.5, 626.0 nm = I S/I = 25, I/IV = 0.4. Band order = IV>III>II> I {Figure A14, dashed trace). M.S. (E.I. 70 eV) 564 {M), 560 {M-4), 546 (M-18), 518 {M-4-42) m/z. (XLIII). Mesoverdin-IX M.E.: 1,3,5,8-Tetramethyl-2,4-diethyl-7' ,72-didehydro-7,y-propanone (73)-porphin-6-propionic acid methyl ester. ( "F "*). 8,13-0 i ethy 1-3,7, 12,17 -tetramethyl-21 22-d i dehydro-23 -o x ocyclohe x a[at]porphyrin-18-propionic acid methyl ester. {"R"). C35H3603N4 560 a.m.u. This compound (XLIII) was formed as a known during the Master's research of Mr. Guteir r ez, as given earli er as a check on possible side products. The majority of the known 'rhodins' yield, upon heating in the presence of mild acid catalysis, the corresponding 'verdins' via dehydrogenation (Fischer and Orth, 1937; Fuhrhop, 1975, 1978; i g 7' 72-didehydro; F", or 2' ,22-didehydro; "R". ) Mesoverdin-IX M.E. {XLIII) UV/VIS {benzene) S = (385) 419.5 {445), (510), (560), 633, 695 nm = I. S/I = 6, I/(IV) = 1.4. Band order= I > (IV) >II>{III). (cf. Stern and Dezelic, 1937a). M S {E. I 14 eV) 560 (M), 487 {M-73) m/z. (XLIV). Dihydro-mesoverdin-IX M E.: 1,3,5,8-Tetramethyl-2,4-diethyl-7' ,72-didehydro-7,y-propanol (73)-porphin-6-propionic acid *see footnote for compound XLI.

PAGE 604

576 methyl ester. ( "F"). 8, 13-Diethyl-3, 7,12, 17-tetramethyl-21 ,22 -didehydro-23-oxycyclohexa[at]porphyrin-18-propionic acid methyl ester. ("R"). C35H380 3 N4 562 a.m.u. Meso-verdin-IX M.E. (XL III) was reacted with NaBH4 as given elsewhere (see text) in order to obtain the corresponding 'oxy-deoxo' derivative (XLIV). Reaction did occur, as witnessed by a drastic alteration in the electronic spectrum from a chlorin or purpurin type (cf. XLIII) to an ETIO-type (IV>III>II>I). However, the presence of a 5th band to lower energy than expected (ca. 670 nm) hampers valid interpretation. Further, the lack of success in obtaining mass spectra indicates a high potential for the co-production of 'oxidationproducts' along with the desired product (XLIV). Thus, the electronic absorption spectral data given below is speculative, at best. Dihydro-mesoverdin-IX M.E. (XLIV). UV/VIS (ethyl ether) S = 408, 508, 538, 582, 640 = I, 670 nm. Band order= (II>670 nm>I). S/I 40, I/IV 0.3. M.S., not determined. (XLV). "Didehydro-deoxo-mesoverdin-IX M.E.: *8,13-Diethyl3,7,12,17-tetramethyl-3-H-benzo[at]porphyrin-18-propionic acid methyl ester. ("R"). C35H3802N4 546 a.m.u. During the Master's research of Mr. Guteirrez, mentioned previously, it was hoped that 'dihydro-mesorhodin-IX ME' (XLII) could be smoothly dehydrated in vitro (g.g. methane or tolyl sulfonyl chloride; cf. Gutei rr.ez, 1986) to the 72 7 3 -didehydro-7 3 -deoxo-ana log of XLI. 'Theoretically,' production of the dehydrated form of XLII would then have allowed catalytic reduction (g.g. H2/Pd) to the desired 6-membered (i.g. cyclopropane) analog of OPE (XXXVII). After decar boxylation, the latter would have provided the purely alkyl known required for recent geochemical isolates (cf. Chicarelli etA}., 1984; *No known nomenclatural analog in the Fischer system.

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577 Wolff et g.J_., 1984) of "pseudo-6-DPEP" compounds (cf. Louda and Baker, 1983). Repeated attempts by Mr. Guteirrez (1986) to affect the 'simple' dehydration of XLII ('oxydeoxo-mesorhodin-ME) were found to lead, as a main (i.g. 50+%) product, to a green product with the expected molecular ion (546 m/z) but which is totally unreactive to moderate catalytic hydrogenation and, via NMR, exhibits only aromatic, aliphatic and pyrrole hydrogens (Guteirrez, 1986). That is, no evidence of an isolated double bond were obtained. Thus, a totally aromatic system, probably the benzo[at]porphyrin structure shown in Figure A15, resulted. Dihydro-deoxo-mesoverdin-IX M.E. (XLV). UV/VIS (benzene) S = (402) 412.5 (435), 535, 568, 612.5, 667.5 nm = I. S/I = 3 3, I/IV = 3.8. Band order= I>II>III >IV (Figure A16). M.S. (E.I., 70 eV) 546 (M), 531 (M-15), 473 (M-73) m/z. (XLVI). Pyrrorhodin-XV: 1,3,5,8-tetramethll-2,4-diethyl-7,ypropanone ( -porphin. ( "F"). 8, 13-Diethyl-2' ,2 -di hydro-3, 7,12,17tetramethyl-2 -oxocyclohexa[at]porphyrin. ("R"). C31H3201N4 476 a.m.u. Pyrrorhodin-XV (XLVI), produced via the oleum catalyzed cyclization of pyrroporphyrin XV as the free acid (see LX), by Professor Treibs was obtained as given under the lead-in to this section. In contrast to mesorhodin-IX ME (XLI), this compound (XLVI) was found to be quite pure via lphplc (see text, cf. compound XLI) and was utilized essentially as received. Pyrrorhodin-XV (XLVI). UV/VIS (benzene) S = 411.0, 513. 0, 551.5, 587.0, 641.5 nm = I. S/I = 11, I/IV = 1.2. Band order= I>IV>III>II. (ethyl ether) S = 406.0, 511.0, 547.0, 587.0, 640.5 nm = I. S/I = 10, I/IV = 1.3. Band order= I>IV>III>II. M.S. (E. I., 70 476 (M), 474 (M-2), 461 (m-15), 459 (M-2-15), 238 (M++), 237 ((M-2) ) 223 ((M-2-28) ++).

PAGE 606

578 (XLVII). 1,3,5,8-Tetramethyl-2 4diethyl-7,y-propanol (7 )-porphin. (11F11). 8,13-Diethyl-2',22'-dihydro3. 7 ,12, 17 -tetramethyl-2 3 -oxycyc lohexa [at l porphyrin. (II R II). c3,H3401 N4. 478 a.m.u. Pyrrorhodin-IX (XLVI) was reacted with NaBH4 in ethanolic ethyl ether as usual (see text). Reaction success was evidenced by conver sion of the electronic spectral type from that of a rhodin (XLVI) to that of a phyllo-modified ETIO-type (see text) and obtaining the calculated molecular (M+ = 478 m/z) and dehydration (M-18 = 460 mz) ions during mass spectral analysis. Dihydro-pyrrorhodin-IX (XLVII). UV/VIS (ethyl ether) S = (375) 400.0, 499.0, 531.0, 571.5, 626.0 nm = I. S/I = 34, !/IV = 0.3. Band order= M.S. (E.I., 70 eV) 478 (M+), 460 (M-H20), 436 (M-42), 216 ((M-4-42)++). ETIO-Nuc leus The term ETIO-nucleus (Figure Ale) or ETIO-type derives from the 4 primary etioporphyrins as defined by the Fischer school (Fischer and Orth, 1937). In essence, each of the 4 'true' etioporphyrins are tetramethyl-tetraethyl-porphyrins differing only by the isomeric placement of these 8 locants (Table Alii). The most common natural etioporphyrin is the number III isomer (LII) and is derivable from the heme derivative mesoporphyrin-IX (LV, i.g. free acid form) via dual decarboxylation (see g.g. Fischer and Orth, 1937; Fuhrhop and Smith, 1975). In the parlance of porphyrin mass spectrometrometry, the ETIOporphyrins correspond to those porphyrins occurring at a nominal mass of 478 14 n (where n is an integer), with 478 m/z being the mass equivalency of a C32 ETIO-porphyrin (cf. Baker, 1966; Baker et QJ., 1967; Yen et gl., 1968).

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579 At present, the primary analytical techniques utilized in porphyrin biomarker application focus upon distinction between DPEP, be they 'true' or 'pseudo' DPEP compounds (cf. Louda and Baker, 1983), and ETIO-series components. Thus, a working knowledge and empirical data bank on the ETIO-type porphyrins is an understood prerequisite to these studies. (XLVIII). Porphyrin: Porphin or porphine. ("F") Porphyrin. ("R"). C20H14N4 310 a.m.u. Porphyrin (XLVIII), the simplest 'porphyrin-type (i.g. fully aromatic) macrocyclic tetrapyrrole, was on-hand in the laboratory of E. W Baker and was originally purchased from the Mad River Chemical Company. According to mass spectral precipts, porphyrin (alt. porphin) can be considered as a ("the") C20 ETIO-porphyrin ( cf. Baker, 1966) and the unsubstituted parent of the series. As a point of interest or curiosity (see text), the electronic spectrum of porphyrin (XLVIII) is of the PHYLLO-type (band order = IV>II>III>I) rather than that of a 'true' ETIO-type (Band order = IV>III>II>I. PHYLLO-type electronic spectra are usually attributed to pertubation of ETIO-type chromophores by alkyl based electron induction (viz donation) at the 'meso' (i.g. methine bridge) positions (Gouterman, 1978; Treibs, 1973) or marked dissymmetry of the rr-cloud due to alkyl-based electron donation at any position (Smith, 1975). Porphyrin (XLVIII), lacking alkyJ substitution, but belonging to the ETIO-series, is therefore a prime example showing that electronic and mass spectral data must coincide before a "type" is given to a geologic isolate.

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Porphyrin (XLVIII). UV/VIS (benzene) S = 396.0, 489.5, 519, 563.2, 616.5 nm = I S/I 300, !/IV = 0.05. Band order = IV>II>III>I. M.S. (E.I., 70 or 12 eV). 310 (M+) m/z. (XLIX). Octamethylporphyrin: 1,2,3,4,5,6,7,8-tetramethylporphin. ("F") 2,3,7,8,12,13,17,18-tetramethyl-porphyrin. ("R"). OMP (trivial). C28H30N4 422 a.m.u. 580 OMP (XLIX) was obtained by the methane sulfonic acid (MSA: see text) demetallation of Mg OMP (XCVII) purchased from Porphyrin Products (Logan, Utah). The resultant free-base, OMP (XLIX), was found to be essentially insoluble in a variety of 'common' porphyrin solvents (g.g. benzene, CH2Cl2 ethyl ether, tetrahydrofuran, etc.) but exhibited a modicum of solubility in freshly distilled chloroform {CHC13). This author attributes the observed insolubility of OMP (XLIX) to near-perfect aromatic stacking due to rr-rr interaction in this perfectly symmetrical molecule, as augmented by the sp2-forced planarity affording van der Waal interaction amongst interdigitated methyl substituents. The observed moderate solubility of OMP (XLIX) in chloroform is attributed to t his solvent's mildly acidic nature which may afford disruption of rr-rr stacking via partial cationization of OMP (XLIX), and allow solvent solute intercalation Octamethylporphyrin (XLIX). UV/VIS S = 398.8, 498.2, 532.8, 567.5, 620.8 nm = I. S/I = 31, I/IV = u.4. Band order= IV>III>II>I M.S. {E.I., 70 eV) 422 (M+) m/z. (L). Octaethylporphyrin: 1,2,3,4,5,6,7,8-0cta ethylpo rphin. ( "F"). 2, 3, 7,8, 12, 13,17, 18-0ctaethylporphyrin. ( "R"). OEP (Trivial). C36H46N4 534 a.m.u. Octaethylporphyrin (OEP, L.) was purchased from Porphyrin Products (Logan, Utah) and, after a lphplc (see text) run which revealed a very high (>99%) degree of purity, was used as received

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581 Octaethylparphyrin (L) UV/VIS (benzene) S = 401.0, 498.5, 531.0, 568.2, 622.0 nm = I. S/I = 30, I/IV = 0.4. Band order = IV>III>II >I. M.S. (E. I., 6 eV) 534 m/z. (E.I 70 eV) 534 (M+), 519 (M-15), 504 (M-15-15) etc (see text) m/z. (LI). Etioporphyrin-!: 1,3,5,7-Tetramethyl-2,4,6,8-tetraethylparphin. ("F"). 2,7,12,17-Tetramethyl-3,8,13,18-tetraethyl-parphyrin. ("R"). C32H38N4 478 a.m.u. Etioporphyrin-! (LI), the mast symmetrical of the etiaparphyrins (viz. actamethyl-actaethyl-parphyrins: see Table AIII), was synthe sized by Dr. J. Gordan Erdman at the Mellon Institute (Pittsburg, Penn.) via the pyrromethene route (see Fischer and Orth, 1937; Fuhrhap and Smith, 1975; Smith, 1975: E. W. Baker, pers. cammun. 1984). As purification during the original preparation ("JGE'') was via HCl/ether partition and 'classic' column chromatography alone ( E. W. Baker, pers. cammun., 1984), and given the shelf age of the product (ca. 25 years), all etiaparahyrin-I (LI) used herein was freshly purified by HCl (2.5 % w/v, aq.)/ethyl-ether partition (see text) and lphplc aver methanal-deactivated silica (see text. cf. Purcell, 1958). Etioporphyrin-! (LI). UV/VIS (benzene) S = 399.5, 498.0, 530.5, 568.5, 622.5 nm = I S/I = 33, !/IV = 0.4. Band order = IV>III > II > I (cf. compound LII) M.S. (E.I., 70 eV) 478 (M+), 476 (M-2), 463 (M15) m/z. (LII). Etioporphyrin-III: 1,3,5,8-Tetramethyl-2,4,6,7-tetraethylparphin. ("F") 2,7,12,18-Tetramethyl-3,8,13,17-tetraethyl parphyrin ("R"). "Natural analytical etioporphyrin" (trivial; Fischer and Orth, 1937). C32H38N4 478 a.m.u. Etioporphyrin-III (LII) was prepared by Dr. J. G. Erdman, as given under the previous compound (LI), except, ostensibly (E. W. Baker, pers. cammun., 1984), by the route of thermal decarboxylation in vacua of mesaparphyrin-IX (LIV) as the free acid (cf. Fuhrhap and Smith,

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582 1975). Purification of etioporphyrin-III (LII) for use herein followed that given above (see compound LI). Etioporphyrin-III (LII). UV/VIS (benzene) S = 399.5, 498.0, 530.5, 568.5, 622.5 nm = I. S/I = 32, I/IV = 0.4. Band order = IV>III>II>I. M.S. (E.I., 6 eV) 478 (M+) m/z. (E.I., 70 eV) 478 (M), 476 (M-2), 463 (M-15), 448 (M-15-15) m/z. (see text). {LIII). Protoporphyrin-IX D.M.E.: 1,3,5,8-Tetramethyl-2,4divinyl-porphin-6,7-dipropionic acid dimethyl ester. ("F"). 3,80ivinyl-13,17-di[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethyl porphyrin. ("R"). C36H3804N4 590 a.m.u. Protoporphyrin-IX O.M.E. (LIII) was made available to this author as an intermediate formed from hemin (equine, Sigma Chemical Co.), by HCl gas catalyzed demetallation in pyridine/methanol/chloroform solu tion gl., 1966; Oinello and Chang, 1978), during the Master's research of Mr. Guteirrez (1987). Protoporphyrin-IX DME (LIII), in the present study, served as a spectrometric standard for free-base etioporphyrins with conjugated vinyl moieties as auxochromes (see text). Protoporphyrin-IX D.M.E. (LIII). UV/VIS (benzene) S = 410.0, 506.5, 540.8, 578.2, 633.0 nm = I. S/I = 28, I/IV = 0.4. Band order = IV>III>II>I. M.S. (E.I., 70 eV) 590 (M+), 517 (M-73) m/z. {LIV). Hematoporphyrin-free base D.M.E.: 1,3,5,8-Tetramethyl2,4-di-(a-hydroxy ethyl)-porphin-6,7-dipropionic acid methyl ester. ("F"). 3,8-dihydroxy (3',8') ethyl-13,17-di[2-(methoxycarbonyl)ethyl]2,7,12,18-tetramethyl-porphyrin. ("R"}. 626 a.m.u. Hematoporphyrin-free base was prepared by Dr. J. G. Erdman during the Mellon Institute studies. Preparation was "most likely" via concentrated sulfuric acid demetallation of hemin (E. W. Baker, pers. commun.) which also leads to hydration of the, originally, vinyl groups and yields the a-hydroxy-ethyl moieties (see Fuhrhop and Smith, 1975).

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583 Hematoporphyrin-free base, as available in the laboratory of Professor Baker (i.g. "JGE," Mellon remnant), was found to be a crude product and probably had suffered the oxidation/breakdown rigors of time. Thus, the sample on hand was first chromatographed, as the diacid, over microcrystalline cellulose, eluting with 25% acetone in petroleum ether (see text). The di-acid of LIV was next esterified with 7% H 2S04 in MeOH and chromatographed over alumina (grade II-III, neutral), eluting with CH2Cl2 Final purification of hematoporphyrinfree base D.M.E. (LIV) was achieved via lphplc over methanol deactivated silica gel (see text) and eluted with 2.5-5.0% (v/v) acetone in petroleum ether. Impurities, at this stage, all (viz. 4 compounds) eluted subsequent to LIV, exhibited 'aberrant' electronic spectra, as compared to regular porphyrins (see text), and, as such, were most likely 'oxidation products' (i.g. uncharacterized, discarded). Hematoporphyrin-free base D.M.E. (LIV). UV/VIS (benzene) S = 408.2, 505.0, 539.2, 577.5, 632.0 nm =I. S/I = 39, I/IV = 0.3 Band order= IV>III>II>I. M.S. (E.I., 70 eV) 626 (M+), 608 (M-H20), 590 (MH20-H20: base peak) m/z. (LV). Mesoporphyrin-IX D.M.E.: 1,3,5,8-Tetramethyl-2,4-diethyl porphin-7,8-dipropionic acid dimethyl ester. ("F"). 3,8-Diethyl13,17-di[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrin. ("R"). C36H4204N4 594 a.m.u. Mesoporphyrin-IX D.M.E. (LV) was prepared by E. W. Baker and co workers (1964) via the catalytic (i.g. Pt) hydrogenation (H2 ) of hemin (viz. Fe(III)Cl chelate of LIII, as the free acid) in aqueous base (0.2N KOH) followed by esterification in methanol with dry HCL(g) as the catalyst. Good supplies (i.g. gram quantities) of the original preparation (Baker et . 1964) were on-hand and an aliquote,

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following cursory chromatographic (lphplc, silica: see text) repurification to remove aging side-products, was utilized herein. 584 Mesoporphyrin-IX D.M.E. (LV). UV/VIS (benzene) S = 401.8, 498.8, 531.5, 569.0, 623.8 nm = I. S/I = 34, I/IV = 0.4. Band order = IV>III>II>I M.S. (E.I., 8 eV) 594 (M+) m/z. (E.I., 70 eV) 594 (M+), 579 (M-15), 521 (M-73) m/z. (LVI). Deuterooorohvrin-IX D.M.E.: 1,3,5,8-Tetramethyl-porphin6, 7-dipropionic acid dimethyl ester. ( "F"). 3, 7-di [2-(methoxycar bonyl)ethyl]-2,8,12,18-tetramethylporphyrin. ("R"). C32H3404N4 538 a.m.u. Deuteroporphyrin-IX D.M.E. (LVI) was prepared by Dr. J. G Erdman ("JGE," Mellon Institute) 'probably' via the devinylation of protohemin followed by demetallation and esterifiction (E. W. Baker, pers. commun., 1985; cf. Fuhrhop and Smith, 1975). Deuteroporphyrin-IX D.M.E. (LVI) was purified, for the reasons and by the methods given under compound LIV, and employed herein as a spectroscopic/ chromatographic standard of an etioporphyrin dimethyl ester containing 2 unsubstituted 'B-positions' (see text). Deuteroporphyrin-IX D.M.E. (LVI) UV/VIS (benzene) S = 401. 2, 497.0, 529, 568.0 (Ia = 595), 621.8 nm =I. S/I =53, I/IV = 0.3. Band order= IV>III>II>I. M.S. (E.I., 70 eV) 538 (M+), 465 (M-73), 392 (M-73-73) m/z. (LVII). Deuteroetioporphyrin-IX: 1,3,5,8-Tetramethyl-6,7-in. ( "F"). 3, 7-D i ethyl-2 ,8, 12, 18-tetramethylporphyri n. ("R"). C28H30N4 422 a.m.u. Deuteroetioporphyrin-IX (LVII, crude) was prepared by Dr. J. G. Erdman ("JGE," Mellon Institute) via the pyrromethene route using, with respect to B-pyrrole positions (see text; cf. Fischer and Orth, 1937), methyl-hydrogen (i.g. M-H) and methyl-ethyl (i.g. M-E) monomers. Following syntheses of the 2 required dipyrrolic dimers (viz. M-H/M-H

PAGE 613

585 and M-E/M-E) further condensation gave the crude tetrapyrrole LVII (E. W. Baker, pers. commun., 1985) plus impurities, is detailed below. The synthesis of LVII by Dr. Erdman took place in the mid-1950's and essentially followed the time-honored Fischer methodology (Fischer and Orth, 1937; cf. Smith, 1975) which employs the thermal condensation of pyrromethenes. Thus, in the condensation of a symmetrical (i.g. R1 = R4 R2 = R3 ) dipyrrole monomer with an asymmetrical (i.g. R1 = R3 R2 = R4 ) dipyrrole, without a method of forced orientation, 4 products are possible (Figure A17). In the present case, these products will be: (a) two isomers of tetramethyl porphyrin (C24, 366 a.m.u.); (b) a single tetramethyl-diethyl porphyrin (viz. LVII, C28, 422 a.m.u.); and (c) a single tetramethyl-tetraethylporphyrin (C32, 478 a.m.u. viz. etioporphyrin-! [LI]; see Table Alii). Except for the isomeric nature of the tetramethyl-porphyrins, all three of these products were indeed found in the preparation of LVII ("JGE" available in the laboratory of Dr. Baker. The mixed product nature of this reaction was most likely known as the original vial was labelled "Deuteroetioporphyrin-crude, JGE, 1958." The electronic spectrum of 'LVII-crude' was found to be that typical of a 'true' ETIO-type rather than that of a "modified-ETIO" (cf. Palmer et 1982). That is, the diunsubstituted, B-positions (i.g. hexa-alkyl), porphyrins, exemplified by the deuteroetioporphyrins (see Fischer and Orth, 1937; Treibs, 1973), exhibit electronic spectra with hypsochromically shifted maxima (ca. nm; this study) and band III and I extinctions which are hypochromic (i.g. lowered, less intense) when compared to the 'true' (viz. acta-alkyl) etioporphyrins (this study: cf. Palmer et . 1982; Treibs, 1935a-b, 1973).

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586 Thus going on the above discourse, it was obvious that LVII, as available, was far from pure. In order to obtain a proper chromato graphic and spectrometric known, it was therefore necessary to affect considerable purification. Fortunately, this was accomplished in 2 steps. First, a rapid chromatography over alumina (grade II-III, neutral) in CH2Cl2 was performed, in order to elute all alkyl porphyrins whilst retaining any oxidized side and/or aging products. Second, the eluate from the 'classic' chromatography was submitted to reverse-phase (RP) lphplc over Cl8 (ODS) bonded 13-24 (8x500 mm) developing with acetone/methanol/water (90/5/5, v/v/v: see text). The resultant chromatogram, shown as Figure A17, reveals that the 3 expected peaks (viz. alkylation paterns, not isomeric forms) were indeed found. As the reverse-phase separation of porphyrins has shown (see g.g. Hajlbrahim et 1978, 1981; Quirke et 1979, 1982; Sundararaman, 1985) and being in-line with the general pattern of RPseparations (see Hamilton and Sewell, 1977; Melander and Horvath, 1980; Snyder and Kirland, 1979), these eluates were the (Figure A17): (a) the tetramethylporphyrin(s) (viz. C24 ETIO-porphyrins, 366 m/z, no isomeric separation, cf. Quirke et 1979); (b) the desired tetramethyl-diethyl-porphyrin (viz. LVII, C28, 422 m/z, Figure AlB); and (c) the tetramethyl-tetraethyl-prophyrin (viz. etioporphyrin-! [LI], see Table Alii). The position of the tetramethyl porphyrin(s) in the resultant chromatogram (Figure A17) was taken as a 'known' for tetra-Bunsubstituted ETIO-porphyrins in the present studies. However, due to the small amounts of final isolates (<5 and the uncertaintity as

PAGE 615

-to isomeric form, a tetramethyl porphyrin 'standard' is not claimed beyond being able to obtain a valid electronic and mass spectrum.* 587 Deuteroetioporphyin (LVII) UV/VIS (benzene) S = 401.0, 497.1, 529, 568.2 (Ia = 594), 621.6 nm = I. S/I = 52, I/IV = 0.3. Band order = IV>III>II>I (Figure AlB). M.S. (E.I., 70 eV) 422 (M+), 407 (M-15) m/z. (E.I., 4.5-6.0 eV) 422 (M+) m/z. {LVIII). Phylloporphyrin. (a) free acid. {b) methyl ester: (LVIIIb) 1,3,5,8,y-Pentamethyl-2,4-diethyl-porphin-7-propionic acid methyl ester. ( "F"). 3 ,8-Diethyl-2, 7,12,15 ,18-pentamethyl-17 [ 2(methoxycarbonyl)ethyl]porphyrin. ("R"). C33H3802N4 522 a.m.u. Phylloporphyrin free acid (LVIIIa) was purchased from Carl Roth laboratories (FRG). The product, as received, was found to be exceedingly impure as shown later by a preliminary electronic spectra check (viz. ETIO not Phyllo type) and subsequent chromatographies. Thus, significant isolation/purification was necessary with this 'product.' In essence, the route followed was: (a) the esterification of LVIIIa, as received, in 7% H2S04-methanol; (b) chromatography over alumina (grade II-III, neutral) eluting with methylene dichloride which afforded, in order of elution; a phylloporphyrin-ME (LVIIIb) enriched fraction, a rhodoporphyrin-ME (LIX) enriched fraction, and a mixture of chlorin-like impurities which were discarded; and (c) the lphplc (see text) of the two main 'enriched' fractions in order to isolate LVIIIb and LIX. Phylloporphyrin-ME (LVIIIb) UV/VIS (benzene) S = 408.2, 504.5, 537.0, 576.5, 630.5 nm = I. S/I = 140, I/IV = 0.1. Band order= IV>II >III>I. M.S. (E.I., 70 eV) 522 (M+), 507 (M-15), 449 (M-73) m/z. *Tetramethylporphyrins (TMP: mixture of 1,3,5,7-and 1,3 ,5,8-TMP; see above). UV/VIS (benzene) S = 401.5, 495, 522, 566, 618 nm = I S/I 40, I/IV = 0.3. Band order= M.S. (E.I., 70 eV) 366 (M+) m/z.

PAGE 616

588 1,3,5,8-Tetramethyl-2,4-diethyl-6carboxyllc acld-porphin-7-propionic acid dimethyl ester. ("F"). 3,8-Diethyl-17,12,18-tetramethyl-13-methoxycarbonyl-17-[2-(methoxycar bonyl)ethyl] porphyrin. ("R"). C34H3804N4 566 a.m.u. Rhodoporphyrin-M.E. (LIX) was isolated as an impurity from a commercial preparation of phylloporphyrin (LVIII), as given above. During the final lphplc purification of LIX a more polar impurity, tentatively identified as rhodoporphyrin-XV-y-carboxylic acid TME, was also found. This impurity, while still exhibiting a RHODO-type electronic spectrum, was noticed to exhibit hyperchromic band III absorption and all bands were bathochromically shifted, with respect to LIX. This second impurity was not characterized further. Rhodoporphyrin-M.E. (LIX). UV/VIS (benzene) S = 408.5, 508, 547.8, 576.2, 635.0 nm = I. S/I = 140, I/IV = 0 2. Band order= III>IV>II>I. M.S. (E.I., 70 eV) 566 (M+), 551 (M-15), 508 (M-58), 493 (M-73) m/z. (LX). Pyrroporphyrin. (a) free acid. (b) methyl ester: (LXb) 1,3,5,8-Tetramethyl-2,4-diethylporphin-7-propionic acid methyl ester. ( "F"). 3 ,8-D i ethyl-2, 7,12, 18-tetramethyl-17[ 2(methoxycarbonyl )ethyl] porphyrin. ("R"). C32H3602N4 508 a.m.u. Pyrroporphyrin, as the free-acid (LXa), was purchased from Carl Roth laboratories (FRG). An electronic spectral check of this product (LXa), as received, revealed a very high amount of impurities. was especially marked, and obvious, in the fact that the band I 'absorption' contained at least 3 maxima and/or overlapping bands. These being 623, 630, and 647 nm indicating, respectively, pyrroporphyrin (LXa), phylloporphyrin (LVIIIa) and meso-pyrrochlorin (XXXI), as found by subsequent analyses. Esterification and purification of the desired product (LXb), and isolation of another 'standard' (XXXI), essentially followed the scheme given previously for phylloporphyrin-M.E. (LVIIIb). In the present

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589 case, the order of elution from neutral alumina (grade II-III) in CH2Cl2 was meso-pyrro chlorin-M.E. (XXXI), pyrroporphyrin-M.E. (LXb) and phylloporphyrin-M.E. (LVIIIb), each being only 'enriched' (i.g. not separate pure fractions) in the compound mentioned. As phyllopor phyrin-M .E. (LVI!Ib) was purified separately, the 'tail' from the above chromatography was discarded and the fractions enriched in XXXI and LXb were purified via lphplc (see text) separately. Pyrroporphyrin-M.E. (LXb). UV/VIS {benzene) S = 404.8, 502. 0, 531.4, 575.8, 622.2 nm = I. S/I = 70, !/IV = 0.2 Band order = IV>III>ii > I M.S. (E.I., 70 eV) 508 (M+), 493 (M-15), 435 (M-73) m/z. Metallo-chelates of Tetrapyrrole Pigments; Other than Chlorophylls, per se Metallo-porphyrins, with regards to biomarker evolution, are the ultimate recognizable products of tetrapyrrole fossilization. Thus, the need for a variety of metalloporphyrin standards (i.g. compounds LXXVIII-CXV, CXVII-CXX) is obvious. The change from biotic chlorophyll or heme into vanadyl DPEP or ETIO-porphyrins was first envisioned as inc luding a transmetallation (i.g. Mg to V=O), defunctionalizations and aromatization by Professor Treibs {1936; cf. Baker and Palmer, 1978). During the 1960's the group of G. W. Hodgson, B. L. Baker and co-workers (see g.g Hodgson et gl. 1960, 1963, 1967, 1969) forwarded the idea that metallo-chlori ns, especially copper, were intermediates between chlorophyll-a {I) and the metallo-(i.g. Ni, V=O) porphyrins of sedimentary bitumen. More recently, Russian workers commonly add the presence of copper chlorins and porphyrins as a universal part of the diagenetic continuum between biotic pigments and the more common geologic metallo (viz Ni, V=O) porphyrins (see Galimov et gl., 1980, 1982, 1983: see text re arti-

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590 facts). However, based on the works of Baker and Smith (Baker and Smith, 1973, 1975a-b; Smith and Baker, 1974}, Baker and Palmer (Baker and Palmer, 1978, 1979a-b; Baker et . 1976, 1977, 1978a-b; Palmer and Baker, 1979, 1980; Palmer et . 1979 and my experiences (Baker and Louda, 1980a-c, 1981a-b, 1982, 1983, 1984, 1986; Louda and Baker, 1981, 1986; Louda et . 1980}, during which not a single verifiable metallo-phorbide or metallo-chlorin, save chlorophylls-a (I) or -c (XIX), could be shown, I doubt seriously the participation of same in the natural diagenesis of tetrapyrroles on a global scale. That is, it is still possible that metallo-chlorins may form in the more acidic (i.g. pH
PAGE 619

591 CIV, CX"I, CXIII, CXV and CXIX) and numerous in vitro derivatizations of geologic free-base porphyrins (see text), that 100% chelation of cu ( e c nat I l 63c t t) 1 h u or u:see ex 1s eas1 y ac 1eved at room temperature (g.g. 24C) under nitrogen, in the dark, for 18-12 hours (see text). Excepting partial aromatization of DOMPP-a (XII) and its 7-propyl-7despropio analog (XIII), yielding LXXXVII and LXXXIX, respectively, no other artifactation was noted {cf. Zelmer and Man, 1983, 1984). In retrospect, it is fortuitous, if not the raison d'etre, that Cu++ chelation by dihydroprophyrins and porphyrins is so facile. That is, there are good evidences that copper tetrapyrroles are indeed part of geochemistry (viz. epigenetic: Baker and Louda, 1984, 1986; Louda and Baker, 1981, 1986; Palmer and Baker, 1978) just as there are probable artifacts given in the literature as fact (i.. reaction of bitumen over activated copper for sulfur removal. cf. Galimov et gl., 1980, 1982, 1983). Nickel and vanadyl porphyrins, the most common geopigments, are included herein for that reason. Nomenclature will not be duplicated in the following sections as this can be found under the appropriate precursor porphyrin ligand, as cited below. In like manner neither the preparation of the tetrapyrrole, with few exceptions, nor the cheiation reaction will be described. These facets of the syntheses can be found in the preceding sections or the main body of the text (viz. Methods/Metals and Chelation) respectively. In all cases, purification or verification of purity was via lphplc or TLC, respectively (see text). All "Oxy-Deoxo" (i.. 9-hydroxy-9-deketo and/or 3-methanol-3-desformyl) derivatives were formed via the sodium borohydride reduction, in

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592 ethanolic ethyl ether (see text), of the corresponding parental 'metallo-carbonyl-tetrapyrrole' LXII from LXI, LXIV from LXIII, etc.). That is, attempts to affect chelation (viz. Cu++) by the corresponding 'oxy-deoxo' free-base often lead to partial reoxidation of hydroxyl moieties yielding, besides the desired product, the parental Cu 'carbonyl-pigment' (Louda, unpublished: VII + Cu++ LXI +LXII). Copper and nickel phorbides (LXI). Cu Pheophorbide-a M.E.: (see VIb. cf Hodgson and Baker, 1967; Jones et g_]_., 1968). C36H3605N4Cu, 667 a.m.u. UV/VIS 401, 421.1, 505, 547, 603.0, 650.0 nm =I. S/I = 1.4, l/IV = 13.6. Band order= I>II>III>IV. M.S. (E.I., 70 eV) 609 (M-58, pyro reaction, see text). (LXII). Cu-9-0xy-9-deo x o-pheophorbide-a M.E.: (see VII). C36H3805N4Cu, 669 a.m.u. UV/VIS (benzene) S = 405.2, 510, (540), (580), 621.5 nm = I. S/I = 3.4, I/IV = 6 1. Band order= I>IV>(II)>(III) M.S., not determined. (LXIIIa). Cu-Mesopyropheophorbide-a M.E.: (see X). C34H3603N4Cu, 611 a .m.u. UV/VIS (benzene) = 404.0, 421.8, 508, 550, 596, 642.8 nm = I, S/I = 1.8, I/IV = 14.8. Band order= I>II>III>IV. M.S., not determined. (LXIIIb). Ni-Mesopyropheophorbide-a M.E.: (see X). C34H3603N4Ni, 606 a.m.u. UV/VIS (benzene) S = (368), (402), 417.6, 498, 538, 593, 640.8nm = I, S/I = 1.4, I/IV = 12.5. Band order= I>II>III>IV (Figure A19). M.S. (E.I., 70 eV) 606/608 (M+), 491 (M-87-28) m/z. {LXIV). Cu 9-0xy 9-deoxo-mesopyropheophorbide-a M.E.: (see XI). C34H3803N4Cu, 613 a.m.u.

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593 UV/VIS (benzene) S = 402.0, 500, (525), 566, 614.0 nm = I. S/1 = 5.0, !/IV= 2.8. Band order= I >IV>(III)>II. M.S., not determined (LXV). Cu-Deoxomesopyropheophorbide-a M.E.: (see XII). C34H3802N4Cu, 597 a.m.u. UV/VIS (ethyl ether) S = 395.2, 494.0, (523), 564.0, 605. 5 nm = I. S/1 = 2.7, !/IV= 12. Band order= I > II >IV>(III). M.S. (E.I 70 eV), 597/599 (M+), 595 (M-2, "aromatization," see text) 522/524/526 (M-2-73/M-73), 298.5/297.5 (M++/(M-2)H) m/z. {LXVIa). Cu-7-Propyl-7-despropio-DOMPP-a: (see XIII) C33H38N4Cu, 553 a.m.u. UV/VIS (benzene) S = 402. 8, 499, 536, 568, 610.8 nm = I. S/I = 3.5, !/IV= 9.1. Band order= I>II >IV>III. M.S. (E.I., 70 eV) 553/551 (M+/(M-2+)), 522 (M-2-29), 510 (M-43) m/z. (LXVIb). Ni-7-Propyl-7-despropio-DOMPP-a: (see XIII). C33H38N4N i, 548 a.m. u. UV/VIS (benzene) S = 399.8, 494, 525, 566, 609. 8 nm = I, S/I = 2 3, I/IV = 10.9. Band order= I > II >IV>III (Figure A20). M .S. (E.I 70 eV) 548/550 (M+), 546/548 (M-2, base), 517/519 (M-2-29), 505/507 (m-43) m/z. (LXVII). Cu Pheophytin-b: (see XV). C55H7006N4Cu, 945 a.m.u. UV/VIS (ethyl ether) S = 438.0, 520 (IV), 582, 628.5 nm = I. S/1 = 2.0, r;rv* = 6.5. Band order= I >II>IV. M.S., not determined. {LXVIII). Cu-3-Methanol-3-desformyl-9-o x y-9-deoxo-pheophytin-b: (see XVI). C55H7406N4Cu, 949 a.m.u. UV/VIS (ethyl ether) S = 409. 2, 505 (IV), (548, III), 635 nm = I. S/I 4, I/IV 5. Band order= I >IV>(III). (Figure A57 dashed). M.S., not determined. Copper chlorins (LXIX). Cu-Chlorin-e6-T.M.E.: (see XXIIIa). C37H4006N4 699 a.m.u *As only 3 visible band were found the ratio "I/IV," in this case, refers to A628 5/A52o

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594 UV/VIS {400), 414.0, 505, {540), {595), 641 nm = I S/I = 1.9, I/IV = 4.8. Band order= I>{II) > IV>{III). UV/VIS (ethyl ether) = {395), 409. 0, 502, {540), {595), 634. 6 nm = I. S/I = 2.5, I/IV = 5 1. Band order= I > (II)>IV>{III). M.S., not determined. (LXX). Cu-Purpurin-18 M.E.: (see XXVIb). C34H3205N4Cu 639 a.m.u. UV/VIS {benzene) = 392, 419.0, 504, 546, 623, 672.0 nm = I. S/I = 1.8, I/IV = 11. Band order= I > II>IV>III. M.S., not determined. {LXXI). Cu-Chlorin-p6-T.M.E.: (see XXVIIb). C36H3806N4Cu, 685 a.m.u. UV/VIS {benzene) = 388, 412.5, 501, (530), (600), 646.5 nm = I. S/I = 2.3, I/IV = 9.8. Band order= I>(II)>IV >(III). M.S., not determined. (LXXII). Cu-Purpurin-7-T .M.E.: (see XXIV). C37H3807N4Cu, 713 a.m.u. UV/VIS (390), 419.8, 507, 542, (620), 669. 5 nm =I. S/I = 2.2, 1/IV = 7.0. Band order= I >(II)> IV>III. M.S., not determined. (LXXIII). Cu-"Oxy-deoxo"-Purpurin-7-T.M.E: (see XXV). C37H4007N4Cu, 715 a.m.u. UV/VIS (402), 414.4, 508, 552, (610), 642.5 nm = I. S/I = 2, I/IV = 10. Band order= I > (II)>IV>III. M.S., not determined. (LXXIV). Cu-Rhodin-g7-T.M.E.: (see XXVIII). C37H3807N4Cu, 713 a.m. u. UV/VIS (benzene) S = 415. 8, 509, 548, 598, 648.8 nm = I. S/I = 6.6, I/IV = 5.2. Band order= I>II>III>IV. M S not determined. (LXXV). Cu-3-Methanol-3-desformyl-rhodin-g7-T.M.E.: (see XXIX) C37H4007N4Cu, 715 a .m.u. UV/VIS (benzene) S = 409.5, 503 (542), (590), 634.6 nm = I. S/I 6, I/IV 2.5. Band order= I > II >IV>III. (cf. Cu Chlorin-e6-TME, LXIX. M.S., not determined. (LXXVI). Co-Chlorin: C20H14N4Co, 367 a .m.u.

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595 Cobalt chlorin (LXXVI), prepared by Dr. E. W. Baker (viz. Mellon Institute studies), was included herein only as a reference for an alternate (i.g. non-copper) metallo-chlorin chromophore. Co-Chlorin (LXXVI): UY/VIS (benzene) S = 392, 415.0, 527, (560), 605 nm =I. S/I = 12, I/IV = 1.4. Band order= I>III>(II). M.S., not determined. (LXXVII). Cu-Mesopyrrochlorin-M.E.**: 571 a.m.u. UV/VIS (ethyl ether) S = 401.6, 502, 528, 564, 614.2 nm = I. S/I = 10, !/IV = 6.5. Band order= I>II>III>IV. UV/VIS (benzene) S = 406.2, 501.2, 531, 566, 617.2 nm = I S/I = 11, !/IV = 7. Band order = I>II>III>IV. M.S. (E.I., 70 eV) 571 (M+), 573 {M+2, aromatization, see text), 512/498/484 (M-59/73/87) m/z. Meta lloporphyri ns of the "DPEP-seri es" (LXXVIII). Ni Phylloerythrin-M.E.: (see XXXII). C34H3403N4Ni, 604 a.m.u. UV/VIS (benzene) S = 413.0, {495), 518, 537(B), 582.2 nm =a. S/a = 4.5, a/B = 3.2. Figure A21). M.S. {E.I., 70 eV) 604/606 {M+), 589 (M-15), 531 {M-73), 302 (M++), 265.5 ((M-73)++) m/z. (LXXIX). Ni-9-0xy-9-deoxo-phylloerythrin-M.E.: (see XXXII). C34H3603N4Ni, 606 a.m.u. UV/VIS {benzene) S = 397.5, B = 516, a= 553.0 nm. S/a = 8.9 a/B = 2.0 (Figure A22). M.S. (E.I., 70 eV) 604 (M-2), 590 (M-18+2), 531 {M-2-73), 517 {M-18+2-73) m/z (see text). (LXXX). Cu-Phylloerythrin-M.E.: (see XXXII). C34H3403N4Cu, 609 a.m.u. UV/VIS (benzene) S = 416.2, (515), (530), 548.0{B), 594.8 nm =a. S/a = 6.4, a/B = 2.6. M.S. (E.I., 70 eV) 609/611 594 (M-15), 536 (M-73), 304.5 (M++), 268 {{M-73)++) m/z. *In this case I/IV, actually !/III, is A605/A527 **Not totally pure as some Cu-phyllochlorin-M.E (see LVIIIb) is in evidence at 565 m/z.

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{LXXXI). Cu-9-0xy-9-deoxo-phylloerythrin-M.E.: (see XXXIII). C34H3603N4Cu, 611 a.m.u. UV/VIS (benzene) S = 404.2, B = 529. 0, a = 563.8 nm. S/a = 20, a/B = 1.2. M.S. (E.I., 70 eV) 611 sporadic, low S/N (pyrolytic). (LXXXII). Ni-2-0xo-phylloerythrin-M.E.: (see XXXVb). C34H3204N4Ni, 618 a .m.u. 596 UV/VIS (benzene) S = 421.0, B = 531, a = 601.8 nm. S/a = 6.6 a/B = 3.3 (Figure A23). M .S. (E.I., 8 eV) 618/620 M S (E.I., 70 eV) 618 (M+), 603 (M-15), 545 (M-73) m/z. (LXXXIII). Ni-2-a-Hydro x y-ethyl-2-desethyl-9-oxy-9-deoxo-phyllo erythrin-M.E.: (see XXXVI). C34H3604N4Ni, 622 a.m.u. UV/VIS (benzene) S = 399.8, B = 516.0, a = 554. 0 nm. S/a = 9.4, a/B = 1.5 (Figure A24). M.S., not determined (pyrolytic). (LXXXIV). Cu-2-0xo-phylloerythrin-M.E.: (see XXXVb). C34H3204N4Cu, 623 a.m.u UV/VIS (benzene) S = 420.8, = (538), B = a= 611.2 n.m. S/a = 7.1, a/B 1 = 3.8. M.S. (E.I., 4-6 eV) 623/625 ( 3Cu/65Cu-M+). (E.I., 70 eV) 623 (M+), 550 (M-73) m/z. (LXXXV). Cu-2-a-Hydroxy-ethyl-2-desethyl-9-oxy-9-deoxo phylloerythrin-M.E.: (see XXXVI). C34H3604N4Cu, 627 a.m.u. UV/VIS (benzene) S = 405.0, B = 529. 8, a = 564.0 nm. S/a = 16, a/B = 1.02. M.S., not determined (pyrolytic). (LXXXVI). Ni-Deoxophylloerythin-M.E : (see XXXVIIb). C34H3602N4Ni, 590 a.m.u. UV/VIS (benzene) S = 396.0, B = 517.0, a= 554.5 nm. S/a = 8.3, a/B = 2.1 UV/VIS (ethyl ether) S = 392. 0, B = a= 550.5. S/a = 8.2, a/B = 2.2. M.S. (E.I., 70 eV) 590/592 (M, 577 (M-15), 519 (M-73) m/z.* (LXXXVII). Cu-Deoxophylloerythrin-M E.: (see XXXVIIb). C34H3602N4Cu, 595 a.m.u. *oata given is for natural isotopic abundance nickel. Synthetic monoisotopic 58Ni-or yielded identical UV/VIS data and the M S. data expected (i.g. M+ = 590 or M+ = 592 m/z, respectively).

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597 Cu-DPE-M.E. (LXXXVII) was prepared in 2 manners, one planned and one not. That is, in the first case, the chelation of cu (see text) by DPEM.E. (XXXVIIb) was carried out and easily yielded the desired (viz. LXXXVII) product. In the second case, Cu-DPE-M.E. (LXXXVII) was found to have formed as an aromatization artifact during the preparation of Cu-DOMPP-a M.E. (LXV: cf. Zelmer and Man, 1983) from the parental free-base phorbide (viz. XII). In the latter case, lphplc separation over methanol-deactivated silica gel in 1.5% acetone/ petroleum ether (see text) easily and cleanly separated these 2 copper complexes (viz. LXXXVII and LXV). Cu-DPE-M.E. (LXXXVII) obtained via either of the above routes yielded identicial physicochemical data, as given below. Cu-DPE-M.E. (LXXXVII). UV/VIS (benzene) S = 402.0, B = 526.0, a = 563. 5 nm. S/a = 11.6, a/B = 1 4 UV/VIS (ethyl ether) S = 398.5, B = 524.0, a= 561.0 nm. S/a = 11.9, a/B = 1.4. M.S. (E.I., 70 eV) 595/597 (M+), 580 (M-15), 522 (M-73) m/z. (LXXXVIII). Vanadyl-Deox ophylloerythroetioporphyrin. V = 0 DPEP: (see XXXVIII). C32C34N4VO, 541 a.m.u. The V = 0 DPEP (LXXXVIII) used herein was an aliquote of that prepared and reported by Dr. E. W. Baker (Baker et 1968). V = 0 DPEP (LXXXVIII). UV/VIS (benzene) S = 410.8, B = 534.0, a= 574.0 nm. S/a = 12, a/B = 1 .3. M.S. (E.I., 4-6 eV) 541 (M+) m/z. (E.I., 70 eV) 541 (M+), 539 (M-2), 526 (M-15), 508-12 (m*, 541-15), 270.5 (M++) m/z. (LXXXIX). Cu-7-Propyl-7-desethyl-DPEP: (see XIII, XXXIX). C33H36N4Cu, 551 a m .u. Cu-7PDE-DPEP (LXXXIX) was not prepared via the chelation of cu by the corresponding free-base (viz. XXXIX) but rather, as given above for compound LXXXVII), as an 'aromatization artifact' (cf. Zelmer and Man, 1984) during the synthesis of the corresponding 7,8 dihydro

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598 compound (i.g. Cu-7PDP-DOMPP-a: LXVI). As with Cu-DPE-ME (LXXXVII)/ Cu-DOMPP-a M.E. (LXV), lphplc separation (see text. cf. LXXXVII) cleanly separated Cu-7-PDP-DOMPP-a (LXVI) from its aromatization product. Cu-7PDE-DPEP (LXXXIX). UV/VIS (benzene) S = 402. 5, B = 526.0, a = nm. S/a = 18, a/B = 1 4. M.S. (E.I., 4-6 eV) 551/553 (M+, 3Cu/65Cu) m/z. (E.I. 70 eV) 551/553 (M+) 549 (M-2), 536 (M-15) 522 (M-29; major loss, see text) m/z. {XC). Ni-DPEP: (see XXVIII). C32H34N4Ni, 532 a.m.u. The Ni-DPEP used herein was an aliquote of that prepared and reported by Dr. E. W. Baker (Baker et gl. 1968). Ni DPEP (XC). UV/VIS (benzene) S = .3 96.0, B = 517.0, a= 554.5 nm. S/a = 8.8, a/B = 2.1. UV/VIS (ethyl ether) S = 392.0, B 514.0, a = 551.5 nm. S/a = 8.0, a/B = 2.1. M.S. (E.I. 4-6 eV) 532/534 M+; 58Ni/60Ni). m/z. (E.I. 70 eV) 532/534 (M+), 517 (M-15), 502 (M-15-15), 502 (m*, 532-15), 266 (M++) m/z. {XCI). Ni-7-Propyl-7-desethyl-DPEP: (see XIII). C33H36N4Ni, 546 a m .u. UV/VIS (benzene) S = 396.0, B = 515.8, a = 553.9 nm. S/a = 8.1, a/B = 2.1 (cf. Figure A25). M.S. (E.I., 4-6 eV) 546/548 (M+) m/z. (E.I., 70 eV) 546/548 (M+), 531 (M-15), 517 (M-29), 273 (M*) 251.5 ((M-43)++) m/z . {XCI I). Ni-2-Methyl-2-desethvl-DPEP, "Abelsonite": C31H32N4Ni, 518 a.m.u. It is quite rare when one is able to claim that the source of an 'authentic' pure standard is Nature itself. However, with the present compound this is the case. 'Abelsonite,' an 'orga nic material' of record (NMNH-143566: Trudell, 1970; Milton et gl, 1978) has been recovered as "fine pink-purp l e metallic patches" (Trudell, 1970) from fissures or grooves of the Parachute Creek member near the Mahogany zone in the Uinta Basin (Green River Formation, Eocene, Colorado-Utah

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U.S.A.: see Mason and Trudell, 1983; Milton et 1978; Trudell, 1970). 599 Since the original proof that "Abelsonite" was a Ni-desmethyl-DPEP (Milton et 1978), its structure has been proven as the 2 methyl-2desethyl form of Ni(C31) DPEP* (Storm et 1984). In the present studies, this author had the good fortune of obtaining an aliquote of the e xact sample (viz. col l ection) of "Abelsonite" which was utilized i n the studies of Dr. Carl y e Storm and co-workers (Storm et 1984). Thus, identical structure is easily, and correctly, I feel, assumed. "Abelsonite" was washed from rock cuttings (i. g core sample) with methylene dichloride, the solvent evaporated in vacuo and chromato graphed (lphplc) over silica (see text). N i -2-Methyl-2 desethyl-DPEP, "Abelsonite" (XCII). UV /VIS (benzene) S = 396.0, B = 517.0, a= 554. 5 nm. 8.7 a/B = 2.2 (cf. A25). M.S. (E.I. 4-6 eV) 518/520 (M+: 5 Ni/60N i = 2.3; calc. 58Ni/ Ni = 2 6; see text) m/z. (E.I., 70 eV) 518 (M+1, 503 (M-15), 488 (M-15-15), 485-489 (m*, 518-15, calc. 488.4) 259 {M+) m/z. Copper Rhodins (XCIII). Cu-Mesorhodin-M.E.: (see XLI). C35H3603N4Cu, 623 a m u. UV/VIS (benzene) S = 409. 5, B = 543. 0, a = 584.8 nm. S/a = 13, a/B = 1.9. M.S. (E.I. 70 eV) 623/625 (M+), 608 (M-15), 550 (M-73), 311.5 (M++), 275 ((M-73)++) m/z. (XCIV). Cu-Dihydro-mesorhodin-M. E .**: (see XLII). C35H3803N4Cu, 625 a.m.u. *In revised nomencl ature ("R"), Abelsonite is: (3-Methyl-3desethyl-17 decarbo x y-131-deo x oporphrinato) nic kel ( II). **"Dihydro, : in this case, implies "ox y-deo xo" (see text).

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600 UV/VIS (benzene) S = 401.2, B = 526, a = 565 nm. S/a = 19, a/B = 1.5. M.S., not determined, per se. (E.I., 70 eV) 607 (M-18, sporadic) m/z. (XCV). Cu-Pvrrorhodin: (see XLVI) C31H300N4Cu, 537 a.m.u. UV/VIS (benzene) S = 410.0+ B = 551, a = 592 nm. S/a = 9.5, a/B = 2 2. M.S. (E.I., 70 eV) 537 (M ), 522 (M-15), 268.5 (M++) m/z. (XCVI). Cu-Dihydro-pyrrorhodin*: (see XLVII) 539 a.m.u. UV/VIS (benzene) S = 399.5, B = 526.0, a = 564.5 nm. S/a = 20, a/B = 1.6. M.S. (E. I., 70 eV) 521 (M-18; M-H20) m/z. Metalloporphyrins of the 'ETIO-series' (XCVII). Mg-Octamethylporphyrin: (see XLVIX). C28H28N4Mg, 444 a.m.u. Mg-OMP (XCVII) was purchased from Mad River Chemical Co., and following chromatographic verification of purity, utilized as a source for OMP, free-base (XLVIX) and the copper chelate (XCIX). Mg-OMP (XCVII). UV/VIS (benzene) S = 409.8, B = 544.8, a= 581.2 nm. S/a = 21, a/B = 1.1. M.S., not determined. (XCVIII). Cu Porphin: (see XLVIII) C20H12N4Cu, 371 a.m.u. UV/VIS (benzene) S = 393.8, B = 516.0, a = 550.0 nm. S/a = 25, a/B = 0.8 M.S. (E.l., 70 eV) 371/373 (M+), 185.5/186.5 (M++) m/z. (XCIX). Cu Octamethylporphyrin: (see XLVIX) C28H28N4Cu, 483 a.m.u. UV/VIS (benzene) S = 398.2, B = 526.0, a = 562.0 nm. S/a = 12, a/B = 2.0. M.S. (E.I., 4-12 eV). 483/485 (M+) m/z. M.S. (E.I., 70 eV) 483 (M+), 468 (M15), 241.5 (M++) m/z. (C). Cu Octaethylporphyrin: (see L) C36H44N4Cu, 595 a.m.u.

PAGE 629

601 UV/VIS (benzene) S = 398.8, B = 525.5+ a = 562.0 nm. S/a = 12, a/B = M.S. (E.I., 4-8 eV) 595/597 (M) m/z. M.S. {E.I., 70 eV) 595 (M ), 593 (M-2), 580 (M15), 565 (M-15-15), 297.5 (M#) m/z. (CI) Cu Etioporphyrin-! : (see LI). C32H36N4Cu, 539 a.m.u. UV/VIS (ethyl ether) S = 394. 5, B = 523.5, a = 560.0 nm. S/a = 10, a/B = 2.2 UV/VIS (benzene) S = 399.5, B = 526.0, a = 562. 5 nm, S/a = 11, a/B = 2 .2. M.S. (E.I., 70 eV) 539/541 (M+), 524 (M-15) m/z. (CII). Cu Mesoporphyrin-IX O.M. E : a .m.u. UV/VIS (benzene) S = 399.8, B = 526. 0 a = 562.9 nm. S/a = 11, a/B = 2.1. M.S. (E.I., 6-8 eV) 655/657 (M+). M .S. (E.I., 70 eV).655 (M+), 653 (M-2), 640 (M-15), 582 (M-73), 509 (M-73-73), 327. 5 (M ) m/z. (CIII). Cu Protoporphyrin-IX O.M.E.: (see LIII). C36H3604N4Cu, 651 a .m.u. UV/VIS (benzene) S = 408.8, B = 534.4, a = 572.2 nm. S/a = 9.8, a/B = 2.0. M.S., not determined. (CIV). Cu Oeuteroporphyrin-D M .E.: (see LVI). C32H3204N4Cu, 599 a.m.u. UV/VIS (benzene) S = 398.4, B = 524.5, a = 560. 0 nm. S/a = 14, a/B = 1.9. M.S., not determined. (CV). Mn (III. ligand= Cl/-OH) Mesoporphyrin-IX O.M.E.: (see LV). C36H4004N4Mn/-Cl-or-OH, 682 or 664 a m.u., respectively. Manganese (+3) mesoporphyrin-IX D.M.E. (XCXV), prepared by Dr. E. W. Baker previously (Baker et gj. 1964), was included herein as an electronic spectral known due to the recent findings of Mn porphyrins in peats and low-ranked humic coals by Professor Raymond Bonnett and co-workers (see Bonnett and Czeckowski, 1981; Bonnett et gj., 1984). Mn(+3) mesoprophyrin-IX O .M.E. (CV). UV/VIS (benzene) S = 360.0, B = 471.2, a= 560 (592) nm. S/a = 9.1, a/B = 0 2. M .S., not determined

PAGE 630

602 {CVI). Vanadyl mesoporphyrin IX D M .E.: (see LV). C36H40Q5N4V, 659 a.m.u UV/VIS {benzene) S = 407.8, B = 534. 4, a= 572.0 nm. S/a = 11, 2.5. M.S. (E.I., 4.5-8 eV) 659 (M+) m/z. M .S. (E.I., 70 eV) 659 (M ), 657 {M-2), 644 (M-15), 628 (M-31), 601 (M-58), 586 572 (M-87), 513 (M-73-73), 329.5 {M++), 277 ({M-105)++ = {M-73-32) ) m/z. {CVII). Vanadyl etioporphyrin-!: (see LI). C32H36N40V, 543 a m u UV/VIS (ethyl ether) S = 403, B = 532, a = 569 nm. S/a = 11, a/B = 2.5. UV/VIS (benzene) S = 407.0, B = 534, a= 571.0 nm. S/a = 11, 2.3. M .S. (E.I., 4-6 eV) 543 (M+) m/z. M S (E.I., 70 eV) 543 (M ), 528 (M-15), 513 {M-15-15), 510-514 (m*: M-15), 271.5 {M++) m/z. {CVIII) Vanadyl etioporphyrin-III: (see LII). C32H36N40V, 543 a.m.u. UV/VIS (ethyl ether) S = 403, B = 532, a = 569 nm. S/a = 11, a/B = 2.5. UV/VIS (benzene) S = 407. 0, B = 534, a= 571.0 nm. S/a = 11, a/B = 2.3. M S (E.I. 4-6 eV) 543 (M+) m/z. M .S. (E.I., 70 eV) 543 (M+) 528 (M-15), 513 (M-15-15), 510-514 (M*: M-15), 271.5 (M++) m/z. (CIX). Ni Mesoporphyrin-IX D .M.E.: (see LV) C36H4004N4Ni, 650 a.m.u. UV/VIS (ethyl ether) S = 389.5, B = 515.0, a = 551.0 nm. S/a = 5.5, a/B = 3.0. UV/VIS (benzene) S = 394.5, B = 519.0, a = 554.5 nm. S/a = 5.4, a/B = 2 9. M.S. (E.I., 70 eV) 650 (M+), 635 {M-15), 577 (M-73), 504 (M-73-73), 325 (M++) m/z. (CX). Ni Octaethylporphy r in: {see L). C36H44N4Ni, 590 a.m.u Three isotopic forms of Ni OEP (CX) were made by the autho r for mass spectrometric studies on the behavior of the 2 main isotopic forms of this metal (viz. 58Ni, 60Ni: see text). These 3 forms were natural isotopic abundance ('Ninat'l,) and each of the two monoisotopic species given above. The electronic and mass spectral data reported below was found to be identical for all 3 forms of Ni OEP, with the obvious e x ception of nominal masses which varied with the nickel isotope(s)

PAGE 631

603 Ni octaethylporphyrin (CX). UV/VIS (ethyl ether) S = 389.5, B = 515.5, a = 550.5 nm. S/a = 4.2, a/B = 2.9. UV/VIS (benzene) S = 393.5, B = 517+5, a=, 552.5 nm. = 4.1, a/B = 3.2. M.S. (E. I. 4-6 eV) 590/592 (M, Ninat L), 590 (M\ 8Ni), 592 (M+, 60Ni) m/z. M.S. (E.I., 70 eV; given for 58Ni) 590 (M+), 575 (M-15) 560 (M-15-15) 295 (M++) m/z. ' (CXI). a.m.u. UV/VIS (benzene) S = 402.8, B = 528.0, a = 561.9 nm. S/a = 15, a/B = }+.4. M.S. (E.I++ 70 eV) 569/571 (M+) 554 (M-15), 496 (M-73), 284.5 (M ), 248 ((M-73) ) m/z. (CXII). Ni Pyrroporphyrin-M.E.*: (see LX) C32H3402N4Ni, 564 a.m.u. UV/VIS (benzene) S = 400.5, B = 523.5, a = 552.5 nm. S/a = 8.4, a/B = 1.8. M.S. (E.I., 70 eV) 564/566 (M+), 549 (M-15), 491 (M-73), 282 (M++), 245.5 ((M-73)++) m/z. (CXIII). Cu Phylloporphyrin-M.E.*: (see LVIII) C33H3602N4Cu, 583 a.m.u. UV/VIS (benzene) S = 406. 0, B = 531.5+ a = 564.5 nm. S/a = 26, a/B = 0.97. M.S. (E.I., 70 eV) 583/585 (M ), 568 (M-15), 510 (M-73), 291.5 (M+), 255 ((M-73)++) m/z. (CXIV). Ni Phylloporphyrin-M.E.*: (see LVIII). C33H3602N4Ni, 578 a.m.u. UV/VIS (benzene) S = 403.8, B = 552.2, a = 558.5 nm. S/a = 13, a/B = 1 4. M.S. (E.I. 70 eV) 578/580 (M+), 563 (M-15), 505 (M-73), 252.5 ((M-73)++) m/z. (CXV). Cu Rhodoporphyrin-M.E.: (see LIX). C34H3604N4Cu, 627 a.m.u. UV/VIS (benzene) S = 407.5, B = 537, a= 580.5 nm. S/a = 11, a/B = 2.3 M .S. (E.I., 4-8 eV) 627/629 (M+) m/z. M.S. (E.I. 70 eV) ++ 627/629 (M+)4612 (m-15) 568.h569 (M-59/-58), 554 (M73), 313.5 (M ). (277 ((M-73) ), 240 ((M-147 +) 7? [M-2-87-58]) m/z. *Each chelate (viz. Cu or Ni) of pyrroporphyrin-ME (LX) or phylloporphyrin-ME (LVIII) contains about 10-15% (via M .S. intensities) of LVIII or LX, respectively.

PAGE 632

604 Benzoporphyrins Benzoporphyrins, most notably their vanadyl chelates, are now known (cf. Baker et gl., 1968; Barwise and Roberts, 1984; Barwise and White head, 1979) to be the 'rhodoprophyrin-like' (see Costantinides and Arich, 1963; Dean and Whitehead, 1963; Fisher and Dunning, 1961; Hodgson et gl., 1963; Howe, 1961; Millson et gl., 1966) pigments found in petroleum and assorted bitumens. The total synthesis of a proper benzoporphyrin known has been carried out and reported by Professor Peter Clezy and co-workers (Clezy et gj., 1977). The parent compound of interest is; 13,17-diethyl7,8,12,18-tetramethylbenzo[2,3]-21H,23H-porphine, which I shall refer to as 'benzoetioporphyrin' herein. The vanadyl (CXVIII) and nickel (CXVII) chelates reported below as standards in the present studies were the most generous gifts of Professor Clezy to the author. An aliquote of the Ni "benzoetioporphyrin" (CXVII) supply was demetalated via methane sulfonic acid catalysis (see text) to yield the free-base (CXVI). The latter was further derivatized (Cu++) to obtain the copper chelate (CXIX). (CXVI). "Benzoetioporphyrin": (see above). C32H32N4 472 a.m.u UV/VIS (benzene) S = 403.5 (478) 504.6, 540.0, 573.5, 630.8 nm = I. S/I = 18, I/IV = 1.3. Band order= III>I>IV>II>IVa (Figure A26). M.S. (E.I., 70 eV) 472 (M+), 470 (M-2), 457 (M-15), 442 (M-15-15), 236 (M++) m/z. (CXVII). Ni "Benzoetioporphyrin": (see CXVI). C32H30N4Ni, 528 a.m.u. UV/VIS (benzene) S = 400.5, B = 528.5, a = 568.5 nT. S/a = 4.0, a/B = 4.4 (Figure A27). M.S. (E.I., 4-6 eV) 528/530 (M ), 526 (M-2} m/z. M .S. (E.I., 70 eV} 528 (M+), 526 (M-2}, 513 (M-15), 498 (M-15-15), 264 (MH} m/z.

PAGE 633

605 (CXVIII). Vanadyl "Benzoetioporphyrin": (see CXVI). C3 2H30N40V, 537 a.m.u. UV/VIS (benzene) S = 415.5, B = 546.5, a = 581.0, 01 = 590.5* nm. S/01 = 7.6, 01/B = 3.1, 01/a2 = 1.05 (Figure A28). M.S. (E.I., 4-6 eV) 537 (M+), 535 (M-2) m/z. M .S. (E.I., 70 eV) 537 (M+), 535 (M-2), 522 (M-15), 520 (M2-15), 507 (M-15-15), 268.5 (M++) m/z. (CXIX). Cu "Benzoetioporphyrin": (see CXVI). C32H30N4Cu, 533 a.m.u. UV/VIS (benzene) S = 405.5, B = 536.0+ a= 574. 5 nm. S/a = 7.7, a/B = 3.1. M.S. (E.I., 4-8 eV) 533/535 (M ), 531 (M-2) m/z. M.S. (E.I., 70 eV) 533 (M+), 531 (M-2), 522 (M-15), 520 (M-2-15), 507 (M-1515), 268.5 (M++) m/z. *a2 01: refer to the.higher and lower energy maxima of this 'bifurcated' a-band (see F1gure A28).

PAGE 634

Table AI:Structural consideration of tctroryrrolo spectrometric koOIHI S. ( 1 ) IINC: ( 2 ) nt 7, 8-D IIIYIHtOI'Oiti'IIYit INS I I a: pheophytin-a lib: tlihyclropheophytln-a I IT: pyrophcophyt in-n IV: 111-oxy-phcophylln-11 v: 9-011-pheophrtln-a VIa: ph C 0 ph 0 1" hi cl C-II Vlb: pheophorhidc-a m: tic VII: 9-0il-phcophorhlclc-n m: VI II: r y r 0 I' h c 0 I' h 0 .. h I cl c n m! I X : 9-0il-pyruphcupho rh ltlc-n m X: mcsopyropheophorh i clo-n He X I : 9 -011-mcsupyrophcophnrhiclo-11 m! X II: omll'l'-o m : He X I IT: 7 -l'nl'-11om P-o XV: phcophytln-h XV I: 3-I'-9-on-r h cop h y t t n-h XV II : phcophorbitlc-b He XV Ill: 3 -mll'-9 -011-ph cophorh I de-h XX I: bactcrlophcophytin-11 XX I I: 2-dJII!IlA-9-011-hac l c.-i ophcophyt ln-11 X X Ill a: chlorin-c6 XXIIIh: chlorln-c6 HIE XX IV : r II .. I' II r I II -7 HI I! tic XXV: 011-purpurin-7 H I E XXVI a: purpurin-Ill He XXVIb: p 11 r p u rl n-I 8 XXV II a: chlorin-p6 XXVI!b: chlorin-p6 Hll! XXV Ill: rhoclin-g7 HI I! X X I X: 3 -P -r h o
PAGE 635

Table flt,continuc d ; RNC: ( 2 ) Rl 112 PORPIIYRINS XXXII: ph y I I o e r y t h r I 11 l:t XXX Ill: !l-Oil-I'E Et XXXIV : 7,8-Jioxy-lli'E HE Et XXXVa: 2-oxn-1'1: flct XXXVb: 2-oxu-I'E m : He flc t X XXV I : 2-CIIIl:llV-!J-011-PE m : oil I! XXXVlla:lli'E Hl XXXV lib :ni'E Et XXXVIII :111'1:1' tic Et X X X I X : 7 -Pill'-llPI!I' l!t XI.: lie H e X 1.1: mcsol'lnd i n-1 X m : {II) lie Et m csorh odin-IX ( I 2 ) He Et X 1 11 ; Oll-mcsorhodin-IX c 1 I l l!t X 1.1 I I : mcsovcrclin-IX Hi { II) J!t XI. IV: 011m c sove rl i n-1 X II : ( I I ) tc I!! X I V : I i d c h y 1 1 o-tl cox o -111 c s u v o nlin-I X m : .It! l!t XI.Vl: p y r r or h o tl i n ( I I ) f!t X I.V II : 0 ll-p y r 1 o rho c l i n ( I I ) l!t X I.V II I : porphyrin('porphin') II II XI.IX: ocl a mcthylporphyrin H e 1.: 0 C t a C l h )' 1 jl 0 I'( I h y I i II Et t:t 1.1: etio porphyrin! lie El 1 .11; etioporphyrin-Ill l l c Et 1 .111: pro t opnq>hyri u 1 X m11 : " v 1 1 v; h c m a t o p u q h )' r i 11 f r" c-I n s c IHII H e nil r: I.V: mc soporphrriuI X ti c Et I.VI: d cutcropurphyriu I X II li e II I.V 11: l curcrocl iupuqdl)'l'in .lc If I.V Ill a; phy II 0(10 qdt)' l j II l c l:t t.V 11 I h: phrlloporphyrin It: tic l!t 1.1 X : rhodoporphyri11 11: H e l!t LX: pyr r o p oqhy r in It: I I C l:t R3 lt4 us J!t Et H e l:t l:t H e H l:t lie I t l!t H e tic l!t l!t tic H e t:t lie l:t He l:t H e l e J!t lc I t Et I I C He: l!t He J!t t c c lit l c II II II Ho H e J!t l:t l!t l!t l c He c J!t N c H e v Ill! U c (I Ill! H e li e l:l H e II . .lc II I lie E I tic H e Et l l c H c 1: t lit! Et li e 116 R7 ----"11"--I' am: ---"1!11 ___ I' am: ----"F"---011, l'nm: ----"11"--I' a ----"11"---l'aHJ! ___ U[:H ___ l'nm: -" I : tl --I' a ----"J:"---Pam: ----"F"---Et ----"1="---l'yl, ----"F"---El I' alii: ---"111"------"112"---I' a I' a HI! ---"1"----PaUl! .... tt ) II _ .. .. l'a.ll ---"K"-I' a --"1."-II ---11111"---II ---" I''----II II II II .ft, I! t II f:l l!t II l!t I! I II Et I' a II! II I' a !II I' a Ill II I' a HI I' a tiE II I' a Ill I' a II I' alii I! I II Et II lie I' a II lie I' a I' a H E II I a Ill II II I' a Ill 118 OII,Hc He ti c .,c tc H e H e II .lc E t ., (' H e .lc .I ., .. ., H e l c l c l c l c (j\ 0 -.....!

PAGE 636

cout luuc.l; I)Huclcl can be seen In Fl1uro AI (7,1-dlhy111'= .. ,.,,.,_ thytyl(-c201141) 4)Exocycllc rl111 structures( 6,y 7,y Jtnaccs);Strttctrcs ''1''-''t. also exist 35 tl't lsoerlc rors shokn for A) b-t B) r 6 H-J l H-C=O H 0 6-cH3 E) y 6 F) y 6 H-1 1 -H H-1 1-H H OH H H I) 7 y UH OH J) 7 y IJo r 6 C) HoLo lo G) I<) 0-CH3 r 6 I I cfc, 0 0 7 y I 0) r 6 l -oH Hi =o H I G-CH3 H) H1= 7 y 1-12= y 6 Vo L) 7 y ll) "' 0 00

PAGE 637

..;.T.::a:..:b:..l:..e;;_..:..A:..--=-r.:..!: !solat:.on o i f:-or.1 mat elut:on :rom cellulose. j.Lr' =--c-'on( ) -.. --. = I I ..... :: :.l .. :.l "' I -= -= c n = = "' = --= I I -= :.l e c .. ... e --e :.. >.. 0 ->.. -:: 11 c ":J -= '--= --.. .. .. :::.. :.l c ::. e 0 c 0 ... :: .. :: ... ... c :1 -:J u :1 ( . -= -= -=-= Eluat e _ -- J =-=-= ::. = ::. Cell-1 PE 134 Cell-: 0 .5 ?;A/ P E 0 : 5o 2.37 ; A /?E 131 :s !.8 0 Ce!.l-3 S.O%A /PE 8-:" 35 33 Cdl-6 5 60 1:s 1..L C :dl-7 10-!.S' A/i'!: z: ..L9 11 Cell-!3 ZS7.A/?E 80 Flush A n -t, 1:1 .,_ l)Estioated using the following 609 I = ... "' o-= -= ".J -.. 30 63.7(Smith anci 76.0(Smith and Benite:,1955) E 1s5:.76(Fuhrhop and 197 5 ) as and for w eight ac:.ds E and Srni:h,1975) l ill. 1 Z:oo Davies,1976). :)PE=petroleurn measures on a volurne-to-volume basis.

PAGE 638

Table A-III:Etioporphyrins,positions of methyl and ethyl substituents. Substituent(!) Isomer Rl R2 R3 R4 RS R6 R7 R8 Etioporphyrin-! Me Et Me Et Me Et Me Et Etioporphyrin-!! Et Et Et Et Etioporphyrin-III Et Et Et Et e Etioporphyrin-IV He Et Et Et E t l)Refer to figure le.Isomers taken from Fischer and Orth(1937;i_.Smith,1975).Me=methyl,Et=ethy l "' ........ 0

PAGE 639

611 a) 2R R1 4R c) R d) R R R e) R R R Figure nuclei of the various tetr3pyrrole pigments employed as ring) bacteriophorbide=3,4-dihydro-phorbide(not shown),b)chlorin,c) 'DPEP', d) 'rhodin' ,e) 'ETIO' and f) 'monoben:oetio (sec text of Appendix A).

PAGE 640

Figure A2: Structure of the hydrazone derivative formed by reaction of pheophytinb with Girard' s "T" reagent(Wetherell and Hendrickson,1959). 612

PAGE 641

-.. E we (J)Q o1, o...c< (j) W'-" o::tJ) -+-' a::c W::J 0::L oro Ub Wa::..O L ro 613 -:-4 I I I 0 10 20 TIMEJmin. Figure A3: RP-LPHPLC chrofllatogram of pheophytin-a (IIa).Conditions;C-18 bonded si1ica(13-with methanol: acetone(95:5,v/v) at 9.8ml/min.(ca.l50psig) with detection at 410nm.Peaks 1-y-are tentativel y typed as dehydro-phyty l -ester analogs of pheophytin-a(IIa:peak (see App A text)

PAGE 642

>._ (/) z UJ ._ z UJ > ._ <( _J UJ a: -50--0 -, 'III"I""IIIIIIIIIIIIIIIJII "111"1 llnpmf"lllllllllllll llllllllljl IIIIII"I""PflfJTIT'F 700 AOO 900 m/z Pigure /\4:J>artinl field desorption mass spectrum of an 'ullomerizcd'preparatio n of phcophytin-a(lla: 870m/z).IO-IIydroxy-pheophytin-a(TV)appcars at 8R6 m/z. Q'\ ....... -1>

PAGE 643

w u z
PAGE 644

1.0 I II I I (b)-I 1\ I I ll w 0.8 u z <( m 0:: ss 0.4 m <( 0.0 tl--.-----,.-----r---===1 350 550 750 350 550 750 WavelengthJ nm f-i gure /\6: T h e e lectr onjc a bsorptio n spectra o f chlorophylls c ------f r a c t i o n s fro m S a r g a s s 1 1 m s r e x t r a c t e d a ) f r e s h o r h)fo l l o w j n g ether. (j\ ....... (j\

PAGE 645

w0.6 u z <[ m 0:::: 80.4 m <[ 617 772 662 ] I 400 500 600 700 800 nm J Figure A7: The electronic absorption spectrum of the crude extract of an iutertidal bacterial mat from Tampa bay,Florida. Solvent;acetone.

PAGE 646

618 0 8 w0. 6 J u z <( m a: 758 OJ <( 02J I I 400 500 600 700 WAV ELENGTHJ nm 800 Figure A8: The electronic absorption spectrum of fraction #4 eluted during the chromatography,over cel lulose. of a bacterial mat extract.Solvent;acetone

PAGE 647

619 1.0 n I ( \ I \ w0. 6 I u I z <[ OJ / a: \ aJ <[ 0 2 \ Figure A9:The electronic absorption spectra of the aqueous methanol(90%Me0H)hypophase(dashed) and the n-hexane e p i p n as e -To 11 owing 1 i qui d : 1 i qui d f r a c t ion at Ion o f cellulose fraction #4.Solvents,as given.

PAGE 648

U1 c ::J OA ())L. Mro II L. +-' I..O .. wz _jo Wo._ >(./) o::cr ow Uet: wo ww ocr 620 I I 0 20 40 60 TIMEJmin. Figure AlO : LPHPLC chromatogram of the purification of a bacteriopheophytin-a containing fraction. A,B and C'=carotenoids; 'D'=bacteriopheophytin-a(XXI); 'E'=pheophytin-a(IIa). Conditions;8x570mm column of C-18 bo nded silica(132 4um ) developed with methanol: acetone(9S:S,v/v) at

PAGE 649

621 7 5 7.0 J w0. 6 u z <[ CD a: aJ <[ l 0 2 I 400 500 600 7 0 0 8 0 0 W AV EL.rNGTHJnm Figure All: The electronic absorption spectrum of purified taken in ethy l ether.

PAGE 650

w0. 6 u z <[ m 0::: 80. 4 OJ <[ 0.2 622 715.8 ] J J J Figure A12 : Th e electronic absorption spectrum the desacetyl-9-oxydeoxo-) o f bacteriopheophytina (XXII) .Solvent; ethyl ether.

PAGE 651

w u z <{ 0 if) C1J <{ J 0.2 l I 350 450 550 650 WAVELFNGTH nm J .Figure M3: The electronic absorption spectrum of 7 -propyl-7-despropio-DPEP(XXXIX) taken in benzene. 623

PAGE 652

Ill u z < ( 1.0 . --.--. ---,----1 -( a) 0 8 -0 6 -Ill 0 4 -n : 0 (/) (I) <.( 0.2-1\_./',_'JV._ 0.01 1--,--, ---,---, -350 1 5 0 n5o W A V E L E N GIII,IItrl It I u z < ( 0 8 -O G -I I ( b) () J I ll. C) Ill Ill < ( 0 2 -\ 0.0l--,.,--.,-----,---, ---j-== Wl< 1 WJ\VELE H < ;III,ttrll rig u r c A I 4 : The c I c c t rclld c a h s o r p t i o n s p ectra o f ( n ) rn c so-rho d i n I X -I HI E ( X I. T) a n d ( h ) t h c 1 o x y d e o x o 1 d c r i v a t j v c d i h y d r o meso -rhodin-TX IH1E( XI.Il),ohtujnc d vja r eductio n ether. (J\ N .j::'-

PAGE 653

H C 3 H 3 C C2H5 II N O.:CCh2 \ 0-CH3 CH3 C2H5 HN (XLV) Figure AlS:Proposed structure for the 'benzo(at)porphyrin' (viz."didehytlro-dcoxo-mesoverdin-IX ME(XLV))ol>taine J via the dehydration of'Jihydro-mesorhodin-IX'. (From: 0"1 N lJ1

PAGE 654

t3 0.6 z < m 0:: 0 tJ) l 0 0 350 450 550 650 750 WAVELENGT HJnm Figure A16:The electronic absorption .spectrum of 'didehydro-deoxo-mesoverdin-IX ME' (XLV) taken in benzene solvent. 626

PAGE 655

H M E E --y--J-;; r ;rO=c H H o:c H H ""'H H H H DP 1 H l M M H H M M M M H E E (::"
PAGE 656

0.8 w 06l l u z <{ 0 if) C1J <{ j \ j l \ I 0.0 350 450 550 6 SO Figure Al8: The electronic absorptio n spectrum of deuteroetioporphyrin-IX(LVII)taken in benzene. 628

PAGE 657

w u z
PAGE 658

w u z <( 0.6 0 (J) aJ
PAGE 659

w u z
PAGE 660

0.6 w u z <{ 0]04 J <[ 0.2-J _. o.o-+-1 -.--.I 350 450 550 650 WAVELENGTH) nm Figure A22: The electronic absorption spectrum of Ni 9-oxydeoxo-phylloerythrin ME(LXXIX) taken in benzene. 632

PAGE 661

0.8 0.6-w u z <{ 0 U) (!) <{ 0.0-+-1 ---,------.----r----;-1-----.-----.-..::====i 350 450 550 650 WAVELFNGTHJnm Figure A23:The electronic absorption spectrum of Ni 2-oxo-phylloerythrin ME(LXXXII) taken in benzene. 633

PAGE 662

w J u z
PAGE 663

0.8 0.6 w u z <( 0 U) co <( J 0.2-0.0 -+-1 ----.,---,-1 350 450 550 650 WAVELENGTHJ nm Figure A25: The electronic absorption spectrum of Ni 7 -ethyl7 -desethyl-DPEP(XCI) taken in benzene.

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w u z
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0.6 w u z <( 0 (j) ClJ <( 0.0 -+-___:_--.--,.--==:;:.----,---:;:=:=:;::==::::; 350 450 550 650 WAVELENGTH) nm Figure A27: The electronic absorption spectrum of Ni 'monobenzoetioporphyrin' (CXVII)taken in benzene. 637

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w u z <{ 0 (}) aJ