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Title:
Electrically charged sol-gel coatings for on-line preconcentration and analysis of zwitterionic biomolecules by capillary electrophoresis
Physical Description:
Book
Language:
English
Creator:
Li, Wen
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects / Keywords:
Protein
Amino acid
Positively charged surface coating
Negatively charged surface coating
Sulfonated sol-gel column
C18-TMS sol-gel column
Dissertations, Academic -- Chemistry -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Novel on-line methods are presented for the extraction, preconcentration and analysis of zwitterionic biomolecules using sol-gel-coated columns coupled to a conventional UV/visible detector. The presented approaches do not require any additional modification of the commercially available standard CE instrument. Extraction, stacking, and focusing techniques were used in the preconcentration procedures. The positively charged sol-gel coatings were created using N-octadecyldimethyl3-(trimethoxysilyl) proplyammonium chloride (C18-TMS) in the coating sol solutions. Due to the presence of a positively charged quaternary ammonium moiety in C18-TMS, the resulting sol-gel coating carried a positive charge. The negatively charged sol-gel coatings were due to the presence of sulfonate groups, which was formed from the oxidation of thiol groups in precursor mercaptopropyltrimethoxysilane (MPTMS) by hydrogen peroxide. Besides MPTMS, tetramethoxysilane (TMOS) and n-octadecyltriethoxys ilane (C18-TEOS) were also used to prepare the sol solution for the creation of the negatively charged coatings. For extraction, the pH of the samples was properly adjusted to impart a net charge opposite to the sol-gel coatings. When a long plug of the sample was passed through the sol-gel-coated capillary, extraction was achieved via electrostatic interaction between the charged sol-gel coating and the charged sample molecules. The extracted analytes were then desorbed and focused via local pH change and stacking. The local pH change was accomplished by passing buffer solutions with proper pH values, while a dynamic pH junction between the sample solution and the background electrolyte was utilized to facilitate solute focusing. The developed methods showed excellent extraction and preconcentration effects on both positively and negatively charged sol-gel-coated columns. On-line preconcentration and analysis results obtained on the sol-gel coated columns were compared with those obta ined on an uncoated fused silica capillary of identical dimensions using conventional sample injections. The described procedure provided a 150 000-fold enrichment effect for alanine on the positively charged sol-gel-coated column. On the negatively charged sol-gel-coated column, the presented sample preconcentration technique provided a sensitivity enhancement factor (SEF) on the order of 3 x 103 for myoglobin, and 7 x 103 for asparagines. The developed methods provided acceptable repeatability in terms of both peak height and migration time.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Wen Li.
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Title from PDF of title page.
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Document formatted into pages; contains 235 pages.
General Note:
Includes vita.

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aleph - 001787201
oclc - 124041525
usfldc doi - E14-SFE0001422
usfldc handle - e14.1422
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SFS0025742:00001


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Electrically charged sol-gel coatings for on-line preconcentration and analysis of zwitterionic biomolecules by capillary electrophoresis
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ABSTRACT: Novel on-line methods are presented for the extraction, preconcentration and analysis of zwitterionic biomolecules using sol-gel-coated columns coupled to a conventional UV/visible detector. The presented approaches do not require any additional modification of the commercially available standard CE instrument. Extraction, stacking, and focusing techniques were used in the preconcentration procedures. The positively charged sol-gel coatings were created using N-octadecyldimethyl[3-(trimethoxysilyl) proply]ammonium chloride (C18-TMS) in the coating sol solutions. Due to the presence of a positively charged quaternary ammonium moiety in C18-TMS, the resulting sol-gel coating carried a positive charge. The negatively charged sol-gel coatings were due to the presence of sulfonate groups, which was formed from the oxidation of thiol groups in precursor mercaptopropyltrimethoxysilane (MPTMS) by hydrogen peroxide. Besides MPTMS, tetramethoxysilane (TMOS) and n-octadecyltriethoxys ilane (C18-TEOS) were also used to prepare the sol solution for the creation of the negatively charged coatings. For extraction, the pH of the samples was properly adjusted to impart a net charge opposite to the sol-gel coatings. When a long plug of the sample was passed through the sol-gel-coated capillary, extraction was achieved via electrostatic interaction between the charged sol-gel coating and the charged sample molecules. The extracted analytes were then desorbed and focused via local pH change and stacking. The local pH change was accomplished by passing buffer solutions with proper pH values, while a dynamic pH junction between the sample solution and the background electrolyte was utilized to facilitate solute focusing. The developed methods showed excellent extraction and preconcentration effects on both positively and negatively charged sol-gel-coated columns. On-line preconcentration and analysis results obtained on the sol-gel coated columns were compared with those obta ined on an uncoated fused silica capillary of identical dimensions using conventional sample injections. The described procedure provided a 150 000-fold enrichment effect for alanine on the positively charged sol-gel-coated column. On the negatively charged sol-gel-coated column, the presented sample preconcentration technique provided a sensitivity enhancement factor (SEF) on the order of 3 x 103 for myoglobin, and 7 x 103 for asparagines. The developed methods provided acceptable repeatability in terms of both peak height and migration time.
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Adviser: Abdul Malik, Ph.D.
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Protein.
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Positively charged surface coating.
Negatively charged surface coating.
Sulfonated sol-gel column.
C18-TMS sol-gel column.
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Electrically Charged Sol-Gel Coatings for On-Line Preconcentration and Analysis of Zwitterionic Biomolecule s by Capillary Electrophoresis by Wen Li A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Abdul Malik, Ph.D. Kirpal S. Bisht, Ph.D. Milton D. Johnston, Jr., Ph.D. Dean F. Martin, Ph.D. Date of Approval: Feburary 3, 2006 Keywords: protein, amino acid, positively charged surface coating, negatively charged surface coating, su lfonated sol-gel column, C18-TMS sol-gel column Copyright 2006, Wen Li

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DEDICATION To my son.

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ACKNOWLEDGMENTS I would like to express my sincere appreciation to my major professor Dr. Abdul Malik, for his instruction, patience, and encouragement that he has shown me during my graduate education at USF. I am also very grateful to the members of the dissertation committee for their support an d assistance. I want to recognize Mr. Rafael Fonseca for his technical support to the CE system. Many thanks to all my colleagues in Dr. Malik’s group for their continuous assistance, encouragement, friendship and many stimulating conversations over the years. This research has been partially funded by a subcontract from a grant by the U.S. Naval Office (N00014-98-1-0848). This financial support is greatly appreciated. The Department of Chemistry is also appreciated for their financial support throughout my study at USF. Finally, I would like to express my gratitude to my parents, my sisters, and my husband. Their continuous support, encouragement and love have made this possible.

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i TABLE OF CONTENTS LIST OF FIGURES....................................................................................................vii LIST OF SCHEMES.....................................................................................................x LIST OF SYMBOLS AND ABBREVIATIONS........................................................xi ABSTRACT...............................................................................................................xiv CHAPTER ONE: CAPILLARY ELECTROPHORESIS: THEORY AND PRACTICE........................................................................................1 1.1 Development of capillary electrophoresi s................ ............. .............. ......1 1.2 Fundamentals of capillary electrophore sis theory.....................................4 1.3 Typical capillary electr ophoresis instrument............ ............. .............. ....10 1.4 Modes of operation and their applicat ions...............................................14 1.4.1 Capillary zone electrophoresis..................................................14 1.4.2 Micellar electrokinetic capillary chromatography....................14 1.4.3 Capillary electrochromatography..............................................17 1.4.4 Capillary gel electrophoresis...... ............. .............. ............. .......19 1.4.5 Capillary isoelectric focusing...................................................20 1.4.6 Capillary isotachophoresis........................................................20 1.5 References for Chapter One.....................................................................21 CHAPTER TWO: REVIEW OF PRECONCENTRATION STRATEGIES FOR CAPILLARY ELECTROPHORESIS...............................................................27 2.1 Introduction..............................................................................................27 2.2 Electrophoresis-based sample preconcentration......................................28

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ii 2.2.1 Normal stacking........................................................................30 2.2.2 Electrokinetic injection/field amplified sample injection.........37 2.2.3 Sweeping...................................................................................41 2.2.4 Capillary isotachophoresis (CITP)............................................45 2.3 Chromatography-based sample preco ncentration techniques..................47 2.3.1 Low-specificity chromatographic preconcentration.................47 2.3.1.1 Preconcentration device.............................................48 2.3.1.2 Application.................................................................53 2.3.2 High-specificity chromatographic preconcentration.................56 2.3.2.1 Immunoaffinity chromatography...............................56 2.3.2.2 MIP technology..........................................................59 2.4 Conclusions..............................................................................................61 2.5 References for Chapter Two....................................................................61 CHAPTER THREE: SOL-GEL TECHNOLOGY AND ITS APPLICATION IN CAPILLARY ELECTROPHORESIS...................................................................70 3.1 Introduction..............................................................................................70 3.2 Fundamentals of sol-gel process..............................................................71 3.3 Characterization of sol-gel materials.......................................................77 3.3.1 The morphology of sol-gel materials........................................77 3.3.2 Study of the chemical bonds within sol-gel structure...............78 3.4 General procedures involved in the preparation of CE columns with sol-gel stationary phases..................................................................79 3.4.1 Pretreatment of the capillary.....................................................79 3.4.2 Sol solution ingredients for the fabrication of the sol-gel stationary phases........................................................................80 3.4.3 Post-gelation treatment of sol-gel stationary phases..................80 3.5 The application of sol-gel technology in CE............................................81 3.5.1 Sol-gel technology for packed columns in CE.......................81

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iii 3.5.2 Sol-gel open tubular CE columns...........................................82 3.5.3 Sol-gel monolithic columns........ ............. ............. .............. ....84 3.6 Sol-gel technology for sample precon centration in CE............................85 3.7 References for Chapter Three...................................................................86 CHAPTER FOUR: POSITIVELY CHARGED SOL-GEL COATINGS FOR ONLINE PRECONCENTRATION OF AMINO ACIDS IN CAPILLARY ELECTROPHORESIS................................................................................................92 4.1 Introduction...............................................................................................92 4.1.1 Column technology...................................................................93 4.1.1.1 Dynamic coatings.......................................................93 4.1.1.2 Chemically bonded coatings......................................94 4.1.2 Positively charged coatings for CE columns............................94 4.2 Experimental............................................................................................95 4.2.1 Equipment.................................................................................95 4.2.2 Chemicals and materials...........................................................97 4.2.3 Preparation of sol-gel open tubular CE columns with positive surface charge.............................................................97 4.2.4 Preparation of samples..............................................................99 4.2.5 Procedures for extraction and preconcentration.......................99 4.3 Results and discussion...........................................................................100 4.3.1 Mechanism of extraction on positively charged sol-gel coatings....................................................................................100 4.3.2 Extraction and preconcentration of amino acid by sol-gel columns...................................................................103 4.4 Conclusions............................................................................................128 4.5 References for Chapter Four..................................................................128

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iv CHAPTER FIVE: NEGATIVELY CHARGED SOL-GEL COLUMN FOR ONLINE PRECONCENTRATION OF ZWITTERIONIC BIOMOLECULES IN CAPILLARY ELECTROMIGRATION SEPARATIONS.......................................................................................................133 5.1 Introduction............................................................................................133 5.1.1 The production of a stable EOF..............................................134 5.1.2 Negatively charged coatings for CE columns.........................135 5.2 Experimental..........................................................................................137 5.2.1 Equipment...............................................................................138 5.2.2 Chemicals and materials.........................................................138 5.2.3 Preparation of the sol-gel coating solutions............................139 5.2.4 Preparation of CE columns with a negatively charged sol-gel coating.........................................................................139 5.2.5 Preparation of samples............................................................140 5.3 Results and discussion...........................................................................140 5.3.1 Sol-gel reactions involved in the coating process...................140 5.3.2 Characterization of the sol-gel coating...................................148 5.3.3 The zeitterionic solute s preconcentration mechanism operated in the negatively charged sol-gel columns...............154 5.3.4 On-line preconcentration of zwitterionic molecules...............157 5.3.4.1 On-line preconcentration of amino acids using sulfonated sol-gel columns.............................157 5.3.4.2 On-line preconcentration of proteins using sulfonated sol-gel columns......................................167 5.4 Conclusions............................................................................................178 5.5 Reference for Chapter Five....................................................................178 APPENDICES………………………………………………………………...……183 ABOUT THE AUTHOR……………………………….…………………….End Page

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v LIST OF TABLES Table 2.1 Summary of approaches available for increasing on-column detection sensitivity in CE 29 Table 3.1 Common tetraalkoxysilane precursors and their physical properties 73 Table 4.1 Names and chemical structures of chemical reagents used in the fabrication of positively charged sol-gel columns101 Table 4.2 Chemical structures and some physical properties of analytes used in the current study102 Table 4.3 Sample extraction and preconcentrations on an electrically charged sol-gel column 113 Table 4.4 Sensitivity enhancement factors for four amino acids achieved on positively charged sol-gel C18-TMS coated columns 114 Table 4.5 Sample extraction and preconcentrations on an electrically charged sol-ge l column by method 2 122 Table 4.6 Sensitivity enhancement factors for four amino acids achieved on positively charged sol-gel C18-TMS coated columns by method 2 12 3 Table 4.7 Repeatability data fo r the preconcentration by C18-sol-gel coated column using amino acids as test solutes 127 Table 5.1 Names and chemical structures of the reagents used in the sol solution to fabricate negatively charged sol-gel sulfonated columns14 1 Table 5.2 MPTMS – tototal sol-gel precursor molar ratio in the coating sol solutions used in the fabrication of negatively charged sol-gel sulfonated columns149

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vi Table 5.3 The effect of sol-gel coating on the repeatability of migration time of a neutral EOF marker, DMSO152 Table 5.4 Chemical structures and some physical properties of the test amino acids in this work159 Table 5.5 Repeatability of sa mple preconcentration on a sulfonated sol-gel column using amino acids as test solutes163 Table 5.6 Sensitivity enhancement factors obtained on a sulfonated sol-gel column using amino acids as test solutes164 Table 5.7 Sensitivity enhancement factors (SEFs) obtained on sol-gel coated columns using proteins as test solutes173 Table 5.8 Illustration of the preconcentration repeatability on a sol-gel sulfonated column using conalbumin as a test sample 175

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vii LIST OF FIGURES Figure 1.1 Classification of the CE techniques3 Figure 1.2 Growth in the number of publication on CE since 1980 till end of 2005 5 Figure 1.3 Representative functional groups on the surface of fused silica capillary 7 Figure 1.4 Representation of the electrical doub le layer 9 Figure 1.5 Schematic representation of a basic capillary electrophoresis instrument11 Figure 1.6 Schematic representation of a MEKC system16 Figure 1.7 Schematic representations of three different types of columns used in CEC18 Figure 2.1 Family tree of sampling procedure for preconcentration in CE31 Figure 2.2 Mechanism of normal sample stacking32 Figure 2.3 Mechanism of sample stacking in which reversed EOF is utilized35 Figure 2.4 Illustration of capillary isoelectric focusing 36 Figure 2.5 Behavior of micelles and neutral analytes during FESI-MEKC40 Figure 2.6 Evolution of analyte zones in CSEI-sweep-MEKC42 Figure 2.7 High-salt stacking with a sample matrix of ionic strength greater than the separation buffer44 Figure 2.8 Representation of capillary isotachophoresis of anions46 Figure 2.9 Schematic illustratio n of the extraction capillary used for in-capillary CE 49 Figure 2.10 Device of SPME for CE and its operation51

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viii Figure 2.11 Microphotograph of the analyte concentrator fabric ated with antibody fragme nts immobilized to glass bead s 58 Figure 2.12 Schematic depiction of the prepar ation of molecular imprints60 Figure 3.1 Overview of a sol-gel process 72 Figure 4.1 Schematic representation of the capillary filling / purging device 96 Figure 4.2 Preparation of a sol-gel column for preconcentration 98 Figure 4.3 Extraction of tryptophan on the sol-gel column 104 Figure 4.4 Illustration of the events during preconcentration and focusing of zwitterionic analytes on a positively charged sol-gel column 106 Figure 4.5 Illustration of the effect of a positively charged sol-gel coating on the preconcentration of alanine 108 Figure 4.6 Effect of sol-gel coating on sample (asparagine) preconcentration 109 Figure 4.7 Effect of sol-gel coating on sample (phenylalanine) preconcentration110 Figure 4.8 Effect of sol-gel coating on sample (tryptophan) preconcentration. 111 Figure 4 9 Method 2 for the preconcentration of zwitterionic analytes on the positively charged sol-gel column steps116 Figure 4.10 Illustration of the effect of sol-ge l coating on sample preconcentration (alanine) by method 2118 Figure 4.11 Illustration of the effect of sol-gel coating on sample preconcentration (asparagine) by method 2119 Figure 4.12 Illustration of the effect of sol-gel coating sample preconcentration (phenylalanine) by method 2120 Figure 4.13 Illustration of the effect of solgel coating on sample preconcentration (tryptophan) by method 2121 Figure 4.14 Juxtaposition of the effect of sol-gel coating on sample preconcentration and a blank run with the same method 125

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ix Figure 4.15 Preconcentration and separation of a mixture of two amino acids on a positively charged sol-gel column using method 2126 Figure 5.1 Electroosmotic flow vs. buffer pH values 150 Figure 5.2 Migration time repeatability for a sol-gel sulfonated column153 Figure 5.3 Illustration of on-line preconcentration using a negatively charged sol-gel column156 Figure 5.4 Illustration of zwitterionic sample preconcentration on a negatively charged sol-gel sulfonated column using arginine as a test sample159 Figure 5.5 Illustration of zwitterionic sample preconcentration on a negatively char ged sol-gel sulfonated column using lysine as a test sample161 Figure 5.6 Illustration of zwitterionic sample preconcentration on a negatively charged sol-gel sulfonated column using asparagine as a test sample162 Figure 5.7 Preconcentration of asparagine on negatively charged sol-gel coated column. 166 Figure 5.8 Preconcentration of conalbumin on a negatively charged sol-gel coated column168 Figure 5.9 Preconcentration of myoglobin on a negatively charged sol-gel coated column169 Figure 5.10 Influence of sol solution MPTMS composition on the preconcentration of myoglobin on a negatively charged sol-gel column171 Figure 5.11 Repeatability of myoglobin preconcentration on a negatively charged sol-gel sulfonated column174 Figure 5.12 Effect of matrix removing media on preconcentration effectiveness using a sol-gel coated column 177

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x LIST OF SCHEMES Scheme 3.1 Sol-gel reaction75 Scheme 3.2 Acid-catalyzed and base catalyzed sol-gel reaction mechanisms76 Scheme 5.1 Illustration of the complete hydro lysis of sol-gel precursors142 Scheme 5.2 Condensations of hydrolysis products from TMSO, MPTMS, and C18-TEOS143 Scheme 5.3 Covalent bonding of the sol-gel coating to fused silica surface145 Scheme 5.4 Deactivation of the sol-gel mediated fused-silica coated surface with PheDMS146 Scheme 5.5 Oxidation of mercaptopropyl group into sulfonic acid moiety by H2O2147

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xi LIST OF SYMBOLS AND ABBREVIATIONS 1,7-DX 1,7-dimethylxanthine 1-MX 1-methylxanthine 4-OHC 4-hydroxycoumarin 7-OHC 7-hydroxycoumarin AFM atomic force microscopy (AFM) AIBN `-azobis(isobutyronitrile) Arg arginine Asp aspartic acid BGE background electrolyte C18-TEOS n -octadecyltriethoxysilane C18-TMS N-Octadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride CGE capillary ge l electrophoresis CIEF capillary isoe lectric focusing CITP capillary isotachophoresis CSEI cation-selective exhaustive injection CZE capillary zone electrophoresis dc inner diameter of the column DMSO dimethylsulfoxide P pressure difference across the column DSC differential scanning calorimetry (DSC) dielectric constant of the buffer E applied electric field ECD electrochemical detectioin EDTA ethylenediaminetetraacetic acid EOF electroosmotic flow ES-MS electrospray mass spectrometry FASI field amplified sample injection FASS field-amplified sample stacking FSCE free solution ca pillary electrophoresis FTIR Fourier transform infrared spectroscopy

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xii Glu glutamic acid viscosity of the buffer HCB high-conductivity buffer HEPES N-(hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) His histidine HPAA hydroxyphenylacetic acid HPLC high performance liquid chromatography L length of the column. Linj average length of the sample, LLE Liquid-liquid extraction LOC limit of detection LPME liquid-phase microextraction MEKC micellar electrokinetic chromatography MIP molecularly imprinted polymer MPA 3-mercaptopropionic acid MPTMS Mercaptopropyltrimethoxysilane MS mass spectrometry NMR nuclear magnetic resonance (NMR) OPA o-phthaldialdehyde PDMS poly(dimethylsiloxane) PEO poly(ethylene oxide) PheDMS phenyldimethylsilane p I isoelectric point PS pseudostationary phase PSG photopolymerized sol-gel PVA poly(vinly acetate) PVC poly(vinyl chloride) PVS poly(vinylsulfonate) q q is the charge of the ionized solute r r is the Stokes’ radius of the solute r-CPA replaceable cross-linked polyacrylamide RSD relative standard deviation SDS sodium dodecyl sulfate SEF sensitivity enhancement factor SEM scanning electron microscopy SPE solid-phase extraction SPME solid-phase microextraction tinj injection time TEM transmission electron microscopy TFA trifluoroacetic acid

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xiii tmc the migration time of a micellar aggregate. TMOS tetramethoxysilane Tris-base tris(hydroxymethyl)aminomethane Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride TTAB tetradecyltrimethyl ammonium bromide VEOF velocity of the electroosmotic flow VEF velocity of the electrophoretic flow VOBS observed migration velocity XPS X-ray photoelectron spectroscopy zeta potential EF electrophoretic mobility EOF electroosmotic mobility,

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xiv Electrically Charged Sol-Gel Coatings for On-Line Preconcentration and Analysis of Zwitterionic Biomolecule s by Capillary Electrophoresis Wen Li ABSTRACT Novel on-line methods are presented for the extraction, preconcentration and analysis of zwitterionic biomolecules usin g sol-gel-coated columns coupled to a conventional UV/visible detector. The presented approaches do not require any additional modification of the commercia lly available standard CE instrument. Extraction, stacking, and focusing techniques were used in the preconcentration procedures. The positively charged so l-gel coatings were created using N octadecyldimethyl[3-(trimethoxysilyl) proply]ammonium chloride (C18-TMS) in the coating sol solutions. Du e to the presence of a po sitively charged quaternary ammonium moiety in C18-TMS, the resulting sol-gel coat ing carried a positive charge. The negatively charged sol-gel coatings were due to the presence of sulfonate groups, which was formed from the oxidation of thiol groups in precursor mercaptopropyltrimethoxysilane (MPTMS) by hydrogen peroxide. Besides MPTMS, tetramethoxysilane (TMOS) and n-octadecyltriethoxysilane (C18-TEOS) were also used to prepare the sol solution for the creation of the negatively charged coatings. For extraction, the pH of the samples was pr operly adjusted to impart a net charge

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xv opposite to the sol-gel coatings. When a long plug of the sample was passed through the sol-gel-coated capillary, extraction wa s achieved via electrostatic interaction between the charged sol-gel coating and the charged sample molecules. The extracted analytes were then desorbed and focused via local pH change and stacking. The local pH change was accomplished by passing bu ffer solutions with proper pH values, while a dynamic pH junction between th e sample solution and the background electrolyte was utilized to facilitate solute focusing. The developed methods showed excellent extraction and preconcentration effects on both positively and negatively charged sol-gel-coated columns. On-line preconcentration and analysis results obtained on the sol-gel coated columns were compared with those obtained on an uncoated fused silica capilla ry of identical dimensions using conventional sample injections. The described procedure provided a 150 000-fold enrichment effect for alanine on the positively charged sol-gelcoated column. On the negatively charged sol-gel-coated column, the presented sample preconcentration technique provided a sensitivity enhancement factor (SEF) on the order of 3 x 103 for myoglobin, and 7 x 103 for asparagines. The developed methods provided acceptable repeatability in terms of both peak height and migration time.

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1CHAPTER ONE CAPILLARY ELECTROPHORE SIS: THEORY AND PRACTICE 1.1 Development of capi llary electrophoresis As a rapid, high-efficiency analytical technique, capillary electrophoresis (CE) techniques have been successfully used in chemical 1-3, biochemical 4-6, biomedical 7-9, pharmaceutical 10-12, environmental13-15, and a host of other areas 16-18. Electrophoresis is defined as “the movement of electrically charged particles or molecules in a conductive liquid medium, usually aqueous, under the influence of an electric field” 19. The separation based on electrophoresis can be tr aced back to 1930’, when Tiselius conducted research on the electrophoretic analysis of colloidal mixtures in 1937 20. In 1948, he was awarded the Nobel Prize in Chemistry due his research in electrophoresis and adsorption analysis as well as breakthrough discoveries concerning the complex nature of the serum proteins. In 1940s and early 1950s, anti-convective media such as paper and gels were introduced to minimize thermal effects caused by application of an electric field to an electrolyte solution 21. Capillary electrophoresis emerged in 1960s. Scientists started performing electrophoresis in narrow diameter tubes to reduce and eliminate problems associated with the using of larger tubes, which include diffusion caused by convection, and Joule heat due to poor heat dissipation, etc. Hjerten used capillary of 300 m internal diameter to carry out electrophoretic separations in 1967 22. In 1974, Virtanen 23 performed electrophoresis in small diameter Pyrex tubing with internal diameter 200 to 500 m. His research is regarded as one of the earliest demonstrations of the advantages of capillary electrophoresis 17. Five years later, Mikkers and co-workers used 200 m i.d. Teflon capillaries for capillary zone electrophoresis. Their research results showed that when electrophoresis was performed in capillary the convection problems had been reduced 24. In 1980s, the inner diameter of the capillary used for electrophoresis was further reduced to 75 m in the work by Jorgenson and Lukacs 25. Commercial instruments for capillary electrophoresis separation technique appeared in 1980s and numerous research groups

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2 were attracted to this research field. By the end of 1990s, capillary electrophoresis separation techniques had reached to some what content of maturity level. Capillary electrophoresis on microchips is an emerging new technology, which has made great strides since late 1990s. This miniaturized analytical technology has the potential to assay hundreds of samples in a very short time (minut es or less). And samples can be prepared on-board for a completed integration of sample preparation and analysis. These features make CE on microchips an attractive technology for the next generation of CE instrumentation26. Based on the column chemistry and operation theory, CE technique perform by different operation modes, including capilla ry zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography 27, capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP). Figure 1.1 shows the classification of electrophoresis techniques. Compared to other separation techniques, such as gas chromatography and high performance liquid chromatography, CE has its unique features. The most significant advantage of CE over other separation techniques is smaller volume of samples and reagents required. With a typical commercia l CE instrument, only a few nanoliters of sample are sufficient for the repetitive analysis 18. This small consumption of reagents enables reduced cost. In addition, CE uses buffer solution as the mobile phase, which imposes much less adverse effect on the environment as hazardous organic solvents which are widely used as mobile phase in HPLC. Secondly, solutes analyzed in CE move through a capillary column as a relatively flat profile, rather than the parabolic flow in HPLC. The sample zones in CE do not spread as much as in HPLC and GC. As a result, greatly enhanced sepration efficiencies can be obtained. Other advantages of CE include faster separation times, ease of automation, and availability for analysis of both polar and nonpolar analytes.

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3 Elect r op h o r esis Slab-Gel Electrophoresis Paper Electrophoresis Capillary Electrophoresis Electrophoresis on Microchip CZE MEKC CECCGE CIEF CITP CZE: capillary zone electrophoresis; MEKC: micellar electrokinetic capillary chromatography CEC: capillary electrochromatography; CGE: capillary gel electrophoresis CIEF: capillary isoelectric focusing; CITP: capillary isotachophoresis Figure 1.1 Classification of the CE techniques

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4 As a relatively new family of techniqu es for the separation and analysis of chemical compounds, capillary electrophoresis has attracted more and more attentions of scientists from various areas since 1990. Du ring the past 25 years, the publications associated with capillary electrophoresis have been increasing dramatically. Based on the searching results with database SciFinder Sc holar, there are 26771 references containing “capillary electrophoresis” as entered published during the year from 1981 to the end of 2005. Figure 1.2 shows the distribution of the references based on publication years. 1.2 Fundamentals of capillary electrophoresis theory Separation in CE depends on the difference in solute migration velocity in an electric field 16-19, 21. Electrophoretic mobility and electroosmotic mobility are two important factors affecting the migration velo cities of charged solutes under the influence of an electric field. The observed migration velocity (OBS )of molecules is the sum of the velocities due to electrophoretic flow and electroosmotic flow, and can be shown as EOF EP OBS (1.1) where, EP is the velocity of the electrophoretic flow EOF is the velocity of the electroosmotic flow, both are in cm/s. Application of voltage across a capillary makes charged solutes migrate through the conductive media toward the oppositely charged electrode, which is termed as electrophoretic flow. It depends on the strength of the applied electric field and electrophoretic mobility of the molecules. Therefore, it is given by EEP EP (1.2) where, E is the applied electric field EP is the electrophoretic mobility.

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5 0 500 1000 1500 2000 2500 3000 19811984198719901993199619992002yearnumber of references Figure 1.2 Growth in the number of publication on CE since 1980 till end of 2005 (based on personal search of SciFinder Scholar 2004, up to end of 2005)

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6 The strength of applied electric field is the ratio of the applied voltage and the length of the separation capillary. The electrophoretic mobilities (EF) of charged analytes are determined by the properties of the analytes and the buffer, and can be described by this equation, r 6 / qEF (1.3) where, q is the charge of the ionized solute is the buffer viscosity r is the Stokes’ radius of the solute. The unit of electrophoretic mobility is cm2/V s. From equation (1.3), it follows that under a certain applied electric field and buffer, the greater the charge-to-size ratio (q/r), the higher the electrophoretic mobility. Electroosmotic flow (EOF), the motion of an electrolyte solution, is another important driving force in CE separation. An alogous to the velocity of electrophoretic flow, the velocity of electroosmotic flow is given by EEOFEOF (1.4) Where, E is the applied electric field EOF is the electroosmotic mobility. In CE, EOF is generated when the following three conditions are met 16, 18, 19, 21: (a) an electric field is applied across the separation column; (b) a conductive buffer solution is placed inside the column; and (c) the inner surface of the column acquires charges due to the existing of the buffer solution. In th e case of fused silica capillary used as the column, the capillary wall is irregularly dist ributed with silanol groups (-SiOH). Figure 1.3 schematically shows surface structure of a fused silica capillary The silica surface acquires a charge when in contact with an electrolyte solution, as expressed by the following equation 28. SiO H SiO-+ H+(1.5)

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7 O Si H O O Si O Si O H O O H Si O O Si O Si O O Si O O H Si O H O O H H Figure 1.3 Representative functional groups on the surface of fu sed silica capillary. Reproduced from Reference29.

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8 The dissociation constant of silanol groups on the fused silica capillary surface is estimated to be pKa 7.5 30. However, it is generally recommended that the point of zero net charge of fused silica is around pH = 2 18, 19, 21. Consequently, the silica surface is negatively charged when an electrolyte solution with pH above 2 is used as the running buffer. The negatively charged silanoate groups attract positively charged cations present in the buffer solution and an electrical double layer is fo rmed. This double layer is composed of a fixed layer which is tightly held by the silanoate groups and a diffuse layer, which is further away from the silanoate groups 19, 21, 29. Under the influence of an electric field, the diffuse layer of cations then migrates toward the cathode. Since the cations are believed to be hydrated, a bulk flow of the whole solution within the capillary is generated 19, 21, 29, 31-33, as shown in Figure 1.4. The electroosmotic mobility, EOF, is given by 4 /EOF (1.6) where, is the dielectric constant of the buffer is the zeta potential is the viscosity of the buffer. Electroosmotic mobility is analogous to electrophoretic mobility, and has the same units, cm2/V s. For neutral compounds, the electroosmotic flow is the general transport mechanism in electrophorectic experiment. For charged compounds, the apparent electrophoretic mobility depends on both electrophoretic and electroosmotic flow. An important feature EOF possesses is its relatively flat flow profile, which contributes to its high separation efficiency compared to parabolic flow profile generated in high-performance liquid chromatography (HPLC) via mechanical pumping. Under typical experimental conditions, electroosmotic flow is generally greater than electrophoretic flow 19, 21. A stable EOF is desired to produce consistent analytical results in CE. If the EOF changes with time, the migration times of the solutes will change, which may result in misleading the id entification and quantitation problems 19.

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9 Fused silica capillary wall N N N N N N N Fixed layer Diffuse layer Plane of shear Figure 1.4 Representation of the electrical double layer. Reproduced from Reference34.

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10 1.3 Typical capillary electrophoresis instrument A typical capillary electrophoresis system consists of a high-voltage power supply, a piece of capillary tube, a detector, sample vials, buffer vials, and a data processing device. Figure 1.5 is a schematic representation of a capillary electrophoresis system. A high-voltage power supply is a prerequisite for electrophoresis to occur. The application of the high-voltage power supply provides high separation efficiency, short separation time, and improved resolution between peaks 19, 35-37. In CE, fused silica capillary is the most widely used as the separation column, although the use of Teflon and Pyrex capillaries have also been reported 38. In many commercial instruments, the capillary is re tained in a cartridge-like device, which is conjunct with cooling system s. Capillaries with 25 – 100 m inner diameter and 30 – 100 cm in length are normally used. To make an optical window for on-column detection, the outer polyimide layer is removed either by scraping or burning. The advantages of the capillary format include effective heat di ssipation and small amount of sample and mobile phase required. On the other hand, since the inner diameter of the capillary is also the optical pathlength when UV/vis absorbance or fluoroscence detector is used, the concentration sensitivity is reduced 18, 19, which is an often-cited drawback of the technique. In capillary electrophoresis, only a sm all volume of sample, usually a few nanoliters, is required. It is suggested that the sample plug length should be no more about 1% of the total capillary length to achieve high column efficiency and resolution 21, 39, 40. Either hydrodynamic injection or electrok inetic injection can be used to introduce samples into CE system. Hydrodynamic injection can be performed by either pressure or gravity. Compared to electrokinetic injection, this method is usually more precise and robust but may not be suitable for the use of packed columns. The average length of the sample, injL, introduced by pressure is given by L 32 / Pt d Linj inj2 c (1.7)

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11 RunInject Autosampler tray Thermostatted compartment Power supplybuffer sample Detector CE column Collector buffer Anode Cathode Figure 1.5 Schematic representation of a basic capillary electrophoresis instrument. Reproduced from Reference17.

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12 where, cdis the inner diameter of the column P is the pressure difference across the column injt is the injection time is the viscosity of the background buffer L is the length of the column. Electrokinetic injection is accomplished by application of a voltage while the inlet of the separation column is placed in the sa mple vial. The sample zone generated by electrokinetic injection is plug-like, but this me thod is normally associated with sampling bias 41. The length of the injected zone, injL by electrokinetic method is given by L / t v ) ( Linj inj EP EOF inj (1.8) Where, injVis the voltage applied for the sample injection EOFis electroosmotic mobility EP is electrophoretic mobility injt is the injection time L is the length of the column. Temperature is an important parameter which should be maintained constant throughout the CE operation to ensure precise analysis. For example, when temperature is increased, the migration time decreases due to the reduced viscosity of the running buffer. Since the viscosity of the solution changes with the temperature, the volume of the injected sample will be different at various temperatures. Thermostatted compartment allows active temperature control for the column, which is important in obtaining reproducible migration times 42-44. Although, absolute temperature has little effect on the separation efficiency, temperature does affect th e resolution in some sp ecific cases. It has been reported recently by Nevado and coworkers 45, during the separation and

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13 determination of omeprazole enantiomers in pharmaceutical preparations, resolution decreased with increase of the temperature due to limited solute-cyclodextrin interaction. A proper choice of background electrolyte provides stable conditions for CE separation. Aqueous buffer solutions are the most commonly used as background electrolytes. An ideal buffer used in CE should possess the following properties: (1) reasonable water solubility, (2) good buffering capacity in the pH range of choice, (3) low absorbance at the wavelength of detection 21. Properties of the bu ffer solution which affect CE separation include its composition pH, and concentration. Under constant capillary temperature, the EO F will decrease with the increase in buffer concentration 46. The pH of the buffer will influence the ioniza tion of the solutes as well as the silanol groups on the surface of fused silica capillary 38, the latter being directly related with the magnitude of EOF. Once a sample has been resolved into va rious compartments, it is necessary to detect the separated zones passing through the column. Most commercial CE instruments are equipped with a UV/visible absorption detector providing wavelength selection from 190 to 700 nm 17, 19, 21. UV/visible detectors are easy to use and applicable to most analytes. For UV/Visible detectors, they are usually used on-column to avoid band broadening 21. However, in this case, effective optical path length of the light equals the inner diameter of the capillary. And as a resu lt, the absorption signal is relatively low. Some molecules lack chromophores and cannot be detected by UV/Visible detectors. In order to visualize these molecules, there are some alternative methods, such as fluorescence 47, 48, electrochemical detectors 48-50 and mass spectrometry 51-53. Laserinduced fluorescence 54 is an important detection option for the analysis of trace level analytes in CE. Since it uses fluorescence of the solutes, derivatization is often needed to chemically label the solute molecules with a flurophore. Electrochemical detection is based on the measurements of the current generated from an oxidation or reduction reaction of the analyte at an electrode surface. This method has high selectivity and sensitivity for some compounds but is us ed to only a limited extent in CE 21. The use of mass spectrometric detection for CE can provide more important information about the compounds. For example, it enables the identification of chemical structures. However, it is relatively difficult to couple CE with an MS detector due to the use of buffer.

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141.4 Modes of operation and their applications 1.4.1 Capillary zone electrophoresis Capillary zone electrophoresis (CZE) is the most widely used and the simplest operation mode among various CE separation modes. When molecules are charged, they can be separated by CZE. In CZE, the separation column is filled with a homogeneous running buffer, and separation occurs in a free solution. Therefore, CZE is also referred to as free solution capillary electrophoresis (FSCE) 19. CZE separation is based on differences in migration velocities of the an alytes. Since the electrophoretic mobilities of cations and anions have opposite directions, they can be separated in the same run. And cations and anions are separated based on their charge-to-size ratios. On the other hand, since neutral analytes do not have electrophor etic mobilities, they are carried through the capillary by EOF at th e same velocity. As a result, they cannot be separated in CZE. Under the influence of a normal electric field (e.g the polarity of the electric field is from positive to negative), the order of elution in CZE is: cations, neutrals, and finally anions. In principle, analytes with different char ge-to-size ratios can be separated by CZE. Many inorganic ions as well as organic ions have been separated. In order to obtain the best separation, optimization of operation conditions has been investigated. Zare and coworkers 55used tetradecyltrimethylammonium bromide (TTAB) as flow modifier and quantitatively anal yzed some low molecular weight carboxylic acids in the form of anions (fomate, acetate, propanoate, butanoate, pentanoate and hexanoate). Macka et al. 56 separated several metallochro mic ligands by CZE and MEKC. 1.4.2 Micellar electrokinetic ca pillary chromatography Unlike in CZE where separation takes place in free solution, micellar electrokinetic capillary chromatography (MEKC) utilizes a micellar pseudostationary phase created from a surfactant or mixed surfactants dissolved in the running buffer above its critical micelle concentration 21. Surfactants contain a hydrophobic and a hydrophilic part in their structure. When the concentration of a surfactant in an aqueous solution exceeds its critical micelle concentration, aggregated structures known as micelles are formed. Due to the presence of the micelles, the analytes partition between

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15 the running buffer and the pseudo-stationary phase, and become sepa rated. Surfactants are normally charged and move inside the cap illary due to their electrophoretic motility under the influence of an electric field. If an analyte is insoluble in the formed micelles, it spends all its time with the running buffer an d migrates inside the capillary at the same speed as EOF. If an analyte is totally soluble in the micelles, it migrates at the same speed as the micelles. For those analytes which partially soluble in micelles, the migration speed will be different from both EOF and the speed of micelles. Thus, it is important that the chosen surfactants are charged in some cases, otherwise, MEKC will not take place. Both chromatographic and electrophoretic principles are represented in MEKC. The advantage of MEKC over CZE is that it provides a mechanism for the separation of neutral as well as ionized solutes 57-60. Figure 1.6 schematically shows the separation principle of a micellar electrokinetic chromatography system. With enhanced selectivity due to the addition of a surfactant into the background electrolyte, recently MEKC has been successfully employed by Ewing and co-workers 61 for the separation of biogenic amines and metabolites in the fruit fly, Drosophila melanogaster Separations of 14 analytes of biological relevance extracted from the fruit fly were performed in 25 mM borate buffer containing 50 mM sodium dodecyl sulfate (SDS) and 2% 1-propanol. Electrochemcial detection (ECD) was utilized. Their results show that the developed borateMEKC-ECD system provides a valu able tool to distinguish and monitor fluctuations in metabolites in the studied subjects and possible application to other animals. Trapp et al. 62 reported efficient MEKC methods with laser induced fluorescence detection for the separation and quantitation of asymmetric dimethyl-L-arginine and Larginine in human plasma samples. The influence of buffer and surfactant concentrations had been investigated and the optimum performance was obtained when 193 mM borate solution with 100 mM deoxycholic acid as the surfactant was utilized. The strong influence of buffer and surfactant solution concentration on the peak resolution demonstrates that both electrophoretic and electrokinetic separation mechanism play important roles into separation process.

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16 Figure 1.6 Schematic representation of a MEKC system. Here, t0 is the migration time of a neutral ‘unretained’ solute, tR is ‘retention’ time in MEKC, tmc is the migration time of a micellar aggregate. Reproduced from Reference 63.

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17 1.4.3 Capillary electrochromatography Capillary electrochromatography 27 effectively combines two major separation techniques: capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). Consequently, it possesses their inherent advantages: high separation efficiency, tunable selectivity and remarkable versatility in separation and may potentially become a viable alternative to HPLC, micro HPLC and CZE 64. CEC uses similar instrumention as CZE, and similar columns as microcolumn liquid chromatography. But unlike CZE, it provides a separation mechanism of both neutral and charged analytes. In a CEC system, the mobile phase flow through the column is maintained by an electric field instead of a high-pressure pumping system used in HPLC. Since the electro-driven flow profile is essentially flat, increased separation efficiency is achieved in CEC. Compared with MEKC, the surfactant-free mobile phases used in CEC make it ideally suited for hyphenation with mass spectrometry 65. In CEC, like in all other chromatograp hic techniques, stationary phase is a critically important element directly affectin g the separation of analytes. Based on the format of the stationary phases, the columns used in CEC are classified into three categories: packed columns 66, 67, monolithic columns68 and open-tubular columns 69, 70. In CEC, the stationary phase has two-fold roles: (1) it provides chromatographic interactions with various types of solute molecules, and (2) the CEC stationary also facilitates the generation of electroosmotic flow through the column. Figure 1.7 is a schematic representation of different types of columns in CEC. The CEC separation of neutral compounds is an analogue to reversed-phase liquid chromatography, while CEC separation of charged analytes is based on a mechanism combining both electrophoresis and reversed-phase liquid chromatography. Duri ng the last decades, CEC, with its unique separation methodology, has achieved great accomplishment in the separation of a wide variety of analytes, such as proteins and peptides 71-75, nucleoside and nucleic acid recognition 76-80, carbohydrates 67, 77, 81-84, and inorganic analytes 1, 85-87. CEC continues to play an important role among analytical techniques, because a wide range of stationary phases and thus operation modes are available. Lin et al. 78, for

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18 packed bed open segment A frits detection window B detection window open-tubular coating C open segment detection window monolith polyimide coating capillary wall open-tubular monolith polyimide coating capillary wall polyimide coating capillary wall Figure 1.7 Schematic representation of three different types of columns used in CEC: (A) a typical packed capillary column (adapted from Reference88); (B) an open-tubular capillary column; and (C) a monolithic capillary column.

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19 example, utilized an open-tubular wall-coated macrocyclic polyamine capillary column separated and determined uridine 5’-monophosphate, guanosine 5’-monophosphate, adenosine 5’-monophosphate, and cytidine 5’-monophosphate in the tested sample. Recently, Ohyama et al.89 has developed a CEC method using 3-(1, 8-naphthalimido) propyl-modified silyl silica gel (NAIP) as the stationary phase to separate caffeine and its two metabolites 1-methylxanthine (1-MX) and 1, 7-dimethylxanthine (1, 7-DX). This method also has been successfully coupled with microdialysis and employed to monitor the caffeine concentration in rat brain. 1.4.4 Capillary gel electrophoresis Capillary gel electrophoresis (CGE) is ad aptation of traditional gel electrophoresis using capillary columns filled with a polymer so lution acting as a molecular sieve. It is usually used for the separation of biopolymers such as proteins and nucleic acids. When the solutes migrate through the sieving medium, larger molecules become hindered more than smaller ones leading thei r separation. CGE is most suitable for the separations of molecules with similar mass/charge ratio but different sizes and shapes. Chemical gels and physical gels are two typical sieving mediums used in CGE. With small mesh spacings (controlled by adjusting the ratio of acrylamide and the crosslinking reagent), poly(acrylamide) is almost exclusively used as a chemical gel. Recently, an alternative sieving matrix, a replaceable cross-linked polyacrylamide (rCPA) was developed for the separation of a wide range of proteins (~ 4 kD to ~300 kD) by sodium dodecyl sulfate (SDS)-CGE 90. When compared with most frequently used sieving matrixes, this developed rCPA was found to be able to permit highest resolutions and faster separation speed for protein separations. A methodology for the separation of some oligonucleotide mixtures by CGE has been developed by Bayer’s research group91. Their method utilized fused silica capillary (100 m i.d.x 50 cm), which was permanently coated with poly(vinyl acetate) (PVA). The gel they used was prepared by dissolving PEG 35000 20% (w/v), 20 mM bis(hydroxyethyl)amino-tris(hydroxymethyl) (B is-Tris), 20 mM boric acid and 20% (v/v) acetonitrile in high-purity water and shaken overnight. Since the PVA coating suppressed the EOF, analytes migrated inside the capill ary exclusively by their electrophoresis. This

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20 CGE system was coupled with electrospray mass spectrometry (ES-MS) as detection unit. Oligonucleotides up to 20 bases in length were successfully separated by the develop CGE/ES-MS system. 1.4.5 Capillary isoelectric focusing Capillary isoelectric focusing (CIEF) separates amphoteric analytes based on their isoelectric points (p I ) in a pH gradient covering the range of p I ’s of the sample 19, 21. In CIEF, the electroosmotic flow is eliminated. The outlet buffer vial contains a catholyte with a high-pH solution, while the inlet buffer vial contains an anolyte with a low-pH solution. Before separation occurs, the capill ary is filled with ampholytes with different p I values to create a pH gradient along th e capillary when a voltage is applied. The analytes migrate through the capillary and rest at their own isolelectric points, at which it moves in neither direction due to a net charge of zero, and accumulate into distinct bands. After this, the focused sample zones are forced through the detector by adding a salt to either the anolyte or catholyte 92, 93. The usefulness of CIEF for protein analys is in biological fluids such as human serum, cerebral spinal fluid, whole cells and cell is well demonstrated 94-98. Crowley and Hayes 99 utilized off-line coupling of CI EF with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) for the analysis of human blood serum. Both p I and mass information were obtained from the collected biological sample. The newly developed platform demonstrates advanced features such faster (hours versus days), more automatable, and simpler compared with traditional techniques. 1.4.6 Capillary isotachophoresis Capillary isotachophoresis (CITP) 100, is an important operation mode in CE. This method is also employed for sample stacking. CITP is performed in a capillary by injecting the sample between two discrete buffer plugs: a leading buffer with a higher mobility ion and a terminating buffer with a lo wer mobility compared to the analytes of interest 19, 21. Anions and cations cannot be separated in the same run in CITP mode. Under the influence of an electric field with a constant current, the anions or cations in the buffers and the sample migrate toward the anode or cathode and arrange themselves in order of mobility. Since electroosmotic flow is usually suppressed, all the buffer and

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21 analyte ions migrate at the same velocity with the leading ions. CITP has long been used as a separation as well as a very effective preconcentration method for inorganic ions 101104. Inorganic ions build a group of compounds with differ ent nutritional value found food and feed samples. Their determination in th ese samples is a specific area of CITP application. Phytic acid in cereal grains, legumes, and feed was determined by CITP 102. The electrolyte system consists of 0.01 M hydrochloric acid and 0.0056 M bis-trispropane (pH 6.1) as the leading electrolyte, and 0.005 M 2-morpholinoethanesulfonic acid as the terminating electrolyte. The described method shows quite good reproducibility, expressed by the relative standard deviation of 3.8%. In addition, CITP has found application in the analysis of pharmaceuticals and drug product formulation 105-108. CITP with conductometric detection was used for the assay determination of tolfenamic, flufenamic, mefenamic, and niflumic acids in drug product formulations 107. The method used 10 mM HCl/20 mM imidazole aqueous solution (pH = 7.1) as the leading electrolyte and 10mM 5,5-diethylbarbituric acid aqueous solution with pH 7.5 as the terminating electrolyte. Analysis can be accomplished in 20 minutes. 1.5 References for Chapter One (1) Hutchinson, J. P.; Zakaria, P.; Bowie, A. R.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2005 77 407-416. (2) Yeung, K.-C.; Lucy, C. A. J. Chromatogr. A 1998 804 319-325. (3) F. Foret. S. Fanali, A. N., P. Bocek Electrophoresis 1990 11 780-783. (4) Lin, C.-H.; Kaneta, T. Electrophoresis 2004 25 4058-4073. (5) Kist, T. B. L.; Mandaji, M. Electrophoresis 2004 25 3492-3497. (6) Britz-McKibbin, P.; Terabe, S. Chem. Rec. 2002 2 397-404. (7) Veuthey, J.-L. Anal. Bioanal. Chem. 2005 381 93-95. (8) Bosserhoff, A.-K.; Hellerbrand, C.; Buettner, R. Comb. Chem. High Throughput Screening 2000 3 455-466.

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22 (9) Wyss, R. J. Chromatogr. B 1995 671 381-425. (10) Bowman, J.; Tang, L.; Silverman, C. E. J. Pharm. Biomed. Anal. 2000 23 663669. (11) Chadwick, R. R.; Hsieh, J. C.; Resham, K. S.; Nelson, R. B. J. Chromatogr. A 1994 671 403-410. (12) Prado, M. S. A.; Steppe, M.; Tarare s, J. F. M.; Kedor-Hackmann, E. R. M.; Santoro, M. I. R. M. J. Pharm. Biomed. Anal. 2005 37 273-279. (13) McDonald, S.; Bishop, A. G.; Prenzler, P. D.; Robards, K. Anal. Chim. Acta 2004 527 105-124. (14) Mahnik, S. N.; Rizovski, B. ; Fuerhacker, M.; Maker, R. M. Anal. Bioanal. Chem. 2004 380 31-35. (15) Brueggemann, O.; Freitag, R. J. Chromatogr. A 1995 717 309-324. (16) Landers, J. P. Handbook of Capillary Electrophoresis ; CRC Press: Boca Raton, FL, 1994. (17) Camilleri, P. Capillary Electrophoresis: Theory and Practice ; CRC Press: Boca Roton, FL, 1993. (18) Altria, K. D. Capillary Electrophoresis Guidbook Principles, Operation, and Applications ; Humana Press: Totowa, NJ, 1996. (19) Baker, D. R. Capillary Electrophoresis ; John Wiley & Sons, Inc.: New York, N.Y., 1995. (20) Tiselius, A. Trans Faraday Soc 1937 33 524-531. (21) Poole, C. F. The Essence of Chromatography ; Elsevier Science: Amsterdam, The Netherlands, 2003. (22) Hjerten, S. Chromatogr. Rev. 1967 9 122-219. (23) Virtanen, R. Zone electrophoresis in a narrow-bore tube employing potentiometric detection ----a theo retical and experimental study ; Helsinki University of Technology: Helsinki, 1974. (24) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979 169 11-20. (25) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 53 53 1298-1302. (26) Dolnik, V.; Liu, S.; Jovanovich, S. Electrophoresis 2000 21 41-54.

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23 (27) Cecchet, F.; Fioravanti, G.; Marcaccio, M.; Margotti, M.; Mattiello, L.; Paolucci, F.; Rapino, S.; Rudolf, P. J. Phys. Chem. B 2005 109 18427-18432. (28) Iler, R. K. The Chemistry of Silica ; John Wiley and Sons, Inc.: New York, 1979. (29) Miller, J. M. Chromatography: concepts and contrasts 2nd. ed.; John Wiley & Sons, Inc.: Hoboken, N.J., 2005. (30) Behrens, S. H.; Grier, D. G. J. Chem. Phys. 2001 115 6716-6721. (31) Corradini, D. J. Chromatogr. B 1997 699 221-256. (32) Mammen, M.; Carbeck, J. D.; Si manek, E. E.; Whitesides, G. M. J. Am. Chem. Soc. 1997 119 3469-3476. (33) Melanson, J. E.; Baryla, N. E.; Lucy, C. A. Trends Anal. Chem. 2001 20 365374. (34) Weinberger, R. Practical Capillary Electrophoresis 2nd ed.; Academic Press: London, UK, 2000. (35) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990 516 33-48. (36) Guttman, A.; Wanders, B.; Cooke, N. Anal. Chem. 1992 64 2348-2351. (37) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1989 61 491-493. (38) Lukacs, K. S.; Jorgenson, J. W. J. High Resol. Chromatogr. 1985 8 407-411. (39) Burgi, D. S.; Chien, R.-L. Anal. Chem. 1991 63 2042-2047. (40) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1989 61 251-260. (41) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988 60 375-377. (42) Kok, W. Chromatographia 2000 51 (suppl.) S1-S89. (43) Faller, T.; Engelhardt, H. J. Chromatogr.A 1999 853 83-94. (44) Mayer, B. X. J. Chromatogr. A 2001 907 21-37. (45) Nevado, J. J. B.; Pealvo, G. C.; Dorado, R. M. R. Anal. Chim. Acta 2005 533 127-133. (46) Bruin, G. J. M.; Chang, J. P.; Kuhlman, R. H.; Zegers, K.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1989 471 429-436. (47) Vogt, C.; Klunder, G. L. Fresenius J. Anal. Chem. 2001 370 316-331.

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24 (48) Timerbaev, A. R.; Buchberger, W. J. Chromatogr. A 1999 834 117-132. (49) Kappes, T.; Hauser, P. C. J. Chromatogr. A 1999 834 89-101. (50) Baldwin, R. P. Electrophoresis 2000 21 4017-4028. (51) Garcia, F.; Henion, J. D. Anal. Chem. 1992 64 985-990. (52) Steiner, F.; Hassel, M. J. Chromatogr. A 2005 1068 131-142. (53) Sawada, H.; Nogami, C. Anal. Chim. Acta 2004 507 191-198. (54) Robson, M. M.; Roulin, S. C. P.; Shar iff, S. M.; Raynor, M. W.; Bartle, K. D.; Clifford, A. A.; Myers, P.; Euerby, M. R.; Johnson, C. M. Chromatographia 1996 43 313-321. (55) Huang, X.; Luckey, J. A.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1989 61 766770. (56) Macka, M.; Haddad, P. R.; Buchberger, W. J. Chromatogr. A 1995 706 493-501. (57) Nishi, H.; Terabe, S. J. Chromatogr. A 1996 735 3-27. (58) Quirino, J. P.; Terabe, S. Science 1998 282 465-468. (59) Wang, Z.; Tang, Z.; Gu, Z.; Hu, Z.; Ma, S.; Kang, J. Electrophoresis 2005 26 1001-1006. (60) Lurie, I. S.; Hays, P. A.; Garcia, A. E.; Panicker, S. J. Chromatogr. A 2004 1034 227-235. (61) Paxon, T. L.; Powell, P. R.; Lee, H.-G.; Han, K.-A.; Ewing, A. G. Anal. Chem. 2005 77 5349-5355. (62) Trapp, G.; Sydow, K.; Dulay, M. T.; Chou, T.; Cooke, J. P.; Zare, R. N. J. Sep. Sci. 2004 27 1483-1490. (63) Watanabe, T.; Terabe, S. J. Chromatogr. A 2000 880 311-322. (64) Mistry, K.; Krull, I.; Grinberg, N. J. Sep. Sci. 2002 25 935-958. (65) Huber, C. G.; Holzl, G. J. Chromatogr. Libr. 2001 62 271-316. (66) Scherer, B.; Steiner, F. J. Chromatogr. A 2001 924 197-209. (67) Zhang, M.; Melouk, H. A.; Chenault, K.; Rassi, Z. E. J. Agric. Food Chem. 2001 49 5265-5269. (68) Hayes, J. D.; Malik, A. Anal. Chem. 2000 72 4090-4099.

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25 (69) Guo, Y.; Colon, L. A. Anal. Chem. 1995 67 2511-2516. (70) Hayes, J. D.; Malik, A. Anal. Chem. 2001 73 987-996. (71) Okanda, F. M.; El Rassi, Z. Electrophoresis 2005 26 1988-1995. (72) Stutz, H. Electrophoresis 2005 26 1254-1250. (73) Shi, Y.; Xiang, R.; Horvath, C.; Wilkins, J. A. J. Chromatogr.A 2004 1053 2736. (74) Li, Y.; Xiang, R.; Wilkins, J. A.; Horvath, C. Electrophoresis 2004 25 22422256. (75) Bandilla, D.; Skinner, C. D. J. Chromatogr. A 2004 1044 113-129. (76) Ohyama, K.; Fujimoto, E.; Wada, M.; Kishikawa, N.; Ohba, Y.; Akiyama, S.; Nakashima, K.; Kuroda, N. J. Sep. Sci. 2005 28 767-773. (77) Allen, D.; Rassi, Z. E. J. chromatogr.A 2004 1029 239-247. (78) Lin, S.-Y.; Chen, W.-H.; Liu, C.-Y. Electrophoresis 2002 23 1230-1238. (79) Lin, S.-Y.; Liu, C.-Y. Electrophoresis 2003 24 2973-2982. (80) Ping, G.; Zhang, W.; Zhang, L.; Schm itt-Kopplin, P.; Zha ng, Y.; Kettrup, A. Chromatographia 2003 57 629-633. (81) Fu, H.; Huang, X.; Jin, W.; Zou, H. Current Opinion Biotech. 2003 14 96-100. (82) Gucek, M.; Pilar, B. Chromatographia 2000 51 S139-S142. (83) Honda, S.; Suzuki, S.; Taga, A. J. Pharm. Biomed. Anal. 2003 30 1689-1714. (84) Que, A. H.; Novotny, M. V. Anal. Chem. 2002 74 5184-5191. (85) Yokoyama, T.; Zenki, M.; Macka, M.; Haddad, P. R. Bunseki Kagaku 2005 54 107-120. (86) Pacakova, V.; Coufal, P.; Stulik, K.; Gas, B. Electrophoresis 2003 24 1883-1891. (87) Fritz, J. S.; Breadmore, M. C.; Hilder, E. F.; Haddad, P. R. J. Chromatogr.A 2002 942 11-32. (88) Colon, L. A.; Maloney, T. D.; Anspach, J.; Colon, H. Adv. Chromatogr. 2003 42 43-106. (89) Ohyama, K.; Wada, M.; Lord, G. A.; Ohba, Y.; Nakashima, M. N.; Nakashima, K.; Akiyama, S.; Lim, C. K.; Kuroda, N. Electrophoresis 2005 26 812-817.

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26 (90) Lu, J. J.; Liu, S.; Pu, Q. J. Proteome Res. 2005 4 1012-1016. (91) Freudemann, T.; Brocke, A. v.; Bayer, E. Anal. Chem. 2001 73 2587-2593. (92) Hjerten, S.; Liao, J.-L.; Yao, K. J. Chromatogr. 1987 387 127-138. (93) Zhu, M.-D.; Hansen, D. L.; Burd, S.; Gannon, F. J. Chromatogr. 1989 480 311319. (94) Righetti, P. G.; Gelfi, C.; Conti, M. J. Chromatogr.B 1997 699 91-104. (95) Rodriguez-Diaz, R.; Wehr, T.; Zhu, M. D. Electrophoresis 1997 18 2134-2144. (96) Righetti, P. G.; Bossi, A. Anal. Chim. Acta 1998 372 1-19. (97) Jin, Y.; Luo, G.; Oka, T.; Manabe, T. Electrophoresis 2002 23 3385-3391. (98) Kilar, F. Electrophoresis 2003 24 3908-3916. (99) Crowley, T. A.; Hayes, M. A. Proteomics 2005 5 3798-3804. (100) Martin, A.; Everaerts, F. Proc. R. Soc. London. A 1970 316 493-514. (101) Valaskova, I.; Havranek, E. J. Chromatogr.A 1999 836 201-208. (102) Blatny, P.; Kvasnicka, F. J. Chromatogr.A 1999 834 419-431. (103) Matejovic, I.; Polonsky, J. J. Chromatogr. 1989 472 441-444. (104) Church, M. N.; Grant, P. M.; Andresen, B. D. Anal. Chem. 1998 70 2475-2480. (105) Urbanek, M.; Pospisilova, M.; Polasek, M. J. Pharm. Biomed. Anal. 2002 28 509-515. (106) Cakrt, M.; Hercegova, A.; Lesko, J.; Polonsky, J.; Sadecka, J.; Skacani, I. J. Chromatogr.A 2001 916 207-214. (107) Polasek, M.; Pospisilova, M.; Urbanek, M. J. Pharm. Biomed. Anal. 2000 23 135-142. (108) Pospisilova, M.; Po lasek, M.; Jokl, V. J. Pharm. Biomed. Anal. 2001 24 421-428.

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27CHAPTER TWO REVIEW OF PRECONCENTRATI ON STRATEGIES FOR CAPILLARY ELECTROPHORESIS 2.1 Introduction Capillary electrophoresis (CE) is a highly efficient separation technique and possesses a number of advantageous features including high overall separation efficiency, short analysis times, and small amounts of re agents or samples required. The application range of CE is possibly the most diverse of all analytical techniques 1-3. With its several operation modes (free zone, micellar, gel, isotacophoresis, and isoelectric focusing), CE can be used to analyze a wide range of compounds from large, complex macromolecules, such as proteins, peptides, nucleotides and nucleic acids, to small solutes, such as organic drugs and inorganic anions and cations. Commercial instruments have been available since 1988. Separations are normally performed employing voltages in the region of 5-30 kV over a piece of fused silica capillary, typically 25-100 m i.d. and 25-100 cm in length. Commercial instruments are commonly equipped with a UV absorbance detector or a diode array detector. Some instruments also offer the capability of fluorescence, laser-induced fluorescence, or electrochemical detection. Most CE separations are performed using an on-column detection mode to prevent loss of separation efficiency due to extracolumn band broadening that usually takes place if an off-column detection cell is used. Consequently, the choice of capillary internal diameter largely governs the sensitivity of the method when UV detector is used as the detection method. However, the necessity of effective dissipation of Joule heating in the CE column, and to preserve high separation efficiency, one is forced to use capillaries with small inner diameter (25 ~ 75 m). Due to the short path length (equal to the inner diameter of the column), on-column UV detection on such small-diameter columns is characterized by low concentration sensitivity, which is the main limitation of C in trace-level analysis and detection of compounds such as toxins, drug residues, or metabolites in biol ogical samples. In order to get the maximum sensitivity, the inner diameter of the capillary may be extended to 100

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28 m if necessary, but at the expense of longer analysis and reduced separation efficiency caused by the necessity to use reduced voltage and/or electrolyte concentration because of the internal heating problems arising from the increased capillary diameter. In order to maximize the detection sensitivity in CE, a range of strategies have been used. Table 2.1 shows some of these strategies including the use of low UV wavelengths, increased capillary diameter, and optimized sampling proced ure. Over the past decades, a series of studies have been undertaken to improve the on-column detection sensitivity in CE. The developed methods (e.g., increasing the bore of the capillary, changing the operation voltage or modifying the capillary) have their own strengths and drawbacks as shown in Table 2.1. Optimizing the sampling procedure to preconcentrate the target analytes is especially attractive since it involves no additional modification of the commercially available standard CE instrument, and it can be easily accomplished by carefully controlling the operation conditions. Commonly used sampling techniques for preconcentration in CE can be divided into two different categories 4: (a) electrophoresis-based sample preparation; and (b) chromatography-based sample preparation. These categories can be further divided into several sub-categories. Figure 2.1 shows the family tree of sampling procedures for preconcentration, and was constructed based on several reviews published during last decade 4-10. 2.2 Electrophoresis-based sample preconcentration The preconcentration method related to electrophoretic processes is based on the differences in mobility and conductivity of sample and running buffer systems. Normal stacking, field amplified sample injection, sweeping and isotachophoresis and dynamic pH junctions are electrophoresis-based sample preconcentration techniques that are commonly used in CE.

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29 Table 2.1 Summary of approaches available for increasing on-column detection sensitivity in CE. Reproduced from Reference1 Action to improved sensitivity Drawbacks to consider Employ low-UV wavelength Increas ed background noise----determine wavelength to give optimum signal-tonoise ratio Increase capillary bore Increased current, reduced EOF giving possible alternation in selectivity Increase injection time Reduction in separation efficiency; excessive time will result in run failure Appropriately use electrokinetic injection Sampling bias for more mobile ions, sample matrix effects on injection amount Increase electrolyte strength Increased current, increased Joule heating, and associated noise Optimize electrolyte composition Effects on selectivity and current Decrease operating voltage Increase in analysis time Decrease temperature Increase in analysis time Modify the capillary Reduction in separation efficiency and resolution, increased cost Derivatize the sample Additional sampling handling Perform indirect detection Extra method development considerations Use wide-bore capillaries EOF profile disturbed, adjustments to rinse and injection times, siphoning effects more pronounced Increase detector slit width Reduc tion in separation efficiency and resolution

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302.2.1 Normal stacking Sample stacking was first used by Ornstein in disc electrophoresis in 1964. It is generally used to describe zone-sharpening effects resulting in sample concentration phenomenon 11. In CE, stacking is one of the most widely used sample preconcentration techniques, in which the velocity of analyte ions is changed by using discontinuous buffers 12-14. The sample is usually dissolved in an electrolyte solution that has a lower conductivity than the running buffer, and the injection is made hydrodynamically. When a high voltage is applied, a higher electric field is developed in the injected sample characterized by lower conductivity than in the more concentrated running buffer because of the higher resistance of the sample z one. As known, electrophoretic velocity is proportional to electric field. Therefore, the analyte ions migrate more rapidly in the dilute sample solution than in the running bu ffer. This feature makes the ionic analysts stack at the boundary between the sample plug and the buffer and forms a narrow “stacked” zone 12, 13, 15 1, 16-20. Under a normal voltage polarity, cations should be stacked at the front of the sample plug, while anions are stacked at the rear of the sample plug. Neutral solutes are not stacked since they are forced through the capillary only by the influence of electroosmotic flow. Figure 2.2 schematically illustrates sample stacking. By using this method, Chien and Burgi 12 preconcentrated phenyl-thiohydantoin-aspartic acid (PTH-Asp), PTH-glutamic acid (PTH-Glu), PTH-arginine (PTH-Arg) and PTH-histidine (PTH-His). Their study showed the possibility of preconcentration and baseline separation of PTH-Asp and PTH-Glu. In clinical application, sample stacking technique was used in capillary electrophoresis-mass spectrometry system by Clench and coworkers 21. The sample they tested was urine, obtained from five male smokers. Their analytes of interest, nicotine and its metabolites in urine were extracted by solid-phase extraction. The solutes then obtained were analyzed using sample stacking capillary electrophoresis-mass spectrometry. The electrolyte they used for CE analysis was 10 mM ammonium formate dissolved in a mixture of 75% acetonitrile and 25% deionized water. The pH of the buffer was adjusted to 2.5 using HCl or formic acid. Results shows sample stacking procedure with MS detection achieved the LODs (limit of detection) of nicotine and cotinine as 0.11 and 2.25 g/mL respectively.

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31 Figure 2.1 Family tree of sampling procedure for preconcentration in CE (based on several reviews published during last decade 4-10).

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32 + + + EOF EOF EOF Inject sample Apply voltage Electrophoresis proceeds Anode Anode Anode Cathode Cathode Cathode Figure 2.2 Sample stacking with a sample dissol ved in a solution, such as deionized water, that has a lower conductivity than the electr ophoresis run buffer. The circles represent a cationic solute. Top: the sample plug is inject ed. Middle: voltage is applied and since the electric field in the sample solution is higher than in the rest of the capillary, the cations rapidly migrate through the sample solution un til they reach the low electric field in buffer, where they slow down and become stacked at the boundary between the solutions. Anions are stacked at the back of the sample plug. Bottom: the stacked ions migrate through the capillary as a zone that is narrower than the sample plug. Reproduced from Reference20.

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33 As shown in Figure 2.2, the migration speed of the analytes is changed by the discontinuous buffer system and the concentration ratio of the sample buffer and support buffer play important roles in achieving optimum separation 14, the preparation and composition of discontinuous buffers greatly affect the preconcentration effect. Studies have shown that discontinuous buffers can be prepared simply by addition of salts into buffer, by dissolving the sample in a low ionic strength buffer, or by adjusting their pH values 19, 20, 22. Aebersold and Morrison 23 reported a method to achieve stacking by pH gradient within the capillary. The sample plug with a high pH was introduced inside the capillary surround ed by buffer at a low pH. This results in the migration of anionic peptides toward the anode when the high voltage is applied. When the analytes reach the acidic (low pH buffer zone), the charge reverses and the migration direction is reversed. The sample is then stacked. A novel strategy for the formation of a sharp pH gradient was developed by Wei and Yeung 22. Their method is based on electrochemical reactions occurring at the ends of a short platinum wire which was inserted into the separation capillary. After the applica tion of a high voltage, OHand H+ were generated at the ends of Pt wire. A pH gradient was then formed along the capillary, while those created ions began to titrate the buffer ions and distribute down the column. It was found that large volume injec tion accompanying sample stacking normally cause some problems such as broadened peaks and increased temperature in the diluted sample zone 24, 25. Meantime, when a large volume of sample is introduced into the separation column for stacking, the solute zone is as wide as the length of the sample plug. In order to minimize the problems caused by the large volume sample injection, several techniques had been developed to achieve a narrow stacked sample band for further analysis. Chien and Burgi 12 introduced a method of stacking, in which a reverse EOF was used to back out the sample matrix after injection into the capillary. The reversed EOF can be generated by switching the polarity of the applied voltage. For instance, if the separation voltage is from an ode to cathode, the sample matrix should be removed out of the capillary by an applied voltage from cathode to anode. This procedure is illustrated in Figure 2.3. First, a large volume of dilute samp le solution (e.g., an injection length of 65 cm in a 100 cm long capillary column) was injected into the column. A negative voltage (from cathode to anode) was then applied to the column ends

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34 to obtain an electroosmotic flow (EOF) toward to the capillary inlet. Consequently, the sample matrix was gradually pushed out of the capillary by the EOF and the anions were stacked at the boundary between sample solution and the background electrolyte. The resulting sample zone was narrow. When th e separation voltage with polarity was opposite to the back-out voltage polarity was applied, CE separation of the sample components was achieved in the capillary under the normal EOF. Chien and Burgi 12 compared the separation efficiency in CE using sample stacking procedure with and without sample ma trix removal. The results showed that the peaks obtained by stacking with sample removal were narrower than those without sample removal. The large volume sample inj ection stacking method with sample matrix removal greatly enhanced the separation efficiency. Using this strategy, Kim and coworkers 17 successfully preconcentrated some protains and an enhancement factor of more than 100 was achieved. A mixture of chlorophenols and chlorophenoxy acids was preconcentrated and separated by Marriott and co-workers 26 using sample stacking strategy. Compared to standard CE analysis, 40and 10fold improvement of sensitivity was obtained by using sample stacking with and without the removal of sample matrix, respectively. Recently, this sample stacking technique was reported to have been used in the determination for metallothioneins in rabbit and eel livers 27. This same research group 28 also used this technique to determine some metal element, such as Cd, Cu, and Zn speciation in fish liver metallothioneins. In addition to the method invented by Chien and Burgi 12 to focus the sample by reversed EOF, capillary is oelectric focusing (CIEF) 29-35 is another widely used focusing technique in CE. In this approach, differences in the isoelectric points (p I s) of analytes are utilized to achieve the focusing effect. First, the separation capillary is filled with a solution of ampholytes. Upon application of an electric field across the capillary, a pH gradient is formed along the length of the ampholyte plug. When zwitterionic analytes are injected into such a capillary, they electrophoretically migrate through the pH gradient of the ampholyte solution. When a charged analyte reaches a location in the ampholyte plug where pH equals the p I value of the analyte, it becomes electrically neutral, and ceases to move via electrophoretic migration. Without the influence of EOF, focused discrete neutral analyte zones line up along the capillary based on their p I values. Figure 2.4

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35 + + + EOF EOF Inject sample hydrodynamically Reverse polarity and apply voltage Reverse polarity Anode Anode Anode Cathode Cathode Cathode + EOF Electrophoresis proceeds Anode Cathode (A) (B) (C) (D) Figure 2.3 Sample stacking of a large sample volume. The circles represent a mixture of two anionic solute. (A) A large volume of sample is hydrodynamically injected. (B) A negative voltage is applied at the capillary inlet, causing the anions to stack and the sample solution to be forced out of the cap illary. Cations and neutrals, not shown, are also forced out of the capillary at the inlet. (C) When most of the sample solution has been removed, the polarity is reversed. (D) The stacked anions are separated by CZE. Reproduced from Reference20.

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36 + -Anolyte CatholyteAnodeCathode pH Gradient Low pH High pH Capillary pI Solutes Figure 2.4 Illustration of capillary isoelectric focusing (CIEF). The cap illary is filled with a mixture of ampholytes and the sample containing zwitterionic solutes. When an electric field is applied, the solutes migrate to the locations in the capillary where the pH = p I for individual solutes, and are focused into narrow zones. Solutes and ampholytes are mobilized to force them through the detector. The anolyte is an acidic soluti on with a pH lower than the p I of the most acidic ampholyte, and the catholyte is a basic solution with a pH higher than the p I of the most basic ampholyte. Reproduced from Reference36.

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37 illustrates the steps of CIEF. CIEF is widely used for analytes with different p I values 3035. This technique is reported to be used with mixtures with very small differences in isoelectric points (as low as 0.01 pH unit). And an enhancement factor of 500 was achieved for polypeptide mixtures resulting from digestion of proteins 34. There are several review papers published recently on the theory and application of CIEF 37-40. 2.2.2 Electrokinetic injection/fi eld amplified sample injection In CE, the sample is introduced into the capillary by different techniques, in which the most commonly used methods are hydrodynamic and electrokinetic injections. Hydrodynamic injection is done either by pres sure or siphoning. Siphon injection is also known as gravity injection, which is perfor med by raising the sample vial above the capillary making the sample to be introduced into the cap illary. Pressure injection is carried out by pressurizing or vacuuming the sample into the capillary. In electrokinetic injection mode, an el ectric field is applied over the capillary length which causes sample to migrate into the capillary by electroosmotic flow and electrophoretic mobility 2, 3, 20, 41, 42. The amount of analytes injected depends on the electrophoretic mobility of the analytes in the sample, electroosmotic flow rate, applied voltage, capillary dimensions, and sample concentrations. Field-amplified sample injection (FASI) is a sample stacking technique operated by electrokinetic injection. The principle of FASI is the same as normal stacking. The main difference between FASI and normal stacking technique is that in normal stacking the focusing process occurs when the separation electric field is applied, while in FASI, the process occurs during injection. In order to produce an amplified electric field on the sample zone, the sample is prepared in a solution of smaller ionic strength than the running buffer. When a high voltage is applied, the electric field over the sample zone is much higher than that in the running buffer, which causes the solutes migrate rapidly into the capillary. When the solutes reach the running buffer zone with lower electric field, they slow down and get concentrated. It was found when the cap illary was switched directly from the running buffer to the dilute sample buffer, the boundary was disturbed and the electric field at the inject point might not be amplified properly, which leads to a lower concentration enhancement than expected. This problem was successfully solved

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38 by Chien and Burgi 43 with introduction of a water plug prior the injection of the dilute sample. With this modification, a high electric field was produced at the injection point and several hundred-fold enhancement was obtained. The advantage of FASI over normal stacking method is that a larger quantity of solute ions and just a small volume of dilute buffer are injected. Consequently, the pr oblems associated with injection of a large volume of dilute buffer solu tion, such as the overall electroosmotic velocity changes, production of laminar flow due to mismatched velocities, are limited. FASI technique has been frequently used in various fields. Its application include the determination of derivatized amino acids 44, small inorganic 45-47 and organic 48, 49 ions in water. In biological applications, pH-mediated sample stacking was used to the analysis of coumarin metabolite s in microsomal incubations 50. The metabolites studied included 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC) and 2hydroxyphenylacetic acid (HPAA). Low limits of detection at the range of 0.1-0.5 M was achieved for those analytes when UV absorbance detection was employed. Yin 45 and Zhang et al. 46 applied this method to concentrate different arsenic species. Velocity difference-induced focusing based on the pH changes in sample and run buffer zones was used in their studies. In Yin’s study, det ection of limits (LOD) obtained was as low as 5.0-9.3 g. L-1 when atomic fluorescence spectrometry was used for detection. Finally, both researchers showed the applicability of their methods to real environmental samples. Sample stacking has been used not only in aqueous CE but also in nonaqueous CE. It was reported by Kim and Chung 51 that methanol was used as the running liquid in nonaqueous CE to preconcentrate and separate ten organic acids in the concentration range of 10-100 nM.. After CE separation they were detected using conventional UV absorption detection. Excellent linear responses and reproducibility in the migration times, and corrected peak areas were obtained. Electrokinetic injection depends on the el ectrophoretic mobilitie s of the solutes as well as the EOF. Solutes with high mobilities are injected in a larger amounts than those with lower mobilites. This phenomenon is called sampling bias, which is one of the drawbacks of FASI. However, sampling bias is not regarded as a serious problem if the samples are all dissolved in the same solutions and the concentrations are similar 20. In

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39 order to determine and concentrate neutral compounds, field-enhanced sample injection micellar electrokinetic chromatography (FESI-MEKC) was used to get sample preconcentrated 52. Figure 2.5 shows the mechanisms and steps contributing to FESIMEKC. As Chien and Burgi 43 described, the introduction of a water plug prior to the sample plug provided a higher electric fi eld. In FESI-MEKC, this water plug also provided a fast pathway for the micelles to carry the neutral analytes. In this process, an anionic surfactant butylacrylate-butyl methacrylate-methacrylic acid copolymers, sodium salt (BBMA), served as the pseudostationary phase, and was used in both sample and buffer solutions. Due to the enhanced electric field in the water plug, the electrophoretic velocities were higher than the bulk electroo smotic flow (EOF). During the electrokinetic injection, micelles carrying the neutral analytes entered the capillary and got stacked in the front portion of the water plug. At the meantime, the water plug was pumped out by the EOF. Once the water was forced out th e capillary, the inlet of the capillary was put inside a vial containing the running buffer and the polarity of the electric voltage is switched. This step is followed by the separation and detection. Using this FESI-MEKC procedure, more than 20and 100fold improvements were obtained for estradiol. Using normal stacking, the same research group also successfully preconcentrated and analyzed neutral compounds (e.g., resorcinol, 1-naphthol, and 2-naphthol) in MEKC 53. For resorcinol, the limit of detection (S/N = 3) was in the order of 10-7 M with UV detection. Sample stacking was first used in CE with low-concentration electrolyte by Chien and Helmer in 199124. Since then, it has been extensively studied over last decade. Besides “field amplified sample injection (FASI)”, it is also called as “head column sample stacking” and “field-amplified sample stacking” (FASS) 11. Overall, sample stacking is an important technique to increase the detection sensitivity in many electrophoresis systems. With the development in microchip-based capillary electrophoresis 54-56, sample stacking techniques have also been used in the microfluidic devices. A few orders of magnitude in sample enrichment have been reported 57.

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40 B1 detector water plug BGS y z x x x x y y y y z z z z x x x y y y z z z y y z z x x x B1 detector detector B1 detector xA B C D Figure 2.5 Behavior of micelles and neut ral analytes during FESI-MEKC. (A) initial situation (water plug, unshaded; micellar separation solution shown as BGS shaded); (B) micelles enter the capillary and carry neutra l analytes emanating from the cathodic vial. The migration order of neutral analytes is dependent on their retention factors to micelles; (C) micelles and neutral analytes stacked at the concentration boundary (shown as B1), voltage is cut and the sample vial is replaced by another BGS vial when the measured current is approximately 97-99% of the predetermined current, voltage is then applied at positive polarity; (D) separation of zones develops. Reproduced from Reference52.

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412.2.3 Sweeping Sweeping analytes in MEKC was introduced by Quirino and Terabe 58-60. It involves the interactions between a pseudostati onary phase (PS) in the separation solution and a sample. The theoretical model of this technique is that the analytes are swept by the pesudostationary phase (micelles, polymers, dendrimers, etc.) which migrates through the sample zone and gets the sample focused. In this technique, the sample is prepared in a solution having a lower, similar, or higher conductivity than the background solution (running buffer). Analytes are preconcentrated because of chromatographic partitioning, complexation, or any interaction between analytes and PS. It is noticed that the length of the sample zone is dependent on the retention factor 58. For both neutral and charged compounds, the length of the sample zone is shorter for analytes with higher retention factors. Sweeping combined with field-enhanced sample injection (FESI), which was named as cation-selective exhaustive injection and sweeping (CSEI-sweep), was used to concentrate naphthylamine. Outstanding improvement in sensitivity as high as millionfold was achieved 60. The procedure of CSEI-sweep is shown in Figure 2.6. At first, the capillary was filled with a high-conductivity buffer devoid of organic solvent (HCB) followed by a short zone of water. The cation ic sample prepared in a low-conductivity solution was then electrokinetically injected into the capillary for a long time (about 10 min). After that, the sample zone was focused by means of sweeping by placing a low-pH buffer solution containing anionic micelles or micellar BGS in the inlet reservoir upon the application of a negative voltage. Separation was achieved by MEKC. The effect of preconcentration is dictated by the strength of the interactions involved. Sensitivity enhancements from tens to several thousand-fold have been achieved. In sweeping, both neutral and charged PS can be used based on the properties of the analytes. For neutral analytes, only charged PS are available for their separation. On the other hand, uncharged PS makes it possible to separate many important charged molecules. In this case, the sample penetrates the neutral PS zone to start the sweeping procedure other than the penetration of PS into the sample zone. Additionally, Landers and coworkers have made significant contribution to sample preconcentration via sweeping, especially for the samples from high salt matrixes 61-63. Contrary to conventional sample stacking procedure, the samples were prepared in

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42 Figure 2.6 Evolution of analyte zones in CSEI-sweep-MEKC: (A) starting situation, conditioning of the capillary with a nonmicellar background buffer, injection of a highconductivity buffer void of organic solvent, and injection of a short water plug; (B) electrokinetic injection at positive polarity (FESI) of cationic analytes prepared in a lowconductivity matrix or water, nonmicellar background buffer found in the outlet end; cationic analytes focus or stack at the interface between the water zone and highconductivity buffer void of organic solvent zone ; (C) injection is stopped and the micellar background solutions are placed at both ends of the capillary, shows the profile of the analytes after FESI; (D) application of voltage at negative polarity that will permit entry of micelles from the cathodic vial into the capillary and sweep the stacked analytes; (E) separation of zones based on MEKC. Reproduced from Reference60

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43 high-conductivity matrixes, which is the ke y to their technique in micellar capillary electrophoresis (MCE), more commonly known as MEKC. Since the separation buffer has a lower conductivity compared with that in sample matrix, a filed amplification occurs within the separation buffer zone, which leads to stacking of the charged micelles at the detector side of the sample matrix and separation buffer interface. The stacked micelles then sweep the neutral compounds through the capillary to the detector. The proposed mechanism of the technique is illustrated in Figure 2.7. The advantage of this method is it allows free optimization of separation buffer parameters including concentration, pH, ionic strength, and organic modifier without affecting the sample stacking method. This novel, robust, and widely applicable technique does not only solve the low sensitivity problem in CE but also the problems associated with the sample preconcentration from high-salt matrixes, which is frequently met in real-life situations. As discussed above, the sweeping techniques used by Quirino used both continuous 58 and discontinuous 60 buffer systems. In contrast, Landers and coworkers 6163 utilized the advantages of discontinuous buffer systems. Since high micelle concentration was used in the sample matrix, a stacking of micelles at the sample/separation buffer interface occurred. Therefore, this technique is also called highsalt stacking. Following the stacking of micelle s, the stacked micelles entered the sample zone and carried or swept the sample through the capillary and got detected. Recently, the principles of high-salt stacking and sweeping has been compared and clarified in a paper by Palmer 64. No matter what kind of buffer system and PS phase is used, the purpose of sweeping is to preconcentrate the anal ytes and analyze them. Britz-McKibbin 65, 66 developed a preconcentration method involving dynamic pH junction and sweeping modes of focusing. Dilute flavins in biological samples with as low as picomolar concentrations have been examined by capillary electrophoresis with laser-induced fluorescence detection. Gavenda and coworkers 67 added sodium dodecyl sulfate (150 mM) into the background buffer as the PS ph ase. Trace amounts of biologically active anthracyclines were analyzed with UV absorption detection. Some basic drugs and their metabolites have been preconcentrated and analyzed by sweeping technique in capillary

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44 Figure 2.7 High-salt stacking with a sample matrix of ionic strength greater than the separation buffer. High-conductivity sample ma trix induces a stacking of the cholate micelles at the interface (t = 1). Analytes (e.g. corticosteroid) experience a reduction in velocity upon encountering the stacked mice lle / sample zone interface (t = 2). The chloride in sample matrix has diffused and results in a neat interface of the sample matrix with the stacking micelles (t = 3). Reproduced from Reference61.

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45 zone electrophoresis using field-amplified sample injection (FASI) by Taylor and coworkers 68. Since sweeping is applicable to both neutral and charged analytes, it is regarded as a highly versatile and effective on-line preconcentration technique in CE 69. 2.2.4 Capillary isotachophoresis (CITP) Capillary isotachophoresis (CITP) 70 can also be employed for sample preconcentration. CITP is accomplished in a capillary where the sample is sandwiched between two discrete buffer plugs: a leading buffer with a higher mobility ion and a terminating buffer having a lower mobility ion than the charged analytes. Upon the application of an electric field with constant current, the ions existing in the sample are distributed into narrow and concentrated zones based on the differences in their mobilities. Figure 2.8 schematically shows the procedure of CITP, in which solute 1 has the highest mobility and solute 3 has the lowest mobility. Transient ITP is a technique in which sample is concentrated and sepa rated by CZE in the same capillary 71, 72. During the separation process, the buffer is changed and ITP does no longer occur. Therefore, the term “transient” is used to name the technique 71. Similar to the principle of CITP, in transient ITP sample is injected between a leading and a tailing electrolyte. After the solutes are concentrated in ITP, separation of the analyte zones occurs. On-column transient ITP technique has been used to preconcentrate and separate various analytes including inorganic 73, 74 75, organic 76, 77 and biomolecules 78. Tu and Lee 73 used chloride ion as the leading ion and dihydrogenphosphate as the terminating ion in ITP to determine the nitrate in seawater. Since the mobility difference for chloride and nitrate ions is small, a zwitterionic surfactant [3-(N, Ndimethyldodecylammonio)propane sulfonate] was added to the background buffer and the selectivity was su ccessfully increased 73. Iodide and iodate in seawater was determined by this technique 75. Several metal cations including La3+, Ce3+, Pr3+, Nd3+, Sm3+, and Eu3+ in low-ppb range have been determined by transient ITP with hydroxyisobutyric acid as the complexing agent to enable the separation of analyte ions that have similar mobilities 74. Shihabi 77 has developed a method in which some water miscible organic solvents such as acetonitrile, acetone and small alcohols were used as a

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46 Inject sample and buffers Terminating Buffer Leading Buffer Terminating Buffer Leading Buffer 321Solute Mixture Apply electric field Separated Solutes Mobility + +Cathode Cathode Anode Anode Figure 2.8 Representation of capillary isotachophoresis of anions. A leading buffer is selected which has anions with higher mobility than any solute anions. A terminating buffer is selected with anions that have lower mobility than any solute anions. When an electric field is applied, the solutes migrate to form individual zones on the basis of their mobilities with solute 1 having the highest mobility and 3 the lowest. The length of each zone is proportional to the amount of solute in the zone. After the solutes migrate into their zones, an equilibrium is established an d the buffers and solutes migrate through the capillary. Reproduced from Reference20.

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47 terminating ion in transient isotachophoresis, while salts existing in samples function as leading ions. This technique has been suggested to be termed as transient “pseudoisotachophoresis” (pseudo-ITP). Compared to tr aditional ITP, pseudo-ITP is much easier to perform. Tyrosine and theophylline were preconcentrated and separated using the developed method. Transient isotachophoresis was also used to perform on-line preconcentration of enantiomers (clenbuterol). Prior to the enantioseparation, ITP was used to concentrate the sample, in which a co unterflow was used to increase the sample loadability of the system and the CZE sepa ration path length. The counterflow was achieved by a counterpressur e that counterbalanced the electrophoretic migration of the compounds. The chiral selector used in this research was dimethyl-cyclodextrin 76. In the area of biology, human insulin, a polypeptide was concentrated by the use of transient ITP 78. 2.3 Chromatography-based sample preconcentrati on techniques Chromatography-based methods repr esent an important category of preconcentration techniques. Compared to electrophoresis-based preconcentration methods, chromatography-based techniques are more selective. Based on the adsorbent, a wide range of analytes can be preconcentrated by these technoliques. As shown in Figure 2.1, it is further divided into two sub-categories as low-specificity and high-specificity chromatographic methods. In this section, principles, mechanisms, applications and the practical aspects of chromatographic-based preconcentration techniques are discussed. 2.3.1 Low-specificity chromatographic preconcentration Solid-phase extraction (SPE) is th e most widely used low-specificity chromatographic preconcentration method. SPE is used to isolate and preconcentrate target analytes from a gas, fluid or liquid sample by passing it through a sorbent bed, thereby allowing their transfer to and retenti on on the solid sorbent. The sorbent with the extracted analytes on it is then isolated from the sample and the analytes recovered by elution with a liquid or fluid, or by thermal desorption 79. In SPE-CE approach, samples are purified and extracted from a liquid mixture onto the solid adsorbent where it is concentrated, and then, the concentrated sample is released from the adsorbent and

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48 analyzed by CE. Various SPE matrices including C2-, C8-, and C18-bonded silica have been reported 80-82. 2.3.1.1 Preconcentration device In-line SPE-CE technique allows the completion of sample purification and preconcentration in a single step. Viberg et al. 80 have developed a miniaturized device to analyze and detect heterocyclic aromatic amines 83 by micro solid-phase extraction coupled to CE (SPE-CE) with nanospray ionization (nESI) mass spectrometric (MS) detection. The schematic illustration of th e extraction capillary for this process was shown in Figure 2.9. As shown in this Figure, it was made of an inlet capillary, an extractor and separation capillary column. The capillary was first conditioned by methanol and water. Sample solution wa s then introduced. Salt and hydrophilic substances were eluted by water and electrolyte, while HAs were extracted by the packed bed. Methanol was then used to elute the HAs out of the capillary for further analysis. Compared to the limit of detection (LODs) obtained from HPLC-atmospheric-pressure chemical-ionisation MS analyses, SPE-CE-nESI-MS technique provided 10-100-fold improvement in LODs for HAs. Because of the use of packing material and the frits, the electroosmotic flow (EOF) usually is disturbed in such a SPE-CE device by the increased back pressure. In addition, in-line SPE methods are often associated with some disadvantages such as the loss of resolution, peak broadening, and peak tailing. In membrane preconcentration (mPC-CE) 84, 85, these drawbacks can be overcome, in which the bed of solid phase are replaced with membranes with conventional LC stationa ry phases. The mPC-CE cartridge was constructed with membrane adsorptive phase inserted inside a piece of Telfon tubing84. The fused silica capillary for CE separation was connected to the ends of the Telfon tubing. Visser et al. 86 developed an interface, through which an SPE and CE parts are coupled together. First, samp le was transferred from the sampling loop to the SPE cartridge by using the loading solvent. After desalting, the extracted sample was desorbed by the elution solvent and transferred to the interface. A hydrodynamic injection was performed when the sample passed the micro-injection vial inside the interface. The

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49 Figure 2.9 Schematic illustration of the extrac tion capillary used fo r in-capillary SPE, CE and nESI. A: The inlet capillary serves as a transfer line to the extractor. B: The extractor is a short packed bed of particles for extraction and purification of the sample. C: In the CE-nESI capillary the sample molecules are separated by CE and electrosprayed into the mass spectrometer. Reproduced from Ref. 80.

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50 interface was then flushed with the CE elec trolyte, after which, the CE separation was started. Solid-phase microextraction (SPME) is a solvent-free sample preconcentration technique developed by Belardi and Pawliszyn 87. In SPME the sample solution is exposed to a sorbent coating applied to a fiber surface and the analytes are extracted by this coating. SPME and SPE are similar. Compared to SPE, no clean-up step is necessary for SPME. With many advantages including simplicity, rapid extraction, solvent-free, SPME has been used with gas chromatography with automation 88-93. SPME-HPLC makes it possible to analyze less volatile or thermally labile compounds 94, 95. Trace impurities such as PAHs, ketones, and alkylbenzenes in aqueous samples have been preconcentrated and analyzed by SPME-HPLC with titania-based material functioning extraction 96. The applications of SPME-CE have been reported by several research groups during last decade 97-100. Li and Weber 99 developed a preconcentration device for CE, which is very simple, inexpensive and solvent waste free. The device was made of a stainless steel extraction rod coated with poly (vinyl chloride) (PVC), a Teflon tube and a microsyringe. The extraction device and operation was illust rated in Figure 2.10. First, the analytes were exposed to the PVC coated rod. Secondly, the extracted analyte was transferred into an aqueous back-extraction solution through the Teflon tube. Finally, the back-extraction solvent containing interested analytes was co llected and injected into CE system to be separated. With this device, it took less th an 30 min to complete extraction, backextraction, and separation of 10 barbiturates. Due to the small sample volume in CE, this device is difficult to couple the CE on-line. Another device developed by Nguyen and Luong 98 utilizes an optical fiber with poly(dimethylsiloxane) (PDMS) coating. Analytes were absorbed in the PDMS coating, and then released into CE system. A piece of 2 cm long heat shrinkable tubing and a piece of 1.5 cm long capillary segment was used to connect the extraction rod and the separation capillary with zero dead volume at the interface. A normal CE separation can be carried out after the releasing the extracted analytes by methanol. The technique described by Nguyen and Luong 98 realized the direct connection between the extraction fiber and the inlet end of separation capillary in CE system, compared with Weber’s off-line method 99. Besides, Whang and Pawliszyn 97

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51 Figure 2.10 SPME device and operation in hyphenation with CE (1) Put the extraction rod in the sample solution for a specific time. (2) Inject 5 L of back-extraction buffer solution into the Teflon tube. (3 ) Put the extraction rod into the Teflon tube. The droplet of the back-extraction buffer solution can be forced to cover the whole surface of the PVC coating by adjusting the position of droplet with the syringe handle before or after placing the rod. (4) Take out the rod after letting it stand horizontally for a specific time. The majority of back-extraction solution will fo rm a droplet near the end of the tube. (5) Collect the solution by moving the droplet spanning the diameter as a piston and transfer the drop to an injection vial. Reproduced from Reference 99.

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52 designed an on-column interface for the coupling of the SPME into CE. Since their device facilitated the direct insertion of the SPME fiber inside the injection end of the CE capillary, the extracted analytes can be comple tely desorbed into the separation capillary. The fiber-in-tube SPME device developed by Jinno et al. 101 is composed of a fiberpacked capillary, which possesses the dual feature of being an extraction medium as well as a separation medium. Liquid-liquid extraction (LLE) and liquid-phase microextraction (LPME), two other techniques are also used for the sample preconcentration in GC, HPLC, and CE as low-specificity chromatogra phic preconcentration methods102-111. Liquid-liquid extraction normally uses a supported liquid membrane (SLM). An organic liquid phase is immobilized in the pores of the support, fo r instance, a membrane. Generally, a waterimmiscible organic solvent including pentane, hexane, diethyl ether, ethyl acetate, chloroform and methylene chloride is used to extract hydrophorbic components from aqueous samples. The basic theory of LLE, its compatibility with CZE, and applications have been discussed and reviewed 109. LLE provides excellent clean-up for sample preparation. On the other hand, the loss of analytes in the evaporation step greatly limits its attractiveness for CZE. The miniaturized verstion of LLE is called liquid-phase microextraction (LPME). Compared with LLE, LPME can achieve higher preconcentration effects using greatly reduced volume of the solvent. A disposable LPME device was developed by PedersenBjergaard and co-workers 106, which consisted of a vial containing the sample solution (donor solution), a porous hollow fiber impregnated with an organic solvent, a screw top with silicone septum, two needles connected to the ends of the hollow fiber. During the extraction, the acceptor solution was inject ed into the hollow fiber with one of the needles the extracted analytes in the acceptor solution were collected by another needle. Chung and coworkers 111 developed a simple and efficient sample preconcentration method for CE using liquid-phase microextraction (LPME). The extraction is completed by transferring the analytes twice between liquid-liquid phases. First, the aqueous sample solution was exposed to a droplet of acceptor (e .g., a thin layer of octanol on the surface). Analytes were extracted from the original sample solution (so-called donor phase) into octanol layer. The pH value of the sample was previously adjusted so that the sample had

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53 higher distribution in the organic solvent. After the equilibrium was achieved, the extracted analytes were back-extracted into the separation buffer (so-called acceptor phase). For this, the pH of the acceptor phase was carefully adjusted so that the analytes had higher solubility than in octanol. After copletion of the LPME procedure, the acceptor phase containing concentrated sample was injected into the CE column for the separation and analysis of the extracted solutes. 2.3.1.2 Application SPE-CE, SPME-CE and LLE-CE and LPME-CE are becoming increasingly popular in analytical chemistry. Their applications include preconcentrating and analyzing a wide range of analytes, generally applied to biological and environmental samples. In order to monitor therapeutic drug, recently Fonge and co-workers 112 used offline SPE to extract tobramycin from human serum. A carboxypropyl bonded phase (CBA) cartridge was used. The extracted analyte was then eluted from the CBA by a mixture of NH3 (25%, w/v in water)-methanol (30:70, vol./vol. in water). After evaporation of the solvent, the analyte was derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA). The tobramycin-OPA/MPA derivative was analyzed by CZE using UV detector at 230 nm. A limit of detection (LOD) of 0.1 g/mL was achieved, which indicates the sensitivity of this method. Lingeman et al. have published several papers based on the applications of SPE-CE, such as the determination of amphoteric compounds in biological samples, including determination of sulfonamides in blood, serum, and urine 113, 114, online dialysis-SPE-CE of acidic drugs in urine and serum 115, the determination of the anticoagulant phenprocoumon in plasma and urine 116. Peptides in the low nanograms per mL range has been extracted and concentrated by method developed by Landers et al. 117 118. In the mean time, peptide mapping was accomplished using SPE-CE system 119. A review by Martinez et al. summarized the application of SPE-CE in the environmental analysis 120. Other applications of SPE-CE in biological area include the determination of insulin de rivatives in urine, serum and plasma by online SPE-CE developed by Visser and co-workers 86, preconcentration, separation and

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54 quantitation of nonsteroidal anti-inflammato ry drugs (NSAIDS) in human urine and serum 121, measurement of urinary free cortisol (UFC) by Rao and co-workers 122. Due to its attractive features such as simplicity, speed, and solvent-free operation, SPME has few applications in CE. The first application of off-line SPME with CE was in biomedical area presented by Li and Weber 99. Barbiturates were extracted and analyzed. Their extraction device was introduced in section 2.3.1.1. The extraction effect was quantified by the preconcentration factor, which was defined as the ratio of the concentration of analyte in back-extraction solvent to the concentration in the sample. The extracted samples were separated in a CE system equipped with a UV detector. The results showed that barbiturate concentrations in the range of 0.1 – 0.3 ppm in urine and about 1 ppm in serum could be determined. In the work by Jinno et al. 101, an on-line fiber-in-tube SPME-CE system has been developed for the separation of four tricyclic antidepressant (TCA) drugs: amitriptyline, imipramine, nortriptyline, and desipramine. After extraction on the fiber, the analytes were desorbed with acetonitrile and then injected into the CE separation capillary. The running buffer for the separation consisted Na2HPO4, -cyclodextrin, and acetonitrile. UV detector was employed. Research results showed that the on-line fiber-in-tube SPME-CE system is a powerful tool to determine analytes in biological matrices. Cifuentes and co-workers123 developed a highly sensitive procedure to detect multiple pesticides including pyrimethanil, pyrifenox, cyprodinil, cyromazine, and pirimicarb at trace levels spiked in different food samples such as grapes and orange juice. In this SPME procedure, commercial extraction fibers were used. Samples were mixed with NaCl solution and the pH was adjusted to 6.0. The extraction process was followed by desorption of the pesticides in methanol. The optimum background electrolyte was determined as 0.4 M acetic acid at pH 4. Mass spectrometry was used as the detector. Their approach achieved the LODs down to 15 ng/mL for the pesticides studied. Another method based on SPME/CE/MS has been developed by Pico et al. 124 to determine some acidic pesticides in fruits. The studied pesticides included ophenylphenol, ioxynil, haloxyfop, acifluorfen, picloram. Commercial SPME fibers were used to extract pesticides from the fruits and mthanol was used to desorb extracted pesticides. The optimum running buffer was 32 mM ammonium formate/formic acid buffer at pH 3.1. The limits of quantification were found in the range of 0.02 5 mg/kg.

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55 In environmental science, SPME and CE hyphenated system was used for the analysis of polycyclic aromatic hydrocarbons 98, phenol 97, and explosives125. The combination of liquid-liquid extraction (LLE) and CZE has been applied to the analysis of drugs from biological fluids, such as plasma, serum, and urine126-128. The advantages of using LLE in sample preparation include: (a) removing inorganic salts from the sample matrix, which interfere the optimal operation conditions for sample injection with field-enhanced sample stacking method in CE; (b) effective removing of plasma and serum proteins which usually deteriorate the peak shapes because of their adsorption to capillary surf ace. Thormann and co-workers 127 determined an antihelminthic drug albendazole (ABZ) in human plasma using CE with on-line UV detector. The sample preparation was conducted via liquid/liquid extraction using dichloromethane. Extract reconstitution wa s performed in N-methylformamide. During CE separation, borate buffer with apparent pH 9.9 in a mixture of methanol and Nmethylformamide (1:3) was used. The limit s of detection (LODs) for ABZ, ABZSO (ABZ’s major metabolite albendazole sulfoxide) and ABZSO2 (ABZSO’s further oxidization product albendazole sulfone) were in the order of 10-7 M. An overview of published methods based on LLE-CZE applicati ons of drugs from biological fluids has been summarized in a review by Pedersen-Bjergaard et al.109, in which the analytes, sample matrix, extraction solvent, reconstituti on solvent, enrichment detection, detection mode as well as limit of quantification we re clearly listed. Pedersen-Bjergaard and coworkers also validated and successfully used the combination of LPME and CE to determine chiral drugs in biological matrices 102, 106, 129. For example, the chiral antidepressant drug mianserin in plasma was determined using LPME-CE technique. First, the analytes were extracted with di-n-hexyl ether and further transferred into an acidic solution. The limit of detection (S /N = 3) was 4 ng / mL when a UV detector was employed 106. Low-specificity chromatographic preconcentration, as shown in its name, allow enhancement of the sensitivity but with low se lectivity. Sometimes, other componenets in the matrix may be preconcentrated in addition to the analytes of interested, which may hinder determination of the target analytes. To avoid this interference, high-specificity chromatographic preconcentration can be used.

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562.3.2 High-specificity chromatographic preconcentration When very selective isolations need to be made, high-specificity chromatography is more appropriate than methods based on low-specificity chromatography. The most frequently used preconcentration techniques based on high-specificity chromatography include immunoaffinity chromatography and molecularly imprinted polymer technology. 2.3.2.1 Immunoaffinity chromatography Immunoaffinity chromatography, so metimes called immunoadsorption chromatography, is based on highly specific interaction of a ligand with its counterpart. Antigen-antibodies and lectin-carbohydroattes are the best representative examples of this kind of interaction. Because an antibody recognizes only a very small part of the antigen, this method is highly specific Normally immunoaffi nity chromatography utilizes an antibody or antibody fragment as a ligand which is immobilized onto a solid support matrix in such a manner that it retains its binding capacity for the analytes. Due to the ability to provide powerful clean-up and selective enrichment, the potential of immunoaffinity chromatography 130 combined with CE has been demonstrated for the determination of analytes at low levels in complex biological matrices 131, 132. The immunoaffinity preconcentration device or enrichment chamber is composed of a solid support with immobilized the antibody. A good solid support should have a maximum surface to immobilize the antibodies and a minimum of hydrostatic resistance. The simplest enrichment chamber utilizes a portion of a capillary containing an immobilized selector on the wall to capture a specific analyte 133. The remaining portion of the capillary is used as the actual electrophoretic separation column. A second type of enrichment chamber uses a membrane placed in a Teflon cartridge, an antibody or receptor is impregnated on the membrane. A third type of on-line immunoaffinity preconcentration system contains HPLC-pakcing beads. In this case, frits are used to retain the packing beads. Because of the high specificity of immunoaffinity chromatographic technique, a substantial increase in concentration of analytes can be achieved, generally in the range of 100-500 folds 134. Kennedy and Cole 135 used capillaries packed with a pe rfused protein G as the chromatographic support. Antibody was loaded on it for create an immunoaffinity stationary phase. This immunoaffinity

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57 preconcentrator could be coupled with CZE either online or off-line. For on-line combination, a flow-gated interface was needed to connect the preconcentration and separation columns, which enabled the direct injection of the desorbed sample plug into the electrophoresis system. For off-line coupling, the desorbed fractions were collected and then injected into the CE system. Using this system, insulin samples was preconcentrated and analyzed. Immunoaffinity chromatography coupled with CE has been widely used to identify and characterize bioanalytes. Here is an example from Guzman 9, 131, 136, 137, who has published several papers since 1990. Immunoreactive gonadotropin-releasing hormone (GnRH) in serum and urine specimens was determined. The controlled-porous glass beads were incubated with FAb peptide, an antibody fragment. A piece of fusedsilica capillary was partially fi lled with the glass beads covalently attached with FAb fragments (e.g., antigen binding fragment of an immunoglobulin G containing the hingeregion cysteine); the beads were retained by two porous frits. This piece of capillary was then fused to two longer capillary columns using a Teflon sleeve and glued with an epoxy resin. Figure 2.11 illustrates the microphotograph of the concentrator. The enriched GnRH was desorbed using a solution containing glycine-HCl buffer pH 2.5. For the separation, a solution composed of sodium tetraborate (80 mM, pH 9.1) and 1% (v/v) acetonitrile was used as the running buffer. When a UV detector was employed, 1 ng/mL was determined as the LOD for the analysis of GnRH 136. Immunoaffinity chromatography was also used by Dalluge and Sander 138 to prepare clinical disease-state marker protein samples prior to capillary electrophoresis sepa rations. In their protocol, the receptor monoclonal anti-cTnI antibodies were covalently immobilized on the pakced bed of porous silica. This procedure was accomplished by putting the pretreated silica particles into phosphate buffer solution cont aining monoclonal anti-cTnI antibodies and allowing the system to stand for 1 h at room temperature. After this, the nonspecifically bound proteins were removed by washing th e silica with Tris-Cl, NaCl, and KCl, containing Tween-20 nonionic detergent, pH 7.2. This was followed by the packing of antibody-loaded silica particles into a piece of capillary and the preconcentration column is ready. When the sample containing cardiac troponin I (cTnI) was passed through the preconcentration column, anti-cTnI selectively extracted cTnI due to their

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58 Figure 2.11 Microphotograph of the analyte concentrator fabricated with FAb antibody fragments immobilized to glass beads. The glass beads were retained between two frits. The analyte concentrator device was connected to two separation capillaries by a Teflon sleeve. The plastic connector was glued to th e separation capillaries by an epoxy resin. The entire fabrication process was monitored by a stereo microscope. Reproduced from Reference136.

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59 complementary structure features. After the sample injection, the column was rinsed extensively with running buffer to remove any nonspecifically bound serum proteins, salts, and other impurities. The target analyte cTnI was then released from the column with the elution buffer consisting of Tris-acetate, -mercaptoethanol, ethylenediaminetetraacetic acid (EDTA), and urea, pH 3.5. The desorbed cTnI was analyzed directly by CE coupled to a UV detector. The limit of detection (LOD) was 2 nmol/L. Selectivity, sensitivity, efficiency, and sample recovery of this method were remarkable. 2.3.2.2 MIP technology MIP is the abbreviation for molecularly imprinted polymer. The synthetic materials made by MIP technology are capable of selectively recognizing specific molecules because of the binding sites created by the template molecules complementing molecules in size, shape and chemical functionality 139. The procedure to make MIPs includes several steps 140. Figure 2.12 schematically shows the preparation of an MIPs. First, an appropriate amount of a functional monomer is mixed with a cross-linker in an organic solvent containing an initiator and a template molecule. After polymerization, the template molecules are removed. The synthesized material has both steric (size and shape) and chemical (spatial arrangement of complementary functionality) memory for the template and is able to selectively rebind im print molecules from sample mixtures. Either the analyte of interest or a structural analogue of it can be the template molecule. For instance, Ratner and co-workers 141 developed a method for imprinting surfaces with protein-recognition sites. With the powerful molecular recognition feature, MIPs have been used in several analytical tools such as liquid chromatography (LC) 142, 143, CE 144-147, solid phase extraction 148, 149, ligand binding assay 150, and sensor technology 151. Since MIPs display characteristics similar to biological receptor, they have been used as selective chiral st ationary phases. De Boer et al. 144 prepared spherical MIP particles for the chiral separation of ephedr ine and salbutamol enantiomers. Owing to its high selectivity, this method might be used for the concentration of samples prior to the CE separation,. However, extensive investigation is needed to reveal the potential application of MIPs in preconcentration methods (online or offline) coupled with CE.

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60 Figure 2.12 Schematic depiction of the preparation of molecular imprints. Reproduced from Reference151.

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612.4 Conclusions Capillary electrophoresis has been drawing increasing attention in a wide range of research fields with its inherent virtues. Its low concentration sensitivity, however, has induced a new research subject in the fields related to CE aimed at accomplishing tracelevel analysis by CE. As shown in this chapter, enrichment of sample has been achieved to reduce the detection limit using various practical methods in CE. Both electrophoresis-based and chromatography-based preconcentration methods have been developed to achieve this goal. Each technology has its own advantages and priorities. Obviously, there is no universal method which is suitable to all situations. In the view of practice, the identification of the most suitable method should be made based on the requirement in specific analytes, the magnitude of sensitivity enhancement, modification of the existing devices, convenience for operation, as well as the costs. With the increasing demands on sensitive detection of analytes in biological matrices, preconcentration technique with high selectivity has shown great potential applications in the future In this dissertation, on -line electrophoresis-based preconcentration methods were developed for amino acids and proteins with sol-gel materials coated fused silica capillaries which were coupled with commercial CE systems. 2.5 References for Chapter Two (1) Altria, K. D. Capillary Electrophoresis Guidbook Principles, Operation, and Applications; Humana Press: Totowa, NJ, 1996. (2) Poole, C. F. The Essence of Chromatography; Elsevier Science: Amsterdam, The Netherlands, 2003. (3) Landers, J. P. Handbook of Capillary Electrophoresis; CRC Press: Boca Raton, FL, 1994. (4) Veraart, J. R.; Lingeman, H.; Brinkman, U. A. T. J. Chromatogr. A 1999, 856, 483-514. (5) Sentellas, S.; Puign ou, L.; Galceran, M. R. J. Sep. Sci. 2002, 25, 975-987.

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62 (6) T.Stroink; Paarlberg, E.; Waterval, J. C. M.; Bult, A.; Underberg, W. J. M. Electrophoresis 2001, 22, 2374-2383. (7) Hempel, G. Electrophoresis 2000, 21, 691-698. (8) Lloyd, D. K. J. Chromatogr. A 1996, 735, 29-42. (9) Guzman, N. A. Anal. Bioanal. Chem. 2004, 378, 37-39. (10) Breadmore, M. C.; Haddad, P. R. Electrophoresis 2001, 22, 2464-2489. (11) Chien, R. L. Electrophoresis 2003, 24, 486-497. (12) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (13) Chien, R. L.; Burgi, D. D. Anal. Chem. 1992, 64, 489A-496A. (14) Burgi, D. S.; Chien, R.-L. Anal. Chem. 1991, 63, 2042-2047. (15) Shihabi, Z. K. J. Chromatogr. A 2000, 902, 107-117. (16) Shihabi, Z. K. Electrophoresis 2000, 21, 2872-2878. (17) Chun, M. S.; Kang, D.; Kim, Y. Microchem. J. 2000, 70, 247-253. (18) Locke, S.; Figeys, K. Anal. Chem. 2000, 72, 2684-2689. (19) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1242-1252. (20) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons, Inc.: New York, N.Y., 1995. (21) Baidoo, E. E. K.; Clench, M. R.; Ma lcolm, R.; Smith, R. F.; Tetler, L. W. J. Chromatogr. B 2003, 796, 303-313. (22) Wei, W.; Xue, G.; Yeung, R. S. Anal. Chem. 2002, 74, 934-940. (23) Aebersold, R.; Morrison, H. D. J. Chromatogr. 1990, 516, 79-88. (24) Chien, R. L.; Helmer, J. C. Anal. Chem. 1991, 63, 1353-1361. (25) Vinther, A.; Soeberg, X. H. J. Chromatogr. A 1991, 559, 27-42. (26) Kruaysawat, J.; Marriott, P. J.; Hughes, J.; Trenerry, C. Electrophoresis 2003, 24, 2180-2187. (27) Alvarez-Llamas, G.; Rodriguez-Cea, A.; Campa, M. R. F. D. L.; Sanz-Medel, A. Anal. Chim. Acta 2003, 486, 183-190.

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63 (28) Alvarez-Llamas, G.; Campa, M. D. R. F. D. L.; Sanz-Medel, A. J. Anal. At. Spectrom. 2003, 18, 460-466. (29) Hjerten, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265-270. (30) Yang, C.; Zhang, L.; Liu, H.; Zhang, W.; Zhang, Y. J. Chromatogr. A 2003, 1018, 97-103. (31) Clarke, N. J.; Naylor, S. Biomed. Chromatogr. 2002, 16, 287-297. (32) Hiraoka, A.; Tominaga, I.; Hori, K. J. Chromatogr. A 2002, 961, 147-153. (33) Spanik, I.; Lim, P.; Vigh, G. J. J. Chromatogr. A 2002, 2002, 241-246. (34) Shen, Y.; Berger, S. J.; An derson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2154-2159. (35) Sheng, L.; Pawliszyn, J. Analyst 2002, 127, 1159-1163. (36) Weinberger, R. Practical Capillary Electrophoresis, 2nd ed.; Academic Press: London, UK, 2000. (37) Jenkins, M.; Ratnaike, S. Clin. Chem. Lab. Med. 2003, 41, 747-754. (38) Hermann, W.; Guenter, S. Clin. Chem. Lab. Med. 2003, 41, 724-738. (39) Marshall, T.; Williams, K. M. Electrophoresis 1998, 19, 1752-1770. (40) Jenkins, M. A.; Guerin, M. D. J. Chromatogr. B 1997, 699, 257-268. (41) Bansal, R.; Chen, H. X.; Marshall, J. T.; Tan, J.; Glaxer, R. I.; Wainer, I. W. J. Chromatogr. B 2001, 750, 129-135. (42) Kakehi, K.; Kinoshita, M.; Kawakami, D. ; Tanaka, J.; Sei, J. ; Endo, K.; Oda, Y.; Iwaki, M.; Masuko, T. Anal. Chem. 2001, 73, 2640-2647. (43) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152. (44) Lada, M. W.; Kennedy, R. T. Anal. Chem. 1996, 68, 2790-2797. (45) Yin, X. B. Electrophoresis 2004, 25, 1837-1842. (46) Zhang, P. D.; Xu, G. W.; Xiong, J. H.; Zheng, Y. F.; Yang, Q.; Wei, F. S. Electrophoresis 2001, 22, 3567-3572. (47) Buscher, B. A. P.; Tjaden, U. R.; Greef, J. V. d. J. Chromatogr. A 1997, 764, 135142. (48) Qin, W.; Li, S. F. Y. Electrophoresis 2003, 24, 2174-2179.

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64 (49) Lunte, C. E.; Scott, D. O.; Kissinger, P. T. Anal. Chem. 1991, 63, 773A-780A. (50) Ward, E. M.; Smyth, M. R.; O'Kennedy, R.; Lunte, C. E. J. Pharm. Biomed. Anal. 2003, 32, 813-822. (51) Kim, B.; Chung, D. S. Electrophoresis 2002, 23, 49-55. (52) Quirino, J. P.; Terabe, S. J. Chromatogr. A 1998, 798, 251-257. (53) Quirino, J. P.; Terabe, S. J. Chromatogr. A 1997, 781, 119-128. (54) Kameoka, J.; Craighead, H. G.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1935-1941. (55) Deng, Y.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1432-1439. (56) Deng, Y.; Henion, J.; Li, J. Anal. Chem. 2001, 73, 639-646. (57) Yang, H.; Chien, R. L. J. Chromatogr. A 2001, 924, 155-163. (58) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-1644. (59) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (60) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023-1030. (61) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-1687. (62) Palmer, J.; Landers, J. P. Anal. Chem. 2000, 72, 1941-1943. (63) Palmer, J.; Burgi, D. S.; Munro, N. J.; Landers, J. P. Anal. Chem. 2001, 73, 725731. (64) Palmer, J. J. Chromatogr. A 2004, 1036, 95-100. (65) Britz-McKibbin, P.; Otsuka, K.; Terabe, S. Anal. Chem. 2002, 74, 3736-3743. (66) Britz-McKibbin, P.; Markuszewski, M. J. ; Iyanagi, T.; Matsuda, K.; Nishioka, T.; Terabe, S. Anal. Biochem. 2003, 313, 89-96. (67) Gavenda, A.; Sevcik, J.; Psotova, J. ; Bednar, P.; Bartak P.; Adamovsky, P.; Simanek, V. Electrophoresis 2001, 22, 2782-2785. (68) Taylor, R. B.; Reid, R. G.; Low, A. S. J. Chromatogr. A 2001, 916, 201-206. (69) Quirino, J. P.; Kim, J.-B.; Terabe, S. J. Chromatogr. A 2002, 965, 357-373. (70) Martin, A.; Everaerts, F. Proc. R. Soc. London. A 1970, 316, 493-514.

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65 (71) F. Foret, S. F., A. Nardi, P. Bocek; Szoko, E.; Karger, B. L. J. Chromatogr. A 1992, 608, 3-12. (72) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem. 1993, 65, 900906. (73) Tu, C.; Lee, H. J. Chromatogr. A 2002, 966, 205-212. (74) Church, M. N.; Grant, P. M.; Andresen, B. D. Anal. Chem. 1998, 70, 2475-2480. (75) Yokota, K.; Fukushi, K. ; Takeda, S.; Wakida, S.-I. J. Chromatogr. A 2004, 1035, 145-150. (76) Toussaint, B.; Hubert, P.; Tjaden, U. R.; Greef, J. V. d.; Crommen, J. J. Chromatogr. A 2000, 871, 173-180. (77) Shihabi, Z. K. Electrophoresis 2002, 23, 1612-1617. (78) Shihabi, Z. K.; M.Friedberg J. Chromatogr. A 1998, 807, 129-133. (79) Pawliszyn, J. Sampling and sample preparation for field and laboratory; Elsevier Science, Amsterdam, The Netherlands, 2002. (80) Viberg, P.; Nilsson, S.; Skog, K. Anal. Bioanal. Chem. 2004, 378, 1729-1734. (81) Schellen, A.; Ooms, B.; Gils, M. V.; Halm ingh, O.; Vlis, E. V. d.; Lagemaat, D. V. d.; Verheij, E. Rapid Commun. Mass Spectrom. 2000, 14, 230-233. (82) Petersson, M.; Wahlund, K.-G.; Nilsson, S. J. Chromatogr. A 1999, 841, 249-261. (83) Steiner, F.; Hassel, M. J. Chromatogr. A 2005, 1068, 131-142. (84) Yang, Q.; Tomlinson, A. J.; Naylor, S. Anal. Chem. 1999, 71, 183A-189A. (85) Rohde, E.; Tomlinson, A. J.; Johnson, D. H.; Naylor, S. Electrophoresis 1998, 19, 2361-2370. (86) Visser, N. F. C.; Hamelen, M. v.; LIngeman, H.; Irth, H. J. Pharm. Biomed. Anal. 2003, 33, 451-462. (87) Belardi, R. P.; Pawliszyn, J. Water Pollut. Res. J. Can. 1989, 24, 179-186. (88) Bigham, S.; Medlar, J.; Kabir, A.; Shende, C.; Alli, A.; Malik, A. Anal. Chem. 2002, 74, 752-761. (89) Alhooshani, K.; Kim, T.-Y.; Kabir, A.; Malik, A. J. Chromatogr. A 2004, 1062, 1-14. (90) Kabir, A.; Hamlet, C.; Malik, A. J. Chromatogr. A 2004, 1047, 1-13.

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66 (91) Kabir, A.; Hamlet, C.; Yoo, K. S.; Newkome, G. R.; Malik, A. J. Chromatogr. A 2004, 1034, 1-11. (92) Arrebola, F. J.; Gonzalez-Rodriguez, M. J.; Garrido Frenich, A.; Marin-Juan, A.; Martinez Vidal, J. L. Anal. Chim. Acta 2005, 552, 60-66. (93) Yu, H.; Xu, L.; Wang, P. J. Chromatogr. B 2005, 826, 69-74. (94) Chen, J.; Pawliszyn, J. Anal. Chem. 1995, 67, 2530-2533. (95) Chan, I. O. M.; Lam, P. K. S.; Cheung, R. H. Y.; Lam, M. H. W.; Wu, R. S. S. Analyst 2005, 130, 1524-1529. (96) Kim, T.-Y.; Alhooshani, K.; Kabir, A.; Fries, D. P.; Malik, A. J. Chromatogr. A 2004, 1047, 165-174. (97) Whang, C.-W.; Pawliszyn, J. Anal. Commun. 1998, 35, 353-356. (98) Nguyen, A.-L.; Luong, J. H. T. Anal. Chem. 1997, 69, 1726-1731. (99) Li, S.; Weber, S. G. Anal. Chem. 1997, 69, 1217-1222. (100) Hernandez-Borges, J.; Cifuentes, A. ; Garcia-Montelongo, F. J.; RodriguezDelgado, M. A. Electrophoresis 2005, 26, 980-989. (101) Jinno, K.; Kawazoe, M.; Saito, Y.; Takeichi, T.; Hayashida, M. Electrophoresis 2001, 22, 3785-3790. (102) Halvorsen, T. G.; Pedersen -Bjergaard, S.; Rasmusse, K. E. J. Chromatogr. A 2001, 909, 87-93. (103) Pedersen-Bjergaard, S.; Rasmussen, K. E. Anal. Chem. 1999, 71, 2650-2656. (104) Pedersen-Bjergaard, S.; Rasmussen, K. E. Electrophoresis 2000, 21, 579-585. (105) Halvorsen, T. G.; Pedersen-Bjergaard, S.; Reubsaet, J. L. E.; Rasmussen, K. E. J. Sep. Sci. 2001, 24, 615-622. (106) Andersen, S.; Halvorsen, T. G.; Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr. A 2002, 963, 303-312. (107) Pedersen-Bjergaard, S.; Ho T. S.; Rasmussen, K. E. J. Sep. Sci. 2002, 25, 141146. (108) Ho, T. S.; Pedersen-Bjerg aard, S.; Rasmussen, K. E. J. Chromatogr. A 2002, 963, 3-17. (109) Pedersen-Bjergaard, S.; Rasmu ssen, K. E.; Halvorsen, T. G. J. Chromatogr. A 2000, 902, 91-105.

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67 (110) Zhu, L. Y.; Tu, C. H.; Lee, H. K. Anal. Chem. 2001, 73, 5655-5660. (111) Choi, K.; Kim, Y.; Chung, D. S. Anal. Chem. 2004, 76, 855-858. (112) Fonge, H.; Kaale, E.; Govaerts, C.; De smet, K.; Schepdael, A. V.; Hoogmartens, J. J. Chromatogr. B 2004, 810, 313-318. (113) Veraart, J. R.; Gooijer, C.; Lingeman, H.; Velthorst, N. H.; Brinkman, U. A. T. J. High Resol. Chromatogr. 1999, 22, 183-187. (114) Veraart, J. R.; Hekezen, J. V.; Groo t, M. C. E.; Gooijer, C.; Lingeman, H.; Velthorst, N. H.; Brinkman, U. A. T. Electrophoresis 1998, 19, 2944-2949. (115) Veraart, J. R.; Groot, M. C. E.; G ooijer, C.; Lingeman, H.; Velthorst, N. H.; Brinkman, U. A. T. Analyst 1999, 124, 115-118. (116) Veraart, J. R.; Gooijer, C.; Lingeman, H.; Velthorst, H. H.; Brinkman, U. A. T. J. Pharm. Biomed. Anal. 1998, 17, 1161-1166. (117) Strausbauch, M. A.; Landers, J. P.; Wettstein, P. J. Anal. Chem. 1996, 68, 306314. (118) Strausbauch, M. A.; Madden, B. J. ; Wettstein, P. J.; Landers, J. P. Electrophoresis 1995, 16, 541-548. (119) Bonneil, E.; Waldron, K. C. J. Chromatogr. B 1999, 736, 273-287. (120) Martinez, D.; Cugat, M. J.; Borrull, F.; Calull, M. J. Chromatogr. A 2000, 902, 65-89. (121) Mardones, C.; Rios, A.; Valcarcel, M. Electrophoresis 2001, 22, 484-490. (122) Rao, L. V.; Petersen, J. R.; Bissell, M. G.; Okorodudu, A. O.; Mohammad, A. A. J. Chromatogr. B 1999, 730, 123-128. (123) Hernandez-Borges, J.; Rodriguez-Delg ado, M. A.; Garcia-Montelongo, F. J.; Cifuentes, A. Electrophoresis 2004, 25, 2065-2076. (124) Rodriguez, R.; Manes, J.; Pico, Y. Anal. Chem. 2003, 75, 452-459. (125) Halasz, A.; Groom, C.; Zhou, E.; Pa quet, L.; Beaulieu, C.; Deschamps, S.; Corriveau, A.; Thiboutot, S.; Ampleman, G.; Dubois, C.; Hawari, J. J. Chromatogr. A 2002, 963, 411-418. (126) Olgemoller, J.; Hempel, G.; Boos, J.; Blaschke, G. J. Chromatogr. B 1999, 726, 261-268. (127) Prochzkov, A.; Chouki, M. ; Theruillat, R.; Thormann, W. Electrophoresis 2000, 21, 729-736.

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68 (128) Heinig, K.; Henion, J. J. Chromatogr. B 1999, 735, 171-188. (129) Andersen, S.; Halvorsen, T. G.; Pe dersen-Bjergaard, S.; Rasmussen, K. E.; Tanum, L.; Refsum, H. J. Pharm. Biomed. Anal. 2003, 33, 263-273. (130) Corradi, M.; Folesani, G.; Andreoli, R.; Manini, P.; Bodini, A.; Piacentini, G.; Carraro, S.; Zanconato, S.; Baraldi, E. Am. J. Respir. Crit. Care Med. 2003, 167, 395-399. (131) Guzman, N. A.; Park, S. S.; Schaufelbe rger, D.; Hernandez, L.; Paez, X.; Rada, P.; Tomlinson, A. J.; Naylor, S. J. Chromatogr. B 1997, 697, 37-66. (132) Heegaard, N. H. H.; Nilsson, S.; Guzman, N. A. J. Chromatogr. B 1998, 715, 2954. (133) Ensing, K.; Paulus, A. J. Pharm. Biomed. Anal. 1996, 14, 305-315. (134) Stroink, T.; Paarlberg, E.; Waterval, J. C. M.; Bult, A.; Underberg, W. J. M. Electrophoresis 2001, 22, 2374-2383. (135) Cole, L. J.; Kennedy, R. T. Electrophoresis 1995, 16, 549-556. (136) Guzman, N. A. J. Chromatogr. B 2000, 749, 197-213. (137) Guzman, N. A. Electrophoresis 2003, 24, 3718-3727. (138) Dalluge, J. J.; Sander, L. C. Anal. Chem. 1998, 70, 5339-5343. (139) Cram, D. J. Science 1988, 240, 760-767. (140) Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645647. (141) Shi, H.; Tsai, W.-B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593-597. (142) Kempe, M. Anal. Chem. 1996, 68, 1948-1953. (143) Haginaka, J.; Takehira, H.; Hosoya, K.; Tanaka, N. J. Chromatogr. A 1999, 849, 331-339. (144) Boer, T. d.; Mol, R.; Zeeuw, R. A. d.; Jong, G. J. d.; Sherrington, D. C.; Cormack, P. A. G.; Ensing, K. Electrophoresis 2002, 23, 1296-1300. (145) Nilsson, K.; Lindell, J. ; Norrlw, O.; Sellergren, B. J. Chromatogr. A 1994, 680, 57-61. (146) Schweitz, L.; Andersson, L. I.; Nilsson, S. Anal. Chem. 1997, 69, 1179-1183.

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69 (147) Lin, J.-M.; Nakagama, T.; X-Z.Wu; Uchiyama, K.; Hobo, T. Fresenius J. Anal. Chem. 1997, 357, 130-132. (148) Bjarnason, B.; Chimuka, L.; Ramstrm, O. Anal. Chem. 1999, 71, 2152-2156. (149) Ferrer, I.; Barcel, D. Trends Anal. Chem. 1999, 18, 180-192. (150) Haupt, K.; Dzgoev, A.; Mosbach, K. Anal. Chem. 1998, 70, 628-631. (151) Andersson, L. I. J. Chromatogr. B 2000, 745, 3-13.

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70CHAPTER THREE SOL-GEL TECHNOLOGY AND ITS APPL ICATION IN CAPILLARY ELECTROPHORESIS 3.1 Introduction The sol-gel process1, 2 generally involves the transiti on of a system from a liquid "sol" phase, into a solid "gel" phase. Sol-gel process provides a versatile approach to the synthesis of inorganic polymers and organic-inorganic hybrid materials 1. The advantageous features of sol-gel technolo gy include (1) mild reaction conditions, (2) possibility to create products of various shap es, sizes, and formats (e.g., monoliths, films, fibers, and monosized particles), (3) ease in the design and fine-tuning of material structure and property through proper selection of sol-gel ingredients. Sol-gel technology’s appearance can be traced back to mid 1800s 1. In early 1960s, contemporary sol-gel processing emerged as a result of specialized requirements for ceramic nuclear fuels 3. Due to its numerous inherent advantages, sol-gel technique has found ever increasing application in a diverse range of fields, such as ceramic industry 4-6, nuclear-fuel industry 7, electronics 8, 9, and chemistry 10, 11 etc.. The application of sol-gel technology for the creation of chromatographic stationary phase started only two decades ago. In 1987, Cortes et al. 12 reported the use of sol-gel technology to create monolithic ceramic beds within small-diameter capillaries and applied such capillaries as separation columns in liquid chromatography (LC). Crego et al. 13developed a method to prepare a sol-gel stationary phase in situ for open tubular liquid chromatography. Using a similar method, Guo and Colon 14, 15 prepared sol-gel based open tubular columns for CEC. The sol-gel technology provided an effective means to chemically bind chromatographic stationary phases to the capillary inner surface and brought new promise to produce stationary phases with high stability and column efficiency in liquid-phase separations. These pioneering works stimulated further developments in the area of sol-gel stationary phases in chromatographic 16-22 and electrophoretic separation 23-29 and sample preparation technologies 30-36.

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713.2 Fundamentals of sol-gel process Figure 3.1 illustrates a typical sol-gel process. Generally, sol-gel process consists of hydrolysis and polycondensation of metal alkoxide precursors. During the process, a liquid like colloidal suspension is formed, which is known as the sol 37. The sol is then transformed into an interconnected network wi th submicrometer size pores and polymeric chains greater than a micrometer, which is called a gel 38. When the liquid is extracted from the wet gel under supercritical conditions, a low density aerogel is produced. If the liquid is removed by thermal evaporation, a material termed xerogel is generated 2. Under elevated temperatures (above 1000 C), the number of pores inside the xerogel is reduced significantly and the density of the material increased substantially. As a result, the porous gel is transformed into a dense ceramic material. In order to properly control the whole sol-gel process and fine-tune the properties of the target product, it is important to understand the chemical reactions involved in the sol-gel process. A typical sol solution ge nerally contains the following chemical components: (1) at least one sol-gel precursor, which is usually a metal alkoxide M(OR)x 39; (2) a solvent to disperse the precursor(s) and a catalyst, which can be an acid 1, 40, 41, a base 42 or a fluoride 43, 44 based on the type of desired end products; and (3) water. Precursors that commonly used in sol-gel pr ocess include silica-based and non-silica-base alkoxides (e.g., metal alkoxides) 39. Many metal elements, such as titanium, aluminum, vanadium, zirconium, and germanium, can be used to prepare metal alkoxides. However, silica based alkoxides are the most widely used precursors due to their well known chemistry, pH stability of Si-C bond, well documented sol-gel methodology, facility of characterizations and commercially available starting materials 45. Table 3.1 lists some common silica based sol-gel precursors. The sol-gel process is a relatively straightforward procedure for the preparation of inorganic or organic-inorganic hybrid materials through hydrolysis of the precursor(s) and alcoholor water condensation of the solgel-active species presen t in the sol solution. The sol-gel-active species may include the alkoxysilane-based precursors as well as any other chemical species reactive to alkoxysilane, silanol and analogous silica species. A polycondensation occurs with the linkage of additional Si-OH tetrahedral to the condensation products, and eventually materials with three-dimensional network

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72 Figure 3.1 Overview of a sol-gel process. Reproduced from Reference 1.

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73 Table 3.1 Common tetraalkoxysilane prec ursors and their physical properties* Name MW bp ( C) nD (20 C) d (20 C) (ctsks) Dipole Moment Solubility Si MeO MeO OMe OMe Si(OCH3)4 Tetramethoxysilane 152.2 121 1.3688 1.02 5.46 1.71 Alcohols Si EtO EtO OEt OEt Si(OC2H5)4 Tetraethoxysilane 208.3 169 1.3838 0.93 1.63 Alcohols Si C3H7O C3H7O OC3H7 OC3H7 Si(OC3H7)4 Tetra-n-propoxysilane 264.4 224 1.401 0.916 1.66 1.48 Alcohols Si C4H9O C4H9O OC4H9 OC4H9 Si(OC4H9)4 Tetra-n-butoxysilane 320.5 115 1.4126 0.899 2.00 1.61 Alcohols Tetrakis(2methoxyethoxy)silane 328.4 179 1.4219 1.079 4.9 Alcohols *Reproduced from Reference 1

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74 structures are produced2. Scheme 3.1 illustrates hydrolysis, condensation and polycondensation of tetramethoxysilane (TMOS) as an example. The catalysts used in sol gel process play an important role. They not only change the reaction speed, the type of the catalyst also affects the structure of the resulting sol gel materials. A generally accepted notion is that acid-catalyzed sol-gel processes are more likely to produce linear polymers because under acidic condition, the hydrolysis of alkoxide precursors undergo faster than the condensation process 46. The mechanism of hydrolysis reaction under acidic conditions involves protonation of the alkoxide group, which is followed by nucleophilic attack by wa ter to form a pentacoordinate intermediate 1, 47. On the other hand, under basic condition, condensation reaction is faster and the rate of the overall sol-gel process is determined by the relatively slow hydrolysis step. In this case, highly condensed particulate structure is more likely to be generated 48. The hydrolysis reaction under basic condition is believed to start with the nucleophilic attack on the silicon atom by the hydroxide anion and form a penta-coordinated intermediate. This step is followed by the substitution of a alkoxide group by a hydroxyl group 1, 47, 49, 50. This is an important feature which enables researchers to manipulate experimental conditions to fine-tune the formation of the end products with desired characteristics. Scheme 3.2 illustrates both acid-catalyzed sol-gel reaction and base-catalyzed sol-gel reaction mechanisms. In addition to the use of catalysts, sol-gel processes can also be initiated by irradiation. It has been reported by Zare and co-workers that the precursor methacryloxypropyltrimethoxysilane is initiated by the application of UV light at 350 nm wavelength 29, 51, 52. Solvents in the sol soluti on play an important role in the formation of a homogeneous sol solution and progression of the gelation procedure1. Artaki et al. systematically investigated the solvent effects on the condensation reaction of the sol-gel process 53. With the aids of Raman spectroscopy, molybdenum chemical reaction, and electron microscopy, the authors concluded that the mechanism of particle aggregation as well as the extent of condensation of the polymeric network is dramatically affected by the presence of organic additives, such as fomamide, dimethyl formamide, acetonitrile and dioxane due their influence on hydrogen bonding and electrostatic interactions,

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75 hy d rolysisSiOCH3 OCH3 H3CO OCH3 4H2O SiOH OH HO OH 4CH3OH SiOCH3 OCH3 H3CO OCH3 SiOCH3 OCH3 HO OCH3 alcohol condensationSiO OCH3 H3CO OCH3 SiOCH3 OCH3 OCH3 CH3OH SiOH OCH3 H3CO OCH3 SiOCH3 OCH3 HO OCH3 water condensationSiO OCH3 H3CO OCH3 SiOCH3 OCH3 OCH3 H2O SiO OH HO OH SiOH OH OH water polycondensation6Si(OH)4SiO O O O SiO O O SiOH HO OH SiOH HO OH SiOH O H HO SiOH O H HO SiOH OH OH Si OH HO OH 6H2O Scheme 3.1 Sol-gel reaction 48

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76 Acid-catalyzed sol-gel reaction mechanism. a. Hydrolysis mechanism SiOR RO RO O R H+Si H O RO RO OR R Si H O RO RO O R HR Si RO RO OR O H H3O+O H H Si RO RO OR O HOR H2OH H H2O b. Condensation mechanism R-Si(OH)3 H+ k 1k-1R-Si(OH)2O H H R-Si(OH)2O H H R-Si(OH)3 k2k-2Si OH OH R O Si OH O H R H3O+ A. Base-catalyzed sol-gel reaction mechanisms a. Hydrolysis mechanism Si OR R O RO RO HOSi O R RO OR HO OR Si OR O R HO OR ROb. Condensation mechanism R-Si(OH)3 OHk 1k-1R-Si(OH)2OH2O R-Si(OH)2OR-Si(OH)3 k2k-2R-Si(OH)2-O-Si(OH)2R OHScheme 3.2 Acid-catalyzed and base catalyzed sol-gel reaction mechanisms

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77 which modulate the nucleophilic substitution mechanism associated with the sol-gel process. In addition to the catalyst type, several other factors, such as water-to-silane ratio, nature of solvent system and the alkoxide precursors, can affect the physical properties of the obtained sol-gel materials 47. In order to get a fine-tuned porosity of the monoliths, the amount of water in sol system should be carefully controlled. Constantin and Freitag reported that there is an optimum content of water (approximately 200%) that facilitates the formation of uniform porous monoliths 54. These authors observed no significant microstructure developments (pores) in the monoliths when the content of water was much less than 200%. On the other hand, wi th water content larger than 300%, sol-gel beads with broad distribution and blocks of non-macrophorous structures were formed. Gelation is followed by the drying step. Du ring this step, the liquid is removed from the interconnected pore network either through extraction to produce an aerogel, ambient conditions to evaporate liquid and generate a xerogel, or extreme thermal condition to obtain ceramic material 2 (Figure 3.1). 3.3 Characterization of sol-gel materials With the development of new analytical and computational techniques for investigation on a nanometer scale, it is poss ible to get more information associated with sol-gel process (e.g., hydrolysis, polycondensa tion, and drying), which enable researchers to have a deeper insight into the fundamental aspects of sol-gel chemistry providing increased scientific understanding. Various techniques have been used to investigate the sol-gel process as well as the properties of the organic-inorganic hybrid materials created though sol-gel process. These techniques in clude nuclear magnetic resonance (NMR), Xray small-angle scattering (XSAS), Rama n spectroscopy, X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), dielectric relaxation spectroscopy (DRS), etc. 3.3.1. The morphology of sol-gel materials To study the surface characteristics and fine structural details of the sol-gel materials, scanning electron microscopy (SEM ) is a powerful tool. With SEM, sample

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78 surface is scanned by a fine electron incident beam which produces an image with great depth of field and an almost three-dimensional appearance. With this feature, SEM is the most widely used technique to evaluate the morphology of sol-gel materials 24, 25, 28, 29, 51, 52, 54-61. Hernandez-Padron et al. 62 reported an SEM morphological study and transparency properties of hybrid SiO2-phenolic resin materials. In the case of sol-gel surface-coated open tubular columns, SEM ca n reveal the uniformity of sol-gel coating thickness and structural defects therein. It is also used to study the effects of various experimental parameters on the properties of produced sol-gel materials 52. In addition to SEM, atomic force microscopy (AFM) 63, X-ray absorption 64 and transmission electron microscopy 65, 66 are also used to investigate the morphology of sol-gel materials. For example, Almeida et al. 64 used extended x-ray absorption to study fine structure and near-edge structure of sili c-titania sol-gel film. Yan et al. 66 used TEM to characterize the magnesium silicate thin films obtained through sol-gel technique. 3.3.2. Study of the chemical bonds within sol-gel structure Techniques like SEM, AFM, etc. provide the picture of sol-gel materials depicting heterogeneous or homogeneous morphologies, while spectroscopic results confirm the existence of chemical bonds within sol-gel structure. To study the chemical bonds in solgel structure, various spectroscopic techniques including Fourier transform infrared spectroscopy (FTIR) 66-68, fluorescence spectroscopy 67 and nuclear magnetic resonance (NMR) 69-72 have been used. Since spectra can be scanned, recorded, and transformed in an extremely rapid pace, FTIR enables the study of sol-gel process in its progression with time. Toyo’oka and co-workers 73 monitored the content of residual silanol groups in sol-gel material as the gelation process progre ssed using attenuated total reflectance (ATR) FTIR hybrid technique. The encapsulation of bovine serum albumin 74 in the sol-gel matrix was confirmed by FTIR by Zuo et al.63. IR was used to characterize capillaries modified with macrocylic dioxopolyamine 75. The typical IR absorptions of NH, C=O, and CH obtained provided the evidence of successful surface modification in capillaries for open-tubular capillary electrochromatography.

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79 The esterification reaction between stearic acid and the epoxy groups of glycidoxypropyltrimethoxysilane was investigated by X-ray photoelectron spectroscopy (XPS) by Zhao and coworkers76. The XPS results provided evidence on the existence of carbon in the reaction product indicating the success in the octadecyl silylation reaction. Another powerful analytical technique, nuclear magnetic resonance (NMR) was used by Rodriguez and Colon 72 to investigate the species present in the sol-gel solution used to modify the inner surface of an open tubular CEC column. It is established that in a sol-gel solution containing more than one precursor, a homogenous hybrid system is usually formed if the monomeric precursors und ergo hydrolysis reactions at similar rates. However, if one of the precursors has much faster rate for the hydrolysis reaction leading to pronounced self-condensation 77, a heterogeneous composition will be produced. Since the properties of the final sol-gel columns ca n be indicated by the species present in the sol-gel solutions prior to the coating proces s, it is very important to understand the characteristics of the sol-gel solution in details. 3.4 General procedures involved in the preparation of CE columns with sol-gel stationary phases Several steps are involved in the prep aration of CE columns with sol-gel stationary phase. The preparation procedures vary depending on the types of the columns and the intended applications. They include pr etreatment of the capillary, fabrication of the sol-gel stationary phases, and the post-gelation treatment of the CE stationary phases. 3.4.1 Pretreatment of the capillary The purpose of capillary pretreatment is to increase the concentration of surface silanol groups. Since sinanol groups on the capillary surface represent the principal binding sites for in situ created sol-gel stationary phases, higher concentration of these binding sties on the capillary surface would facilitate the formation of highly secured solgel stationary phases through chemical bonding with the capillary inner walls. Alkali solutions are used to clean the capillary surface in addition of some organic solvents 14, 15. In the reported one-step synthesis of monolit hic silica column by Constantin and Freitag 54, the pretreatment of the bare fused silic a was accomplished by flushing with NaOH, then, with HCl, and followed by rinsing with purified water. The similar pretreatment

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80 method was used by other research ers to prepare sol-gel open-tubular 76 and monolithic columns 55-57. Hayes and Malik 24, 25 reported the use of hydrothermal treatment of the inner surface of the fused sili ca capillary for the preparation of both sol-gel monolithic and sol-gel open tubular columns. The purpose of hydrothermal pretreatment was explained as cleaning the capillary inner surface and increasing the surface concentration of silanol groups to effectively anchor the in situ created sol-gel stationary phases, and it is also being used by other research groups78, 79. 3.4.2 Sol solution ingredients for the fabrication of the sol-gel stationary phases In addition to the typical components in the sol solution (e.g., precursor(s), a solvent system, a catalyst and water), the actual operations for creating sol-gel stationary phases often involve the use of various additives to provide the desired end products. A porogen is often used in the sol solution, especially in creating a porous monolithic bed. Toluene was found to be a suitable porogen for photopolymerized sol-gel monolithis for CE 29, 52. Poly(ethylene oxide)(PEO) and polyethylene glycol 80 were also used as porogens by different researcher 58, 60, 81. Porogens generally play a dual role: they serve as a thorough-pore template and as a solubilizer of silane reagent. Deactivation reagents represent another important type of sol solution additives used to derivative residual silanol groups on the stationary phase, and thereby reduce harmful adsorptive effects of the latter on CE separation. Hayes and Malik reported the use of phenyldimethylsilane (PheDMS) as a deactivation reagent for both open-tubular and monolithic sol-gel columns 24, 25. The deactivation reagent reacts with the residual silanol groups on the stationary phase resulting in the reduction of chromatographically harmful adsorption sites on the stationary phase. The effect of the deactivation was evaluated by the comparing the column performance obtained on columns prepared with and without the addition of deactivation agent. 3.4.3 Post-gelation treatment of sol-gel stationary phases In order to minimize or eliminate the volume shrinkage during the fabrication of sol-gel stationary phase, especially for solgel monoliths, post-gelation treatment is needed. Various techniques have been developed to accomplish post-gelation treatment.

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81 In the case of open-tubular columns, organic solvents were used to flush the sol-gel stationary, followed by equilibrating the columns with running buffers 14, 15, 76. It was found by Zuo and co-workers 63 that aging under moist conditions at lower temperatures benefits the encapsulation of biological macromolecules. While an accelerated rate of aging may lead to cracks on the dried gel. 3.5 The application of so l-gel technology in CE Like all other chromatographic systems, in CE the column is a fundamentally important component. Column technology, therefore, can be regarded as the key to the success of CE. A careful review of the published papers devoted to the fabrication of CE columns over the last decade shows that sol-gel technology shows a promising direction in CE column technology. It is applicable to the preparation of CE columns in three different formats: open-tubular columns with sol-gel surface coatings, capillary columns packed with micro/submicro particles, and capillary columns with monolithic beds. 3.5.1 Sol-gel technology for pa cked columns in CE Sol-gel technique has been used in three distinct areas in packed columns for CE. They are: (1) preparation of micrometer and submicrometer size sol-gel particles used to pack the capillary, (2) creation of sol-gel frit in packed columns, and (3) preparation of sol-gel entrapped chromatographic packing material. Retaining end frits are commonly used in CE packed columns to keep the particulate packing material inside the capillary. Traditionally, on-column frits are produced by sintering of silica based packing materials by heating a short segment of the packed bed with a flame or applying lowvoltage resistive heating for a short period of time. Consequently, the particles of the pa cking material in this segment become connected with neighboring particles and the cap illary wall at their contact points to form a permeable barrier and retain the stationary phase. These methods to produce on-column frits put a high thermal stress on the protec tive polyimide coating of the fused silica capillary and may lead to fragility, variable permeability, and destruction of the chemical bonds in the frit region 82-85. Since sol-gel process can occur under mild conditions, it is a good alternative method to prepare frits for packed columns 22, 86, 87. The frits prepared

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82 by sol-gel technology have been proved to possess good mechanical strength, and high pH and solvent stabilities 88. Piraino and Dorsey studied the performance of several types of frits, including sintered frits, photopolymerized frits, and frits made by sol-gel process. Their research results suggested that capillaries with sol-gel frits showed the greatest electroosmotic mobility 89. It is believed that the use of frits is associated with a number of problems, such as bubble formation during CE operation 90, column fragility 91, variable permeability and related shortcomings 84, 85. To avoid the use of frits, sol-gel technology has been used to entrap the bonded stationary particles inside the capillary 86. The methodology involved the preparation of a sol-gel solution cont aining tetraethoxysil ane, ethanol, and hydrochloric acid followed by addition of packing particles to create a suspension. This suspension containing packing particles was th en introduced into the capillary to generate a packed column. Another approach to fabricate packed columns with sol-gel process was developed by Tang et al. 92. Based on their method, the capillary was first packed with the chromatographic particles prior to th e application of sol so lution. A sol solution was then introduced into the particle packed cap illary. After the conversion of the sol to a gel, the sol-gel bonded packed colu mn was dried using supercritical CO2. 3.5.2 Sol-gel open tubul ar CE columns In open tubular columns, the stationary phase is bonded to or spread as a coating on the inner surface of the capillary, whic h constitute an important category of CE columns. They represent an alternative to packed columns and free from the problems caused by the use of frits in traditional packed columns. In order to provide sufficient retentive properties and sample capacity, open tubular columns should have thick stationary phase coatings. However, with conventional fabrication methods, it is usually difficult to achieve thick and stable coatings. Research works devoted to solving these problems include four main directions 93. They are (1) using thick, immobilized organic polymer coatings to improve the column phase ratio, (2) creating etched inner surface of the capillary with bonded organic ligands to provide higher surface area and enhanced solute/stationary phase interactions, (3) us ing dynamic nanoparticles as pseudostationary phase, and (4) coating the capillary with sol-gel technology. Based on experimental

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83 results, researchers 14, 15 found that columns prepared by sol-gel technology had enhanced surface area, improved hydrolytic stability, and enhanced retentive characteristics. Compared with traditional methods, sol-gel approach is much simpler. Using sol-gel precursors containing an alkyl chain (such as C6, C8, C16, and C18), sol-gel technology allowed the preparation of stationary phase coatings within fused silica capillaries, where these chemically bonded alkyl chains served as retentive moieties for the reversed-phase separation of uncharged polycyclic aromatic hydrocarbons, aromatic ketones and alcohols 14, 15, 24, 94, 95. Wu et al. 96 developed a reversed-phase open-tubular column coated with a solgel stationary phase containing an amine moiety. An enhanced EOF in an acidic buffer and reduced adsorption of peptides on the capillary wall were achieved. These columns provided fast separation fro peptide samples: six peptides were baseline resolved within 3 min. Hayes and Malik 24 developed a positively charged sol-gel ODS stationary phase for open-tubular column providing reversed electroosmotic flow. The key precursor used by was (N-octadecldimethyl[3-(trimethoxy silyl)propyl]ammonium chloride). The methoxysilyl groups enabled the creation of sol-gel network structure and attachedment of the created sol-gel stationary phase onto the capillary wall. The presence of the octadecyl group reinforced the chromatographic interactions between organic analytes and the sol-gel stationary phase. The quaternary amine group chemically incorporated in the stationary phase structure provided a positively charged capillary surface which is in contrast with the electrical properties of bare fused silica capillary surface that carries a negative charge. Constantin and Freitag 94 introduced ion exchange groups into open-tubular column by sol-gel process. The ion exchange groups were the results from the addition of (pentafluorophenyl)dimethylchlorosilane into sol solution. The obtained open-tubular column was successfully used for the separation of amino acids. The use of fluorinated stationary phase for the separation of fluorinated organic compounds and halogenated organic compounds was demonstrated by Narang and Colon 97. The open-tubular column bearing sol-gel-deived fluorinated stationary phase were

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84 prepared by using a sol solution containi ng tridecafluoro-1,1, 2,2-tetrahydrooctyltriethoxysilane (F13-TEOS), TEOS, ethanol, hydrochloric acid and water. Wang and Zeng 75, 98 used 3-(2-cyclooxypropoxyl)propyl-tri-methoxy silane as a bridge to connect 1,4,7,10-tetraazacyclotridecane-11,13-dione and 2,6-dibutyl-cyclodextrin to TEOS. By doing this, macrocyclic polyamine derived-and -cyclodextrin derived stationary phases were attached onto the sol-gel matrix. The obtained opentubular columns were used to separate isom eric nitrophenols and benzenediols, isomeric aminophenols, diaminobenzenes, dihydroxybenzenes, and biogenic monoamine neurotransmitters. 3.5.3 Sol-gel monolithic columns In chromatographic science, monolithic columns are referred to as continuous bed columns, fritless columns or rod columns 99. Compared to the tradit ional packed columns, monolithic columns have many advantages such as ease in construction, higher surface area and porosity, improved mass transfer, absence of retaining end frits, and elimination or significant reduction of certain operation problems inherent in packed columns due to the presence of the retaining end frits 12. Compared to open-tubular columns, the solute molecules do not have to diffuse a long distance through the liquid mobile phase to reach the stationary phase in monolithic columns. Preparation of monolithic columns using so l-gel technology provides a variety of important advantages over other methods. Cortes et al. 12 reported the pioneering research work using sol-gel technology to create silica-based monolithic beds inside fused capillary in 1987. Fujimoto pub lished a detailed procedure fo r the preparation of sol-gel monolithic columns for CE 99. First, the capillary was filled with the sol solution for 20 hours at 40 C. The formed material was then washed with water and treated with ammonium hydroxide solution for 24 hours at 40 C. After thermal conditioning, the chromatographic moiety C18 was bonded to the sol-gel ma trix with a 10% solution of dimethylocadecylchlorosilane. Tanaka and co-workers 60, 100 also fabricated sol-gel monolithic columns suibable for both HPLC and CE. These researchers used a two-step procedure: (1)a sol-gel silica monolithic bed was created inside the capillary (2) a surface-derivatization reaction was

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85 used to chemically bind the desired chromatographic ligand to the surface of the porous silica monolith. The sol solution they used included TMSO as the precursor, PEO as the porogenic agent and acetic acid as the catalyst. Hayes and Malik 25 developed a method to prepare sol-gel monolithic column for CEC in a single-step procedure. These researchers used octadecyl group as the organic component of the stationary phase facilitating the solute/stationary interactions during CE separation. In this method, al l the process including the form ation of sol-gel monolithic matrix, the introduction of the organic moiety C18 and the deactivation of the unreacted silanol groups were accomplished in one step, which provided a much simpler and faster method for the preparation of so l-gel ODS monolithic CE column. 3.6 Sol-gel technol ogy for sample preconcentration in CE The organic-inorganic hybrid materials synthesized by sol-gel technology have been explored applications in sample preparation, such as sample preconcentration. Malik and co-workers introduced sol-gel coated fibers and capillaries for solid-phase microextraction (SPME) and capillary mi croextraction (CME or in-tube SPME) 30, 34, 35. The sol-gel coated capillary was also used for the sample preconcentration in high performance liquid chromatography (HPLC) 33 and CE 101, 102. Quirino and co-workers 102, 103 developed a photopolymerized sol-gel (PSG) material for sample preconcentration in CE. The porous PSG monolith has a high masstransfer rate, which promotes preconcentration of dilute samples. In this method, (methacryloxypropyl)trimethoxysilane was used as the precursor, toluene as the porogenic agent. The photopolymerization reaction was initiated by exposing the fused silica capillary filled with sol-gel solution to 365 nm light. The obtained sol-gel monolith acted as a solid-phase extractor as well as a separation stationary phase. The preconcentration effect was calculated in terms of peak heights, up to 100-fold increase for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the peptide mixture when UV detection was used. Oguri and Toyo’koa et al. 101 developed a method of on-line preconcentration prior to on-column derivatization CEC. A sol-gel octadecasiloxane capillary column was created using thermal sol-gel reaction of tetr aethyl orthosilicate to capture ODS particles

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86 in a piece of capillary. A standard biogenic amine solution consisting of histamine, methylhistamine, and serotonin were effectively concentrated at the inlet site of the capillary column by filed-amplified sample stacking, a gradient effect, or both. An increased sensitivity by a factor of 1000-fold greater than that of normal on-column derivatization CEC was obtained when a fluorescence detector was equipped. 3.7 References fo r Chapter Three (1) Brinker, C.; Scherer, G. Sol-Gel Science; Academic Press: Boston, MA, 1990. (2) Hench, L. L.; West, J. K. Chem. Rev. 1990, 90, 33-72. (3) Klein, L. C.; Pope, E. J. A.; Sakka, S.; Woolfrey, J. L. Sol-gel processing of advanced materials; The American Chemical Society: Westerville, OH., 1998. (4) Wu, C.-S. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 1690-1701. (5) Zhu, Q.; Qiu, F.; Quan, Y.; Sun, Y.; Liu, S.; Zou, Z. Mater. Chem. Phys. 2005, 91, 338-342. (6) Nogami, M.; Nagasaka, K. J. Mater. Sci 1991, 26, 3665-3669. (7) Tel, H.; Eral, M.; Altas, Y. J. Nucl. Mater. 1998, 256, 18-24. (8) Villanueva-Ibanez, M.; Luyer, C. L. ; Parola, S.; Marty, O.; Mugnier, J. J. Sol-Gel Sci. Technol. 2004, 31, 277-281. (9) Bhattacharya, S. K.; Tummala, R. R. J. Mater. Sci.: Mater. Electron. 2000, 11, 253-268. (10) Schubert, U. New. J. Chem. 1994, 18, 1049-1058. (11) Marxer, S. M.; Robbins, M. E.; Schoenfisch, M. H. Analyst 2005, 130, 206-212. (12) Cortes, H. J.; Pfeiffer, C. D. ; Richter, B. E.; Stevens, T. S. J. High Resol. Chromatogr. Chromatogr. Commun. 1987, 10, 446-448. (13) Crego, A. L.; Diez-Masa, J. C.; Dabrio, M. V. Anal. Chem. 1993, 65, 1615-1621. (14) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511-2516. (15) Guo, Y.; Colon, L. A. J. Microcol. Sep. 1995, 7, 485-491. (16) Wang, D.; Chong, S. L.; Malik, A. Anal. Chem. 1997, 69, 4566-4576.

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87 (17) Cervini, R.; Day, G.; Hibberd, A.; Sharp, G. Chem. Aust. 2001, 68, 16-18. (18) Zeng, Z.; Qiu, W.; Xing, H.; Huang, Z. Anal. Sci. 2000, 16, 851-854. (19) Shende, C.; Kabir, A.; Townsend, E.; Malik, A. Anal. Chem. 2003, 75, 3186-3198. (20) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (21) Ishizuka, N.; Kobayashi, H.; Minakuchi, M. H.; Nakanishi, K.; Hirao, K.; Hosoya, K.; Ikegami, T.; Tanaka, N. J. Chromatogr. A 2002, 960, 85-96. (22) Channer, B.; Uhl, P. U.; Eueby, M. R.; McKeown, A. P.; Lomax, H.; Skellern, G. G.; Watson, D. G. Chromatographia 2003, 58, 135-143. (23) Hayes, J. D.; Malik, A. J. Chromatogr. B 1997, 695, 3-13. (24) Hayes, J. D.; Malik, A. Anal. Chem. 2001, 73, 987-996. (25) Hayes, J. D.; Malik, A. Anal. Chem. 2000, 72, 4090-4099. (26) Sakai-Kato, K.; Kato, M.; Toyo'oka, T. Anal. Chem. 2002, 74, 2943-2949. (27) Sakai-Kato, K.; Kato, M.; Toyo'oka, T. Anal. Biochem. 2002, 308, 278-284. (28) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Zare, R. N. J. Sep. Sci. 2002, 25, 3-9. (29) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Kato, M.; Zare, R. N. Anal. Chem. 2001, 73, 3921-3926. (30) Bigham, S.; Medlar, J.; Kabir, A.; Shende, C.; Alli, A.; Malik, A. Anal. Chem. 2002, 74, 752-761. (31) Kabir, A.; Hamlet, C.; Yoo, K. S.; Newkome, G. R.; Malik, A. J. Chromatogr. A 2004, 1034, 1-11. (32) Kabir, A.; Hamlet, C.; Malik, A. J. Chromatogr. A 2004, 1047, 1-13. (33) Kim, T.-Y.; Alhooshani, K.; Kabir, A.; Fries, D. P.; Malik, A. J. Chromatogr. A 2004, 1047, 165-174. (34) Alhooshani, K.; Kim, T.-Y.; Kabir, A.; Malik, A. J. Chromatogr. A 2004, 1062, 1-14. (35) Chong, S. L.; Wang, D.; Hayes, J. D.; Wilhite, B. W.; Malik, A. Anal. Chem. 1997, 69, 3889-3898. (36) Li, X.; Zeng, Z.; Gao, S.; Li, H. J. Chromatogr. A 2004, 1023, 12-25.

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88 (37) Davis, J. T.; Rideal, E. K. Interracial Phenomena; Academic Press: New York, 1961. (38) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY., 1953. (39) Mehrotra, R. C. J. Non-Cryst. Solids 1990, 121, 1-6. (40) Sharp, K. G. J. Sol-Gel Sci. Technol. 1994, 2, 35-41. (41) Sanchez, J.; Rankin, S. E.; McCormick, A. V. Ind. Eng. Chem. Res 1996, 35, 117129. (42) Yoda, S.; Ohshima, S.; Kamiya, K.; Kawai, A.; Uchida, K.; Gotoh, A.; Ikazaki, F. J. Non-Cryst. Solids 1996, 208, 191-198. (43) Pavan, F. A.; Gobbi, S. A.; Moro, C. C.; Costa, T. M. ; Benvenutti, E. V. J. Porous Mater. 2002, 9, 307-311. (44) He, L.; Powers, K.; Baney, R. H.; Gower, L.; Duran, R. S.; Sheth, P.; Carino, S. R. J. Non-Cryst. Solids 2001, 289, 97-105. (45) Ogoshi, T.; Chujo, Y. Compos. Interfaces 2005, 11, 539-566. (46) Tilgner, I. C.; Fischer, P.; Bohn en, F. M.; Rehage, H.; Maier, W. F. Microporous Mater. 1995, 5, 77-90. (47) Collinson, M. M. Crit. Rev. Anal. Chem. 1999, 29, 289-311. (48) Sarwar, M. I.; Ahmad, Z. Eur. Polym. J. 2000, 36, 89-94. (49) Corriu, R. J. P.; Leclercq, D. Angew. Chem. Int. Ed. Engl. 1996, 35, 1420-1436. (50) Buckley, A. M.; Greenblatt, M. J. Chem. Educ. 1994, 71, 599-602. (51) Kato, M.; Dulay, M. T.; Bennett, B. D.; Quirino, J. P.; Zare, R. N. J. Chromatogr. A 2001, 924, 187-195. (52) Kato, M.; Sakai-Kato, K.; Toyo'oka, T.; Dulay, M. T.; Quirino, J. P.; Zare, R. N. J. Chromatogr. A 2002, 961, 45-51. (53) Artaki, I.; Zerda, T. W.; Jonas, J. J. Non-Cryst. Solids 1985, 81, 381-395. (54) Constantin, S.; Freitag, R. J. Sol Gel. Sci. Technol. 2003, 28, 71-80. (55) Kang, J.; Wistub a, D.; Schurig, V. Electrophoresis 2002, 23, 1116-1120. (56) Chen, Z.; Hobo, T. Anal. Chem. 2001, 73, 3348-3357.

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89 (57) Chen, Z.; Hobo, T. Electrophoresis 2001, 22, 3339-3346. (58) Breadmore, M. C.; Shrinivasan, S.; Wolf e, K. A.; Power, M. E.; Ferrance, J. P.; Hosticka, B.; Norris, P. M.; Landers, J. P. Electrophoresis 2002, 23, 3487-3495. (59) Breadmore, M. C.; Shrinivasan, S.; Karlinsey, J.; Ferrance, J. P.; Norris, P. M.; Landers, J. P. Electrophoresis 2003, 24, 1261-1270. (60) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; SOga, N.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (61) Morishima, K.; Bennett, B. D.; Dula y, M. T.; Quirino, J. P.; Zare, R. N. J. Sep. Sci. 2002, 25, 1226-1230. (62) Hernandez-Padron, G.; Rojas, F.; Garcia-Garduno, M.; Peza, C. Emerging Fields in Sol-Gel Science and Technology; Kluwer Academic Publishers: Norwell, Mass, 2003. (63) Zuo, X.; Wang, K.; Zhou, L.; Huang, S. Electrophoresis 2003, 24, 3202-3206. (64) Almeida, R. M.; Marques, M. I. d. B.; Orignac, X. J. Sol Gel. Sci. Technol. 1997, 8, 293-297. (65) van. Hoydonck, P. G. A.; AWuyts, W. A.; Vanaudenaerde, B. M.; Shouten, E. G.; Dupont, L. J.; Temme, E. H. M. Eur. Respir. J. 2004, 23, 189-192. (66) Yan, Y.; Hoshino, Y.; Kuan, Z.; Chaudhuri, S. R. Chem. Mater. 1997, 9, 25832587. (67) Brennan, J. D.; Hartman, J. S.; Ilnicki, E. I.; Rakic, M. Chem. Mater. 1999, 11, 1853-1864. (68) Fidalgo, A.; Ilharco, L. M. J. Non-Cryst. Solids 2001, 283, 144-154. (69) Pursch, M.; Jaeger, A.; Schneller, T.; Brindle, R.; Albert, K.; Lindner, E. Chem. Mater. 1996, 8, 1245-1249. (70) Jones, S. A.; Wong, S.; Burlitch, J. M.; Viswanathan, S.; Kohlstedt, D. L. Chem. Mater. 1997, 9, 2567-2576. (71) Delattre, L.; Babonneau, F. Chem. Mater. 1997, 9, 2385-2394. (72) Rodriguez, S. A.; Colon, L. A. Appl. Spectrosc. 2001, 55, 472-480. (73) Sakai-Kato, K.; Kato, M.; Nakakuki, H.; Toyo'oka, T. J. Pharm. Biomed. Anal. 2003, 31, 299-309. (74) Halvorsen, T. G.; Pedersen-Bjergaard, S.; Reubsaet, J. L. E. ; Rasmussen, K. E. J. Sep. Sci. 2001, 24, 615-622.

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90 (75) Wang, Y.; Zeng, Z.; Xie, C.; Guan, N.; Fu, E.; Cheng, J. Chromatographia 2001, 54, 475-479. (76) Zhao, Y.; Zhao, R.; Shangguan, D.; Liu, G. Electrophoresis 2002, 23, 2990-2995. (77) Re, N. J. Non-Cryst. Solids 1992, 142, 1-17. (78) Allen, D.; El Rassi, Z. Analyst 2003, 128, 1249-1256. (79) Allen, D.; Rassi, Z. E. Electrophoresis 2003, 24, 408-420. (80) Nilsson, J.; Speg el, P.; Nilsson, S. J. Chromatogr. B 2004, 804, 3-12. (81) Martin, J.; Hosticka, B.; Lattimer, C.; Norris, P. M. J. Non-Cryst. Solids 2001, 285, 222-229. (82) Behnke, B.; Johansson, J.; Zhang, S.; Bayer, E.; Nilsson, S. J. Chromatogr. A 1998, 818, 257-259. (83) Rebscher, H.; Pyell, U. Chromatographia 1994, 38, 737-743. (84) Hilder, E. F.; Klampfl, C. W.; Macka, M.; Haddad, P. R.; Myers, P. Analyst 2000, 125, 1-4. (85) Robson, M. M.; Roulin, S. C. P.; Shar iff, S. M.; Raynor, M. W.; Bartle, K. D.; Clifford, A. A.; Myers, P.; Euerby, M. R.; Johnson, C. M. Chromatographia 1996, 43, 313-321. (86) Dulay, M. T.; Kulkarni, R. P.; Zare, R. N. Anal. Chem. 1998, 70, 5103-5107. (87) Schmid, M.; Baml, F.; Khne, A. P.; Welsch, T. J. High Resol. Chromatogr. 1999, 22, 438-442. (88) Zhang, X.; Huang, S. J. Chromatogr. A 2001, 910, 13-18. (89) Piraino, S. M.; Dorsey, J. G. Anal. Chem. 2003, 75, 4292-4296. (90) Carney, R. A.; Robson, M. M.; Bartle, K. D.; Myers, P. J. High Resol. Chromatogr. 1999, 22, 29-32. (91) Bosch, S. E. v. d.; Heemstra, S.; Kraak, J. C.; Poppe, H. J. Chromatogr. A 1996, 755, 165-177. (92) Tang, Q.; Xin, B.; Lee, M. L. J. Chromatogr. A 1999, 837, 35-50. (93) Malik, A. Electrophoresis 2002, 23, 3973-3992. (94) Constantin, S.; Freitag, R. J. Sep. Sci. 2002, 25, 1245-1251.

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91 (95) Freitag, R.; Constantin, S. J. Sep. Sci. 2003, 26, 835-843. (96) Wu, J.-T.; Huang, P.; Li, M. X.; Qian, M. G.; Lubman, D. M. Anal. Chem. 1997, 69, 320-326. (97) Narang, P.; Colon, L. A. J. Chromatogr.A 1997, 773, 65-72. (98) Wang, Y.; Zeng, Z.; Guan, N.; Cheng, J. Electrophoresis 2001, 22, 2167-2172. (99) Fujimoto, C. J. High Resol. Chromatogr. 2000, 23, 89-92. (100) Tanaka, J.; Nagayama, H.; Kobayashi, H.; Ikegami, T.; Hosoya, K. J. High Resol. Chromatogr. 2000, 23, 111-116. (101) Oguri, S.; Tanagaki, H.; Hamaya, M.; Kato, M.; Toyo'oka, T. Anal. Chem. 2003, 75, 5240-5245. (102) Quirino, J. P.; Dulay, M. T.; Zare, R. N. Anal. Chem. 2001, 73, 5557-5563. (103) Quirino, J. P.; Dulay, M. T.; Bennett, B. D.; Zare, R. N. Anal. Chem. 2001, 73, 5539-5543.

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92CHAPTER FOUR POSITIVELY CHARGED SOL-GEL COATINGS FOR ONLINE PRECONCENTRATION OF AMINO ACIDS IN CAPILLARY ELECTROPHORESIS 4.1 Introduction Capillary electrophoresis (CE) represents a class of highly efficient electro-driven liquid phase separation techniques that are often complementary to HPLC 1. Compared to HPLC, CE provides good mass detection limits especially when the sample volume is limited. But in some cases, samples are not volume limited and concentration detection limits are more important 2, 3. CE separation techniques provide poor concentration sensitivity compared with liquid chromatography, especially when on-column UV detection technique is used to prevent loss of separation efficiency due to extracolumn band broadening that usually takes place in the detection cell when an off-column detection scheme is used. Generally, low concentration sensitivity of CE techniques is a consequence of the small sample injection volu me and the short optical path length of the capillary 4. Several preconcentration techniques including electrophoresis-based 5-19 and chromatography based 20-26 methods, have been developed to enhance the concentration sensitivity of CE separations. In this chapter, a positively charged solgel coating for on-line preconcentration of amino acids zwitterionic biomolecules in general is presented. The preconcentration methods based on the utilization of capillar y with such a sol-gel coating do not require any additional modification of the commercially available standard CE instrument. Extraction, stacking, and focusing techniques were used in the preconcentration procedures. With zwitterionic properties, amin o acids can carry either a positive or a negative charge when the pH of the samples is properly adjusted. Therefore, the electrostatic interaction between the analytes and CE column can be used to facilitate the extraction procedure.

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934.1.1 Column technology Unlike HPLC, CE does not require high-pressure pumping systems for mobile phase delivery. The typical high-voltage operation in CE is associated with electroosmotic pumping mechanism that serves as the driving force for mobile phase flow through the CE column. The pressure-free operations in CE provide the technique with some significant advantages over HPLC, and makes CE operation practically free from particle size, column length and availa ble maximum pressure limitations inherent in HPLC. Thus, CE opens up real possibilities to achieve extremely high separation efficiencies and column performances in liquid-phase separation. However, materialization of this great potential of CE will require effective solution of a number of problems mostly in the area of column technology development. The silanol groups on the fused silica ca pillary surface are responsible for the generation of electroosmotic flow (EOF) and the adsorption of sample ions. In some cases, surface coatings are used to screen th ese active sites to suppress or reduce EOF, or to reverse the direction of EOF 27-29. Capillary wall coating techniques normally can be classified into two categories: dynamic coating methods and chemical bonding methods 13. 4.1.1.1 Dynamic coatings Dynamic coatings are produced by sequential adsorption of various kinds of coating reagents on the surface of a fused silica capillary 1, 3, 30. The advantages of this type of coating technique made simplicity in column preparation by using the rinse function of standard CE instrumentation, an d the dynamically modified capillaries exhibit improved EOF reproducibility compared to an unmodified fused silica capillary. Therefore, this method is widely used to coat capillaries for the separation of various analytes 30-34. Various types of coating agents have been used 1, including alkylamines and diamines 27, 28, 35, 36, neutral and ionic polymers 29, 37, 38, cationic surfactants 28, 39, and polyelectrolyte multilayers 40, 41. For instance, Bendahl and colleagues 41 developed a simple procedure for the generation of dynam ic coatings by noncovalent adsorption of ionic polymer Polybrene and poly(vinylsulfonate) (PVS). The dynamic coating produced by their method was proved to be stable over a wide pH range of 2-10. Fast separation of

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94 basic test compounds was achieved at low pH. Recently, Chang and co-workers 42 used commercially available coating reagent EOTrolTM to dynamically modify the capillary for CE. The coating procedure involved pretreatment of the capillary with 1 M NaOH and water under pressure (20 psi) for 10 min and 1 min, respectively. And this pretreatment process was followed by the dynamic coating process with EOTrolTM in running buffer at a concentration of 0.15% w/v. The coatings allowe d reproducible EOF modulation in the cathodal direction and rapid separation of isofoms of three model proteins (bovine serum albumin, transferrin, a1-antitrypsin). 4.1.1.2 Chemically bonded coatings A number of procedures have been employed to prepare chemically bonded coatings, which are not usually destroyed due to rinsing of the capillary with buffer solution. The coating techniques include surface derivatization using silane chemistry 4345. By this technique, both halo and alkoxy silanes can be coupled to fused silica capillary surface through one or more siloxane bonds 46-48. Poly(ether) chains with different length or different substituent groups can also be covalently bonded to a sublayer generated by the silanization reaction 1. In addition, organic polymerization-based techniques 49-52 represent another important method to generate chemically bonded coatings. Chemically bonded coatings can be obtained by cross-linking of prepolymers or by polymerization of monomers on the capillary surface. Sol-gel approach 53-59 providing organic-inorganic hybrid materials have been attracting more and more interests in the column technology due to its various advantageous features. 4.1.2 Positively charged coatings for CE columns In a CE column with a positively charged inner surface, the diffuse component of the electrical double layer contains anions and generates an anodic electroosmotic flow instead of the cathodic EOF produced by untreated fused silica capillary characterized by a negatively charged surface. Both dynamic coating techniques and chemical bonding methods have been used in or der to obtain positively charged coatings on the capillary surface. Positively charged sites have been generated using a cryptand-containing polysiloxane 60 or quaternary ammonium group 61-65. In Lee and co-workers 60 prepared

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95 positively charged quaternary amine surface coating using iodopropyltrimethoxylsilane and poly(4-vinylpyridine-co-butylmethacrylate). Iodopropyltrimethoxylsilane was first used to bond onto the capillary surface through a siloxane bond, and poly(4vinylpyridine-co-butylmethacrylate) was immobilized through a reaction between the carbon-iodo bonds and the pyridine groups in the polymer, producing quaternary amine moieties. The positively charged quaternary amine effectively provided a reversed EOF compared to an untreated fused silica capillary. Columns with positive surface charges obtained in this way were successf ully used to separate proteins. As mentioned in Chapter Three, sol-gel columns offer many advantages over conventional columns for the separation in GC 66, 67, HPLC 53, 54, 56, 68, and CE 53-55, 69. In addition, sol-gel coated capillaries have been employed for the isolation and preconcentration of a wide variety of polar and nonpolar analytes by solid-phase microextraction analysis 70, 71. This chapter, describes pos itively charged sol-gel coatings created by using N-octadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride as a sol-gel precursor. The obtained positively charged sol-gel columns were used for online preconcentration and CE analysis of amino acids. 4.2 Experimental 4.2.1 Equipment All sample preconcentration and CE experiments were performed on a Bio-Rad BioFocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA) equipped with programmable, multiwavelengt h UV/visible detector. The BioFocus 3000 operating software system (version 6.0) was used to collect and process the CE data. A Barnstead model 04741 Nanopure deionized water system (Barnstead/Thermodyne, Dubuque, IA) was used to prepare deionized water, ~ 17 M /cm. A homemade gas pressure-operated capillary filling / purging device53 was used for coating the fused-silica capillary. Figure 4.1 schematica lly illustrates the filling / purging device. A Microcentaur model APO 5760 centrifuge (Accurate Chemic al and Scientific Corp., Westbury, NY) was used for centrifugation of the sol solutions. A Fisher model G-560 Vortex Genie 2 system (Fisher Scientific, Pittsburgh, PA) was used for thorough mixing of various chemical ingredients in the sol-gel coating solution. A Chemcadet model 5984-50 pH

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96 F u se d silicacapillary Flow control valve Flow control valve Pressurization chamber Sol solution vial Gas flow outlet Threaded detachable cap inert gas supply Figure 4.1 Schematic representation of the home made capillary filling / purging device, adapted from Reference 53

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97 meter (Cole-Palmer Instrument Co., Chicago, IL) equipped with a TRIS-specific pH electrode (Sigma-Aldrich, St. Louis, MO) was used to measure the buffer and sample pH. 4.2.2 Chemicals and materials Fused-silica tubing of 50-m i.d. was purchased from Polymicro Technologies (Phoenix, AZ) for the preparation of sol-gel coated columns. Sample vials (600L), HPLC grade methylene chloride, methanol, and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). Tetramethyl orthosilicate (99+%) and trifluoroacetic acid (99%) were purchased from Aldrich (Milwaukee, WI). Tris (hydroxymethyl)aminomethane hydrochloride (reagent grade) and amino acids (DLalanine, DL-asparagine, DL-phenylanlaine and DL-tryptophan) were purchased from Sigma (St. Louis, MO). N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride and phenyldimethylsilane were purchased from United Chemical Technologies, Inc. (Bristol, PA). 4.2.3 Preparation of sol-gel open tubular CE columns with positive surface charge Sol-gel open tubular C18-TMS columns with a positive surface charge were prepared according to the procedure described by Hayes and Malik 54 Briefly, the sol solution was prepared by thoroughly mixing appropriate amounts of the following ingredients: (a) two sol-gel precursors {tetramethoxysilane (TMOS) and NOctadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride (C18-TMS)}, (b) a deactivation reagent [phenyldimethylsilane (PheDMS)], and (c) a sol-gel catalyst [trifluoroacetic acid (TFA) (containing 5% water)]. A piece of previously cleaned and hydrothermally treated fused-silica capillary was first sealed at one end using an oxyacetylene flame. The prepared sol solution was then introduced into the capillary from its open end, creating a pressurized gas pocket at the sealed end. This filling process was carried out under 40 psi of helium pressure using a homemade filling device 53. Because of the presence of a pressurized gas pocket at the sealed end of the capillary, the inner surface of the gas-containing part of the capillary remained untouched by the sol solution. Figure 4.2 illustrates the process. After a 20-min in-capillary residence time, the

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98 polyimide coating capillary wall pressure sol solution sol-gel coating optical windowA B C D E Figure 4.2 Preparation of a sol-gel column for preconcentration adapted from Ref.55 A: a piece of hydrothermally pretreated fused silica capillary. B: sealing one of the capillary ends via an oxyacetylene torch. C: filling part of the capillary with the sol solution by helium pressure. D: depressurization of the capillary followed by opening of the sealed end of the capillary through the open end. E: preparation of a UV detection window after condition.

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99 filling gas pressure was released to allow the pressurized gas pocket to expel the sol solution from the capillary through its open end. The sealed end was cut open and the capillary was further purged with helium fr om the previously sealed end, allowing a segment of the capillary at this end to re main untouched by the sol solution. Thus, a segment of the capillary at the open end was coated, leaving at the initially sealed end an uncoated segment (~25 cm). Once the remaining steps of the column preparation are complete, this uncoated segment can be used to create an optical window for on-column UV detection. After coating and purging, both ends of the capillary were sealed with an oxy-acetylene torch and the capillary was further conditioned in a GC oven at 150C for 2 hours. Following thermal treatment, the sealed capillary ends were cut open. The column was purged under 40 psi helium pressure for an additional 30 min and then sequentially rinsed with 100% acetonitrile, deionized water and desired running buffer. The UV detection window was created by burning the extenal polyimide coating on the uncoated segment of the capillary. 4.2.4 Preparation of samples DL-alanine, DL-asparagine, DL-phenylalanine and DL-tryptophan purchased from Sigma (St. Louis, MO) were dissolved in deionized water to make the test samples. 4.2.5 Procedures for extrac tion and preconcentration After the sol-gel coated column was installed on the CE system, it was first filled with the running buffer using gas pressure. The inlet end of the column was then inserted into the sample vial. Samples were hydrodynamically injected for 3 min at 100 psi. Under these conditions, the pH of the sample soluti on was kept above the isoelectric point (pI) of the test amino acid to impart a net negative charge to the solute amino acids (Table 4.2). Electrostatic interaction between the positively charged sol-gel coating and negatively charged amino acid molecules led to their extraction on the sol-gel column. To show the extraction effect of the sol-gel coat ing, the sample matrix was removed from the column either by purging with deionized wate r or by reversed electroosmotic flow. After this, the inlet end of the capillary was retu rned back to the buffer reservoir, which

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100 contained an acidic running buffer. The extracted analytes were then desorbed by this acidic running buffer and analyzed under the effect of a high applied electric field. 4.3 Results and discussion Table 4.1 lists the names and chemical structures of sol-gel solution ingredients used in this research to create a positively charged sol-gel coating. The sol-gel coprecursor, C18-TMS, possesses a number of important structural features. The methoxysilyl groups in the C18-TMS and TMOS are sol-gel active, and they participate in the formation of the sol-gel polymeric network through hydrolysis and polycondensation reaction 53-55, 72. Its quaternary amine moiety is responsible for the positive charge on the sol-gel coating, which not only provides the basis for electrostatic interaction between sol-gel coating and analytes in samples but al so supports reversed electroosmotic flow in the CE column. In addition, the octadecyl chain like a pendant group is capable of providing the chromatographic interactions. Although a number of other chemicals including poly(diallyldimethylammonium chloride) 64, chitosan 73 and cyrptandcontaining polysiloxane 60 have been used by different research groups to generate a positively charged coating surface, in the present study, C18-TMS was used. This sol-gel approach to achieve this goal was introduced by our group and is characterized by its simplicity and effectiveness. 4.3.1 Mechanism of extraction on po sitively charged sol-gel coatings The mechanism of extraction in a CE column with a positively charged sol-gel C18-TMS coating is based on the electrostatic interaction. The structures of amino acid test solutes used in the current study together with their dissociation constants are shown in Table 4.2. Due to their zwitterionic pr operties, amino acids can bear a net positive charge, a net negative charge, or be electrically neutral in different pH environments. At pH values above its isoelectric point, an amino acid will possess a net negative charge. Because of the electrostatic interactions, the negatively charged species will get extracted by the positively charged C18-TMS coating on the inner surface of the CE column.

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101 Table 4.1 Names and chemical structures of chemical reagents used in the fabrication of positively charged sol-gel columns Regent Function and Reagent Name Reagent Structure Sol-gel precursors: Tetramethoxysilane (TMOS) OCH3Si OCH3OCH3H3CO N-Octadecyl-dimeth yl[3-(trimethoxysilyl)propyl] ammonium chloride (C18-TMS) N+H3C-(H2C)17C H3CH3(CH2)3Si OCH3OCH3OCH3 Deactivation reagent: Phenyldimethylsilane (PheDMS) Si CH3CH3H Catalyst: Trifluoroacetic acid (TFA) F3C C OH O

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102 Table 4.2 Chemical structures and some physical properties of analytes used in the current study Name Structure pKa of -COOH Group 60a pKa of -NH3 + Group60a Isoelectric point* Alanine O NH2C H3OH 2.3 9.7 6.0 Asparagine O O N H2NH2OH 2.0 8.8 5.4 Phenylalnine O NH2OH 1.8 9.1 5.5 Tryptophan O NH2NH OH 2.4 9.4 5.9 Data were calculated according to the procedure described in Reference74.

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103 Unlike Quirino and Terabe 13, 75-78, who used MEKC-based sweeping of the analytes from dilute sample solutions by employing surfactants, we used an acidic buffer to elute the extracted amino acid analytes from the sol-gel surface coating and collect them in the form of a compressed zone. This was accomplished in a simple step during CE operation without using any micellar so lution. The use of surfactants in MEKC mobile phase often makes the technique difficult to interface with different detection systems, most notably with mass spectrometry. The presented sol-gel approach to sample preconcentration does not use any surfactants, and thereby elegantly overcomes a serious shortcoming inherent in MEKC approach. 4.3.2 Extraction and preconcentration of amino acid by sol-gel columns Based on reported stacking and sweeping methods8, 9, 13, 75-82, the maximum volume of the sample that could be injected into the CE system is the volume of the column itself. In the case of very dilute sa mples, even such a sample volume may not be enough to enrich a detectable amount of the analyte after preconcentration. In the sample preconcentration technique described in this chapter, the sample volume that can be used for analyte enrichment is not limited to one co lumn volume. It allows for the injection of multiple column volumes of sample. By continuously passing the sample solution for an extended period through the sol-gel coated capillary possessing enhanced surface area and appropriate surface charge, the analytes can be extracted from a large volume of the sample. The pH of the sample solution sh ould be carefully chosen, so that the zwitterionic analyte of interest assume a net electric charge which is opposite to the surface charge on the sol-gel coating. The extracted analytes can be further focused into a narrow band by manipulating the buffer pH. Figure 4.3 illustrates the enrichment of the tryptophan sample (pI = 5.9) using extraction on a capillary with positively charged sol-gel coating followed by focusing of the extracted analytes. At the end of an extended period of sample injection (3 min), the sample matrix was pushed out of the column by a flow of deionzied water (A) or the running buffer (B). After this, a high voltage was applied and CZE was performed using an acidic buffer (50 mM Tris-HCl, pH 2.2). No sample peak could be detected, if a voltage was

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104 -3 -2 -1 0 1 2 3 4 5 6 7 01020min.mAU. A B Figure 4.3 Extraction of tryptophan on the sol-gel column. Experimental conditions: solgel coated column 75 cm x 50 m; the effective length of the column is 70.4 cm; mobile hase 50 mM Tris-HCl (pH 2.2); injection 3 min at 100 psi; running voltage V = -15 kV; wavelength of UV detector 200 nm; sample 10 M tryptophan (pH 7.67). After injection, the sample matrix was removed by (A) deio nized water 3 min at 100 psi, (B) mobile phase 3 min at 100 psi.

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105 applied after rinsing the column with the lo w-pH buffer (Figure 4.3B) capable of washing the extracted analytes off the sol-gel coating. On the other hand, when the sample matrix was removed by deionized water (pH 7.0) (Figure 4.3 A), the extracted amino acid molecules remained attached to the positively charged capillary surface due to electrostatic attractive forces between negatively charged solute ions and the positively charged capillary surface. Deso rption of the extracted amino acid and its focusing into a narrow zone was accomplished by using a high electric field (V = 15 kV) and a low-pH buffer, as illustrated in Figure 4.4. The whole procedure consisted of three steps: (a) extraction, (b) removal of the sample matrix and (c) desorption and enrichment of the extracted analyte using a low-pH running buffer and a high electric field. In the first step, the column was filled with sample solution. Negatively charged analytes were extracted on the positively charged inner surface of the sol-gel column. This process was followed by the removal of sample matrix with deionized water. In the third step, a high electric voltage (V = -15 kV) was applied between the ends of the sol-gel capillary, using pH 2.2 Tris-HCl (50 mM) as the running background electrolyte. The cathode was on the inlet side and a node on the outlet side of the capillary. Under the applied electric field, an anodic EOF was generated in th e CE capillary with positively charged sol-gel coating. Once the acidic running buffer (50 mM Tris-HCl pH 2.2) came in contact with the front of the extracted solute zone, it reversed the net charge of the amino acid molecules, providing a repulsive mechanism for their desorption from the capillary surface. EOF moved the desorbed analyte molecules forward, gradually desorbing more and more amino acid molecules and focusing them into a narrow zone.

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106CathodeAnode 1. Filling the capillary with the sample 2. Sample matrix is removed by water 3. Applying a high electric voltage V= -15kV 4. Analytes are focused by an acidic buffer EOF CathodeAnode EOF EOF cation anion neutral analyte Figure 4.4 Illustration of the events during preconcentration and focusing of zwitterionic analytes on a positively charged sol-gel column: (1) sample was passed through the column under helium pressure (e.g., 100 psi for 3 min); (2) sample matrix was removed from the column by water under helium pressure (e.g., 100 psi for 3 min); (3) application of high electric voltage (e.g., V = -15 kV); (4) the acidic running buffer desorbed the extracted analytes and carried them to the detector.

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107 Figure 4.5, 4.6, 4.7 and 4.8 show the electropherograms of four amino acids alanine, asparagine, phenylalanine and tryptophan preconcentrated from 10 M aqueous on a positively charged sol-gel column using an extended injection time (3 min). To show the enrichment effect, two samples of the same amino acid at two different concentration levels were analyzed on an uncoated fused silica capillary column with the identical dimensions using conventional hydrodynamic injections. One of the samples had the same concentration as the one used in the preconcentration experiment, and the other sample had at least 1000-fold higher concentrations of the amino acids. From these figures, it is evident that the sample is greatly preconcentrated when analyzed on a sol-gel column. For example, a 10 M alanine sample solution was preconcentrated on the solgel column and a peak of more than 6 mAU was obtained (Figure 4.5A). With conventional mode of injection on an uncoated capillary column, an alanine solution (100 mM) only gave a peak height of less than 2 mAU (Figure 4.5B). Figure 4.5 C shows that with an uncoated capillary column and conventional injection, no peak was detected for 10 M alanine sample solution. Similarly, the sol-gel column preconcentrated a 10 M tryptophan sample and gave a peak height of more than 6 mAU in Figure 4.8(A). While with the uncoated fused silica capillary and with conventional mode of hydrodynamic injection, no peak was obtained for 10 M tryptophan in Figure 4.8(C). Using an uncoated capillary, we also ran a tryptophan sample of 1000 times higher concentration (10 mM). As a result, a peak with a little more than 10 mAU in height was obtained shown in Figure 4.8(B). Based on these results, the limit of dete ction values (LOD, S / N = 3) were calculated and the results are presented in Table 4.3. We can see that with the positively charged sol-gel C18-TMS coated column, the presented preconcentration method provided significantly lower LODs of these amino acids. The most effective preconcentration result was obtained for alanine. LOD of alanine was reduced from 10.2 mM on an uncoated column to 139 nM on the sol-gel coated column, which corresponds to an enrichment factor of more than 73,000 times.

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108 Alanine-4 -2 0 2 4 6 8 10 12 0102030min. mAUA B C Figure 4.5 Illustration of the effect of a positively charged sol-gel coating on the preconcentration of alanine. The sample concentrations of A, B, and C was 10 M, 100 mM and 10 M. pH of the prepared samples: 7.7. Electropherogram A was obtained using the sol-gel coated column (75 cm x 50 m). The effective length of the column was 70.4 cm; mobile phase, 50 mM Tris-HCl (pH 2.2). Hydrodynamic injection for 3 min at 100 psi. UV detection at 200 nm. Sample matrix was removed by deionized water for 3 min at 100 psi, running voltage V = -15 kV. Electropherograms B and C were obtained on an uncoated column (75 cm x 50 m) with the same mobile phase. The effective length of the column is 70.4 cm. Hydrodynamic injection at 10 psi sec., running voltage V = +15 kV. UV detection at 200 nm.

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109 Asparagine -4 -2 0 2 4 6 8 10 12 0102030min. mAUA B C Figure 4.6 Effect of sol-gel coating on samp le (asparagine) preconc entration. The sample concentrations of A, B, and C was 10 M, 50 mM, and 10 M. Operation conditions are the same as shown in Figure 4.5.

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110 Phenylalanine-4 -2 0 2 4 6 8 10 12 0102030min. mAUA B C Figure 4.7 Effect of sol-gel coating on sample (phenylalanine) preconcentration. The sample concentrations of A, B, and C was 10 M, 10 mM and 10 M. Operation conditions are the same as shown in Figure 4.5.

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111 Tryptophan-4 -2 0 2 4 6 8 10 12 0102030 min.mAUA B C Figure 4.8 Effect of sol-gel coating on sample (tryptophan) preconcentration. The sample concentrations of A, B, and C was 10 M, 10 mM and 10 M. Operation conditions are the same as shown in Figure 4.5.

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112 In order to calculate the sensitivity enha ncement factor (SEF), we employed peak areas as well as the corrected peak areas (the ratio between peak area and the migration time) using the following equation 75. factor dilution ration preconcent without obtained parameter peak ration preconcent with obtained parameter peak SEF The sensitivity enhancement factors (SEF) for the studied amino acids are presented in Table 4.4. The SEF values were different for different samples. In addition, the observation that the migration time of the sample running in an uncoated column was much longer than that of obtained in sol-gel coated column is due to the different electroosmotic flow in coated and uncoated columns. When a low-pH acidic buffer is used as the mobile phase in an uncoated column, a significant portion of the silanol groups on the fused silica surface remain protonated by the acidic mobile phase, resulting in a decreased surface charge, and hence reduced EOF. Experiments with a neutral marker, DMSO, showed that when the pH of the running buffer was 2.22, the electroosmotic mobility in the untreated fused silica column was 1.02 x 10-4 cm2/V.s. On the other hand, an acidic running buffer practically did not influence the positive charge on the sol-gel surface of the column. This is explained by the fact that dissociation of the quaternary amine group anchored to the surface coating practically remains unaffected by this pH change. The direction of electroosmotic mobility obtained on the sol-gel coated column using the same buffer and DMSO as an EOF marker had a value of 4.02 x 10-4 cm2/V.s., being reversed in direction and about four times greater in magnitude than the EOF in the uncoated column under identical operating conditions.

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113 Table 4.3 Sample extraction and preconcentrations on an electrically charged sol-gel column Aa Bb Cb Sample Concentration M LOD nM (S/N=3) Concentration mM LOD M (S/N=3) Concentration M LOD (S/N=3) Alanine 10 139 100 10,170 10 N/Ac Asparagine 10 98 50 864 10 N/A Phenylalanine 10 141 10 195 10 N/A Tryptophan 10 115 10 203 10 N/A aA: column (75 cm x 50 m) with a positively charged sol-gel coating, the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.2). Samples were injected hydronamically for 3 min at 100 psi. Running voltage V= -15 kV. Wavelength of UV detector: 200 nm. bB and C: uncoated column (75 cm x 50 m), the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.2). Samples were injected hydronamically for 10 secpsi. Running voltage V= +15 kV. Wavelength of UV detector: 200nm. c N/A, not applicable.

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114 Table 4.4. Sensitivity enhancement factors for four amino acids achieved on positively charged sol-gel C18-TMS coated columns SEF by Method 1 Sample By Height By Area Alanine 55,374 61,048 Asparagine 3,596 1,817 Phenylalanine 995 1,730 Tryptophan 928 1,496

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115 4.3.3 Preconcentration of amino ac ids with the removal of sample solutions by reversed EOF Remarkably high sample-enrichment factors were achieved using the samplepreconcentration method mentioned above. However, it might be possible to further improve this SEF, if we consider the following. Deionized water was used during the sample matrix removal step after extraction for an extended period of time. Undoubtedly, this led to the elution, and therefore loss of portion of the analytes extracted on the sol-gel column. To prevent this loss, the following experiment was designed. The procedure is illustrated schematically in Figure 4.9. The sample solution was passed through the sol-gel column for an extended injection period (3 min at 100 psi). The positively charged sol-gel coating extracted the anions in the sample solution. Next, a high voltage (+15 kV) was applied with anode in the inlet side and cathode in outlet end. In this stage, a number of processes occurred. One of them is that the anions in sample solution migrated to anode while cations migrated toward the cathode side by electrophoretic flow. In addition, the electroosmotic flow was generated towards the capillary inlet. EOF, being stronger than the electrophoreitc flow of the ions, forced the sample matrix to move toward the capillary inlet and leave the capillary from the inlet end. At the same time, because the running buffer was acidic, on contact it reversed the electric charge of the extracted analytes and provided an effective mechanism for their desorption from the positively charged surface coating in the column via mutual repulsion. During this process, the analyte was focused at the boundary of the sample solution and the running buffer. The current was observed carefully to decide the time when the voltage polarity needed to be reversed. While the column was filled with sample solution, the current was low due to the low conductivity of the dilute sample solution. With more and more sample matrix being pushed out of the column, more and more running buffer filled in the column, the current increased because of the higher conductivity of the media filling the column. Just before the current soared up quickly, the polarity of the voltage was reversed. The focused sample zone was carried by the resulting electroosmotic flow towards to the outlet of the capillary and detected by the UV/vis detector. It can be noticed that instead of mechanically rinsing the column with deionized water, a reversed electroosmotic flow was applied in conjunction with a low-pH buffer to remove the sample matr ix. Unlike the water-rinsing procedure

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1161. Filling the capillary with the sample 2. Applying a high electric voltage V=+15kV Anode Cathode 3. After a high electric voltage V=+15kV has been applied for a certain time. Anode Cathode EOF 4. Switching the polarity of the high electric voltage to V=-15kV. CathodeAnode EOF EOF cation anion neutral analyte Figure 4.9 Method 2 for the preconcentration of zw itterionic analytes on the positively charged sol-gel column steps. 1. Sample was passed through the column under helium pressure (e.g. 100 psi for 3 min). 2. High voltage was applied (V= +15 kV), acidic running buffer came into the column, where extracted analyte desorption occurred via the local pH change, which caused the redistribution of ions inside the column. 3. Under the reversed EOF, the sample matrix was remo ved from the capillary, and the positively charged analytes were focused at its anodic end. 4. The polarity of the electric field was switched to V=-15 kV, the focused analytes were carried to the detector.

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117 described in the previous section, this method prevented the loss of analytes that have already been extracted on the sol-gel column. The sample preconcentration results obtained by this method are shown in Figure 4.10, 4.11, 4.12 and 4.13. The results show that with this method, (hereafter referred to as Method 2), the sample preconcentration effect is more significant even compared with the results obtained by the method we desc ribed in the previous section (hereafter referred to as Method 1). This indicates that in Method 1, when the sample matrix was removed by water, some analytes loss took place. However, with Me thod 2, the sample matrix was pushed out of the capillary by reversed electroosmotic flow, and the amount of analyte loss was greatly reduced. The LOD (S / N = 3) and SEF by Method 2 were calculated and are listed in Table 4.5 and Table 4.6. Comparing these data with those listed in Table 4.3, we can see that for samples of equal concentration the LODs were greatly reduced with Method 2. For example, with tryptophan as the test sa mple, Method 2 allowed to lower the LOD to 24.5 nM from 115 nM that was achieved by Method 1. The enhancement in sensitivity is more than five times. Comparing these data with the results obtained from a bare fused silica column, the sensitivity enhancement factor were calculated and shown in Table 4.6. It can be observed that the best results for both preconcentration methods belong to the same amino acid, alanine. This can be explained from its smaller size compared with other amino acid samples. According to Beer-Lambert Law, the amount of absorbed light is proportional to the product of sample concentration and its molar absorptivity coefficient. Since the inner surface area of the column is constant, the smaller the analyte, the more amino acids can be extracted on the same area of the sol-gel column. After they are desorbed from the column, the smaller molecule possesses a higher concentration. From Table 4.4, we note two important points: (a) both methods greatly increased the detection sensitivity, and (b) Method 2 is more effective compared with Method 1 because it reduced the sample loss during the sample matrix removal step.

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118 Alanine-5 0 5 10 15 20 25 0102030min. mAU A B C Figure 4.10 Illustration of the effect of sol-gel coating on sample preconcentration (alanine) by Method 2. The sample concentrations of A, B, and C were 10 M, 100 mM and 10 M. pH values for all samples were 7.7. Electrophoregrams marked with A were obtained using sol-gel coated column (75 cm x 50 m), the effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.2). Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V=-15 kV. UV detection at 200 nm. Electrophoregrams marked with B and C were obtained on an uncoated column (75 cm x 50 m) with the same mobile phas e, the effective length of the column was 70.4 cm. Hydrodynamic injection for 10 sec psi. Running voltage V=+15 kV. UV detection at 200 nm.

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119 Asparagine -5 0 5 10 15 20 25 30 35 0102030 min. mAU A B C Figure 4.11 Illustration of the effect of sol-gel coating on sample preconcentration (asparagine) by Method 2. The sample concentrations of A, B, and C were 10 M, 50 mM and 10 M. Operation conditions are the same as described in Figure 4.10.

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120 Phenylalanine-5 5 15 25 35 45 55 65 75 85 95 0102030 min. mAUA B C Figure 4.12 Illustration of the effect of sol-gel coating sample preconcentration (phenylalanine) by Method 2. The sample concentrations of A, B, and C were 10 M, 10 mM and 10 M. Operation conditions are the same as described in Figure 4.10.

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121 Tryptophan-5 5 15 25 35 45 55 65 75 0102030 min. mAUA B C Figure 4.13 Illustration of the effect of sol-gel coating on sample preconcentration (tryptophan) by Method 2. The sample concentrations of A, B, and C were 10 M, 10 mM and 10 M. Operation conditions are the same as described in Figure 4.10.

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122 Table 4.5 Sample extraction and preconcentrations on an electrically charged sol-gel column by Method 2. Aa Bb Cb Sample Concentration M LOD, nM (S/N=3) Concentration mM LOD, M (S/N=3) Concentration M LOD (S/N=3) Alanine 10 60.7 100 10,170 10 N/Ac Asparagine 10 47.3 50 864 10 N/A Phenylalanine 10 23.3 10 195 10 N/A Tryptophan 10 24.5 10 203 10 N/A aA: column (75 cm x 50 m) with a positively charged sol-gel coating, the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.22). Samples were injected hydronam ically for 180 seconds at 100 psi. Running voltage V= -15 kV. Wavelength of UV detector: 200 nm. bB and C: uncoated column (75 cm x 50 m), the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.22). Samples were injected hydronamically for 10 sec*psi. Running voltage V= +15 kV. Wavelength of UV detector: 200 nm. cN/A: not applicable

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123 Table 4.6 Sensitivity enhancement factors for four amino acids achieved on positively charged sol-gel C18-TMS coated columns by Method 2a SEF by Method 2 Sample By Height By Area Alanine 153,770 66,782 Asparagine 16,773 21,427 Phenylalanine 11,248 63,469 Tryptophan 6,326 10,754 a Operation conditions are as same as shown in Figure 4.10.

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124 Figure 4.14 represents experimental data showing the preconcentration of alanine by Method 2 (trace A), which was compared with a blank run (trace B). This experiment was designed to verify whether the peaks obtained by the described preconcentration methods were artifacts of system peaks. The absence of such a peak in the blank run clearly indicates that the peak in (A) is not a system peak and confirms the real possibility of performing sample preconcentration using the described methods. Unlike Method 1 (which includes extraction and focusing operations only), Method 2 includes an additional step allowing electrophoretic migration of the extracted charged analytes. This provides a real opportunity to achieve separation of the extracted analytes by Method 2. Figure 4.15 highlights this point and illustrates the practical utility of Method 2 by providing an example of online preconcentration and separation of two amino acids: tryptophan and asparagine. As can be seen in Figure 4.15, the two preconcentrated amino acids are more than baseline separated with a wide gap between them. The reproducibility of the sample preconcentration methods was examined by a series of experiments and shown in the terms of the relative standard deviation (RSD) of migration time and peak height. Table 4.7 shows the experimental and calculation results. Quite good repeatability in migration times was obtained with both preconcentration methods for all test analytes. The RSD values in terms of migration time are no more than 3.7%. The RSD values in the range of 3.8% to 28%were obtained for peak height repeatability. The presented data reveals that in both cases, Method 2 provided significantly better repeatability than Method 1. The sample matrix removal procedure in Method 1 probably caused the inferior reproducibility for some solutes in Method 1.

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125 Alanine -5 0 5 10 15 20 25 0102030 min. mAU A B Figure 4.14 Juxtaposition of the effect of sol-gel coating on sample preconcentration by Method 2 and a blank run with the same method. Trace (A) is 10 M alanine. Sample pH value was 7.7. Trace (B) is a blank run. Sol-gel coated column (75 cm x 50 m), the effective length of the column was 70.4 cm ; mobile phase 50 mM Tris-HCl (pH=2.2). Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V=-15 kV. UV detection at 200 nm.

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126 -5 -3 -1 1 3 5 7 9 0102030 min. mAU.A B a b Figure 4.15 Illustration of the preconcentration and separation of a mixture of two amino acids on a positively charged sol-gel column us ing Method 2. pH value of the sample was 7.7. Electropherograms were obtained using sol-gel coated column (75 cm x 50 m), the effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl (pH=2.2)/ACN 50/50. Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V=-15 kV. UV detection at 200 nm. Electropherogram marked with A was obtained by preconcentrating and separating an amino acid mixture, in which peak a is tryptophan and peak b is asparagine. Electropherogram B was obtained by running a blank.

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127 Table 4.7 Repeatability data fo r the preconcentration by C18-sol-gel coated column using amino acids as test solutes Method 1a Method 2b Analytes av. tR (min) RSD (%) (n=4) av.height (mAU) RSD(%) (n=4) av. tR (min) RSD (%) (n=4) av.height (mAU) RSD(%) (n=4) Alanine 4.37 2.77 5.80 27.70 17.67 1.62 20.80 10.10 Asparagine 4.36 1.32 5.73 13.81 12.08 0.95 26.53 9.45 Phenylalanine 4.44 1.11 7.57 3.83 14.43 1.73 94.04 5.91 tryptophan 4.45 2.27 6.61 9.18 9.56 3.70 86.26 15.93 a Experimental conditions: same as in Figure 4.5. bExperimental conditions: same as in Figure 4.10.

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1284.4 Conclusions For the first time, we have shown on-column extraction and preconcentration effect offered by positively charged sol-ge l column in capillary zone electrophoresis. Using a positively charged sol-gel coating, a 1 50,000 fold enrichment effect was obtained for alanine. The newly developed methods do not limit the volume of the injected sample and they do not require modification of a standard CE system to achieve the preconcentration effect. They allow large-volume injection of the sample for an extended period of time, and are very effective in enriching trace concentrations of zwitterionic solutes. Large sensitivity enhancement factors (SEF) on the order of 105 were obtained in the study. Further sensitivity enhancement should be possible in a number of ways by (a) performing the preconcentration step without the removal of sample buffer; (b) using thicker sol-gel coatings or monolithic beds; and (c) derivatizating the amino acids with proper derivatization reagents. 4.5 References for Chapter Four (1) Poole, C. F. The Essence of Chromatography; Elsevier Science: Amsterdam, The Netherlands, 2003. (2) Weinberger, R. Practical Capillary Electrophoresis, 2nd ed.; Academic Press: London, UK, 2000. (3) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons, Inc.: New York, N.Y., 1995. (4) Albin, M.; Grossman, P. D.; Moring, S. E. Anal. Chem. 1993, 63, 489A-497A. (5) Shihabi, Z. K. Electrophoresis 2002, 23, 1612-1617. (6) Burgi, D. S.; Chien, R.-L. Anal. Chem. 1991, 63, 2042-2047. (7) Chien, R. L. Electrophoresis 2003, 24, 486-497. (8) Chien, R. L.; Burgi, D. D. Anal. Chem. 1992, 64, 489A-496A. (9) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (10) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141-152.

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129 (11) Shihabi, Z. K.; M.Friedberg J. Chromatogr. A 1998, 807, 129-133. (12) Shihabi, Z. K. Electrophoresis 2000, 21, 2872-2878. (13) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (14) Quirino, J. P.; Kim, J.-B.; Terabe, S. J. Chromatogr. A 2002, 965, 357-373. (15) Palmer, J. J. Chromatogr. A 2004, 1036, 95-100. (16) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-1687. (17) Breadmore, M. C.; Shrinivasan, S.; Karlinsey, J.; Ferrance, J. P.; Norris, P. M.; Landers, J. P. Electrophoresis 2003, 24, 1261-1270. (18) Britz-McKibbin, P.; Mark uszewski, M. J.; Iyanagi, T. ; Matsuda, K.; Nishioka, T.; Terabe, S. Anal. Biochem. 2003, 313, 89-96. (19) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1729-1735. (20) Viberg, P.; Nilsson, S.; Skog, K. Anal. Bioanal. Chem. 2004, 378, 1729-1734. (21) Pedersen-Bjergaard, S.; Rasmussen, K. E.; Halvorsen, T. G. J. Chromatogr. A 2000, 902, 91-105. (22) Guzman, N. A.; Park, S. S.; Schaufelberger, D.; Hernandez, L.; Paez, X.; Rada, P.; Tomlinson, A. J.; Naylor, S. J. Chromatogr. B 1997, 697, 37-66. (23) Heegaard, N. H. H.; Nilsson, S.; Guzman, N. A. J. Chromatogr. B 1998, 715, 2954. (24) Stroink, T.; Paarlberg, E.; Waterval, J. C. M.; Bult, A.; Un derberg, W. J. M. Electrophoresis 2001, 22, 2374-2383. (25) Li, S.; Weber, S. G. Anal. Chem. 1997, 69, 1217-1222. (26) Cole, L. J.; Kennedy, R. T. Electrophoresis 1995, 16, 549-556. (27) Chiari, M.; Melis, A. Trends Anal. Chem. 1998, 17, 623-632. (28) Melanson, J. E.; Baryla, N. E.; Lucy, C. A. Trends Anal. Chem. 2001, 20, 365374. (29) Millot, M. C.; Vidal-Madjar, C. Adv. Chromatogr. 2000, 40, 427-466. (30) Phinney, K. W.; Sander, L. C. Chirality 2005, 17(Suppl.), S65-S69. (31) Lurie, I. S.; Bethea, M. J.; McKibben, T. D.; Hays, P. A.; Pellegrini, P.; Sahai, R.; Garcia, A. D.; Weinberger, R. J. Forensic Sci. 2001, 46, 1025-1032.

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130 (32) Lurie, I. S.; Panicker, S.; Hays, P. A.; Garcia, A. D.; Geer, B. L. J. Chromatogr.A 2003, 984, 109-120. (33) Lurie, I. S.; Hays, P. A.; Parker, K. Electrophoresis 2004, 25, 1580-1591. (34) Steiner, S. A.; Wats on, D. M.; Fritz, J. S. J. Chromatogr.A 2005, 1085, 170-175. (35) Righetti, P. G.; Gelfi, C.; Verzola, B.; Castelletti, L. Electrophoresis 2001, 22, 603-611. (36) Baryla, N. E.; Lucy, C. A. J. chromatogr.A 2002, 956, 271-277. (37) Horvath, J.; Dolnik, V. Electrophoresis 2001, 22, 644-655. (38) Chiari, M.; Cretich, M.; Damin, F.; Ceriotti, L.; Consonnt, R. Electrophoresis 2000, 21, 909-916. (39) Melanson, J. E.; Baryla, N. E.; Lucy, C. A. Anal. Chem. 2000, 72, 4110-4114. (40) Graul, T. W.; Schlenoff, J. B. Anal. Chem. 1999, 71, 4007-4013. (41) Bendahl, L.; Hansen, S. H.; Gammelgaard, B. Electrophoresis 2001, 22, 25652573. (42) Chang, W. W. P.; Hobson, C.; Bomberger, D. C.; Schneider, L. V. Electrophoresis 2005, 26, 2179-2186. (43) Hsu, J. C.; Chen, W.-H.; Liu, C.-Y. Analyst 1997, 122, 1393-1398. (44) Kirkland, J. J.; Glaj ch, J. L.; Farlee, R. D. Anal. Chem. 1989, 61, 2-11. (45) Liu, C.-Y.; Chen, W.-H. J. Chromatogr.A 1998, 815, 251-263. (46) Strelec, I.; Pacakova, V.; Bosakova, Z.; Coufal, P.; Guryca, V.; Stulik, K. Electrophoresis 2002, 23, 528-535. (47) Belder, D.; Deege, A.; Husmann, H.; Kohler, F.; Ludwig, M. Electrophoresis 2001, 22, 3813-3818. (48) Wan, H.; Ohman, M.; Blomberg, L. G. J. Chromatogr.A 2001, 924, 59-70. (49) Burt, H.; Lewis, D. M.; Tapley, K. N. J. Chromatogr.A 1996, 739, 367-371. (50) Bentrop, D.; Kohr, J.; Engelhaardt, H. Chromatographia 1991, 32, 171-178. (51) Nakatani, M.; Shibukawa, A.; Nakagawa, T. J. Chromatogr.A 1994, 672, 213-218. (52) Kohr, J.; Engelhaardt, H. J. Microcol. Sep. 1991, 3, 491-495.

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131 (53) Hayes, J. D.; Malik, A. J. Chromatogr. B 1997, 695, 3-13. (54) Hayes, J. D.; Malik, A. Anal. Chem. 2001, 73, 987-996. (55) Hayes, J. D.; Malik, A. Anal. Chem. 2000, 72, 4090-4099. (56) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511-2516. (57) Guo, Y.; Colon, L. A. J. Microcol. Sep. 1995, 7, 485-491. (58) Guo, Y.; Imahori, G. A.; Colon, L. A. J. Chromatogr.A 1996, 744, 17-29. (59) Colon, L. A.; Maloney, T. D.; Anspach, J.; Colon, H. Adv. Chromatogr. 2003, 42, 43-106. (60) Huang, M.; Yi, G.; Bradshaw, J. S.; Lee, M. L. J. Microcol. Sep. 1993, 5, 199-205. (61) Minnoor, E.; Liu, Y.; Pietrzyk, D. J. J. Chromatogr. A 2000, 884, 297-309. (62) Hsieh, Y.-Y.; Lin, Y.-H.; Yang, J.-S.; Wei, G.-T.; Tien, P.; Chau, L.-K. J. Chromatogr. A 2002, 952, 255-266. (63) Zhang, M.; Yang, C.; Rassi, E. E. Anal. Chem. 1999, 71, 3277-3282. (64) Liu, Q.; Lin, F.; Hartwick, R. A. J. Chromatogr. Sci. 1997, 35, 126-130. (65) Smith, J. T.; Rassi, Z. E. J. High Resol. Chromatogr. 1992, 15, 573-578. (66) Wang, D.; Chong, S. L.; Malik, A. Anal. Chem. 1997, 69, 4566-4576. (67) Shende, C.; Kabir, A.; Townsend, E.; Malik, A. Anal. Chem. 2003, 75, 3186-3198. (68) Chen, Z.; Uchiyama, K.; Hobo, T. J. Chromatogr. A 2002, 942, 83-91. (69) Wu, J.-T.; Huang, P.; Li, M. X.; Qian, M. G.; Lubman, D. M. Anal. Chem. 1997, 69, 320-326. (70) Chong, S. L.; Wang, D.; Hayes, J. D.; Wilhite, B. W.; Malik, A. Anal. Chem. 1997, 69, 3889-3898. (71) Bigham, S.; Medlar, J.; Kabir, A.; Shende, C.; Alli, A.; Malik, A. Anal. Chem. 2002, 74, 752-761. (72) Malik, A. Electrophoresis 2002, 23, 3973-3992. (73) Sun, P.; Landman, A.; Hartwick, R. A. J. Microcol. Sep. 1994, 6, 403-407. (74) Mathews, C. K.; Holde, K. E. v.; Ahern, K. G. Biochemistry 3rd edition, 3rd ed.; Benjamin Cummings: San Francisco, CA, 2000.

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132 (75) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023-1030. (76) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-1644. (77) Quirino, J. P.; Terabe, S. J. Chromatogr. A 1997, 791, 255-267. (78) Kim, J.-B.; Quirino, J. P. J. Chromatogr.A 2001, 916, 123-130. (79) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1242-1252. (80) Nuez, O.; Moyano, E.; Puignou, L.; Galceran, M. T. J. Chromatogr.A 2001, 912, 353-361. (81) Hjerten, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265-270. (82) Quirino, J. P.; Terabe, S. J. High Resol. Chromatogr. 1999, 22, 367-372.

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133CHAPTER FIVE NEGATIVELY CHARGED SOL-GEL COLUMN FOR ONLINE PRECONCENTRATI ON OF ZWITTERIONIC BIOMOLECULES IN CAPILLARY ELECTROMIGRATION SEPARATIONS 5.1 Introduction Capillary electrophoresis (CE), characterized by high column efficiency and separation speed, has been successfully used in a wide range of areas 1-15. While CE provides a number of significant advantages, its use in conjunction with on-column UV detection results in low concentration sensitivity due to short optical path length. To improve the concentration sensitivity in capillary electrophoresis, a number of strategies have been developed such as sample preconcentration, use of alternative capillary geometry, and using other detection modes 16. The first strategy has attracted a great degree of interest since it neither involves any modification of commercially available CE instrument nor does it increase the cost associat ed with using alternative detection modes. In Chapter Four, we have shown the possibility of using a positively charged solgel coating for on-column preconcentration and analysis of amino acids using UV detection. In this approach, the sample pH values were initially maintained above the amino acid isoelectric points. Under these conditions, amino acids (zwitterionic analytes, in general) carried a net negative charge. When such a sample was passed through a capillary column with a positively charged su rface, electrostatic interaction between the solutes and the coating led to effective extraction of the amino acids. The extracted analytes were further desorbed by a local pH change and analyzed by CE. This chapter describes a method for the creation of a negatively charged sol-gel coating in a fused silica capillary and dem onstrates how such a sol-gel column can effectively combine principles of capillary microextraction 17 with those of stacking and focusing techniques 18, 19 to increase the sample concentration sensibility in CE. Another important issue discussed in this chapter is providing a stable EOF in CE. It is well known that among CE operation mo des, such as capillary zone electrophoresis

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134 (CZE), capillary electrochromatography (CEC) and micellar electrokinetic chromatography (MEKC) 20, 21, electroosmotic flow (EOF) plays an important role by serving as the driving force for the mobile phase flow through the column. In CE separation, if EOF changes with time, the migration times of the solutes will change. This may lead to inaccurate results in identification and quantitation 21. Therefore, a stable EOF is highly desired for consistent analytical results in CE separations 21. 5.1.1 The production of a stable EOF As mentioned in previous section, the origin of EOF lies on the electrical double layer formed on the capillary inner wall. Under high electric field, the counter ions on the diffuse layer of the double layer begin to move, and drag the solvated water molecules with them, which give rise to the bulk move ment of the liquid in the columns. In fused silica capillary columns, commonly used in CE separations, the electrical double layer forms as a result of the deprotonation of the silanol groups present on the inner surface of the capillary wall 21, 22. The concentration of deprotonated silanol groups residing on the inner surface of capillary is a major factor that determines the magnitude of EOF 23. In addition, various operational parameters such as the concentration, pH, viscosity of the buffer solution, type of electrolyte used to prepare the buffer solution, and operation voltage also affect the the magnitude of EOF 21, 24. For silanol groups on the fused silica cap illary surface, the pKa value is estimated at around 7.5 25 However, in contact with buffer solutions with pH above 2, some of these silanol groups start dissociating into negatively charged silanate. The dissociation of the silanol groups increases with an increase in buffer pH resulting in an increased EOF, which may cause poor migration time reproducibility in CE when fused silica capillary severs as the separation column A second cause for the migration time fluctuation is the absorptive interaction between the column and solutes like proteins and peptides. Adsorption of solu tes on the capillary surface not only affects consistency in their migration times, but also produces poor separation efficiencies. In fact, not a single mode of binding contributes to protein adsorption 26-29. It is commonly assumed that adsorption is associated with the electrostatic interactions between the net charge on the

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135 molecule and the silica surface 23, hydrogen bonding with OH, NH or CO groups, as well as hydrophobic interaction 30. To minimize the migration time fluctuation problem, several strategies have been developed to prepare columns with stable EOF in CE. One of the most commonly used approaches is to create an appropriate coating on the fused silica capillary inner surface to derivatize or shield the silanol groups. These coatings can be either electrically neutral or carry net charges. In the case of neutral co ating, the separation will only depend on the electrophoresis due to the suppression of EOF. With a charged coating, a relatively stable EOF may be generated if the coating material is carefully selected. Poly(viny1 alcohols) (PVA) with a molecu lar weight about 50 000 were applied by Gilges et al. to modify the fused s ilica surface dynamically and permanently for the separation of charged molecules such as proteins 31. The dynamic thin polymeric coatings were formed by adsorption of PV A on the capillary surface when a PVA solution was passed through the capillary for a short time before actual separations. The generation of PVA coatings is based on the fact that poly(vinyl alcohol) becomes water insoluble by thermal treatment at temperatures of up to 160 C. These PVA coatings successfully suppressed the EOF. Other neutral polymers used as coating materials include methylcellulose 32, poly(methylglutamate) 33, polyethylene glycols 34, 35 and Ucon 36, 37. Angulo and co-workers 38 developed a CE analysis method to study kin17 protein-DNA affinity by using a nonpermanent poly(ethylene oxide) (PEO) based coating to avoid adsorption of kin17. Their coating pr ocedure was as follows: the capillary was first pretreated with NaOH, HCl and Milli-Q water. Then A 0.2% w/v PEO solution in 0.1 M HCl was used to flush through the capillary to achieve reprotonation of the capillary with water and HCl. After each run, the coating was regene rated by a series of rinses with water, HCl as well as a solution of PEO. It is reported that this coating procedure was optimized to provide a residual and stable EOF. 5.1.2 Negatively charged coatings for CE columns Depending on the desired direction of EOF and the properties of analytes, either positively or negatively charged coatings have been created 39-45. In a CE column with a

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136 positively charged inner surface, the diffuse component of the electrical double layer contains anions and generates an anodic el ectroosmotic flow instead of the cathodic EOF produced by untreated fused silica capillary characterized by a negatively charged surface. To generate positively charged sites on the inner surface of column, cryptand-containing polysiloxane 40 and quaternary ammonium group 39, 41-46 have been used. The commonly used negatively charged coatings are prepared using sulfonatecontaining materials 40, 47. Lee and co-workers 40 created sulfonic acid groups on the capillary surface by in situ copolymerization. In their method, the capillary surface was first treated with 7-oct-1-enyltrimethoxysilane. Then, 2-acryloyl-amido-2-2methylpropane sulfonic acid and acrylamide mixtures were copolymerized with `azobis(isobutyronitrile) (AIBN) as an initiator. The most important advantage to use sulfonate-containing materials is that sulfonic acid group, unlike the silanol group, can stay dissociated under low pH conditions. Therefore, a CE column with a surface coating containing the sulfonic acid moiety is expected to provide a stable cathodic EOF within a wide pH range. It should be noted that EOF in an untreated fused silica capillary changes with pH since the silanol groups represent a weak acid whose dissociation is greatly affected by pH changes. Recently, Wiedmer and co-workers 48, 49 developed a method to coat fused silica capillary with anionic liposomes in the pr esence of N-(hydroxyethyl)piperazine-N’-(2ethanesulfonic acid) (HEPES) as background electrolyte solution. The coating was done by rinsing the pretreated capillary with liposomes in HEPES background electrolyte solution for 10 min at a pressure of 930-940 mbar. The coating solution was allowed to stay inside the capillary for 15 min and washed with background electrolyte solution for 10 min to remove unbound liposomes. The cap illary modified with the liposomes coating was successfully used to separate uncharged steroids as model compounds. Among various methods to control EOF in CE, sol-gel approach is a new direction in achieving this goal 36, 41-43, 50-52. For example, Hayes and Malik 36 used sol-gel based technology to prepare a Ucon-coated fu sed silica capillary column in CE. The coating procedure involves (1) preparation of sol solution containing proper ingredients, (2) filling the capillary with the sol solution, and (3) formation of sol-gel network inside the capillary. The obtained solgel coated column was successfully used to separate test

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137 samples (e.g. basic proteins, and nucleotides). Allen and El Rassi 50 developed a two-step preparation method to create positively char ged sol-gel monolithic column in CE, in which the sol-gel silica backbone was first prep ared, and followed by the introduction of organic moieties that contributed to the positive charge on the obtained columns. A similar pathway was reported by these authors to produce sol-gel monolithic columns with cyano or cyano/hydroxyl functional groups 53. By sol-gel technology 54, 55, an in situ created sol-gel layer is chemically bonded to the capillary surface, greatly increasing columns’ operational stability. In the meantime, sol-gel chemistry makes it possible to synthesize organic-inorganic hybrid materials with advanced properties. Columns prepared by sol-gel technology offers many advantages over their conventional counterparts in GC 56, 57, HPLC 58, 59, and CE 36, 42, 43, 51, 60. In this chapter, we described the use sol-gel chemistry to create a sulfonic acidcontaining sol-gel coating on the inner surface of a fused silica capillary. The electrostatic interaction between the analytes and the capillary surface was used to extract analytes from dilute sample solutions. Extraction tec hniques including solid-phase extraction (SPE) 61 and liquid-liquid extraction (LLE) 62 have been used for sample preconcentration in CE. Compared with electrophoresis-based preconcentration methods, extraction-based approaches are more selective, and a wide range of analytes can be preconcentrated by these methods. Here, coupling of the preconcentrator with CE usually requires modification of the instrument, and may be considered as a drawback. In the present study, microextraction and dynamic pH junction were combined to increase sample preconcentration sensitivity in CE using a commercial instrument without any modifications. Sulfonic acid (pKa ~2) 63 is fully ionized over a wide range of pH 40. Therefore, it is less influenced by the change of pH environment compared with silanol groups (pKa ~ 7.5) 25 on fused silica capillary. Thus, the created sol-gel coating not only provided a more stable EOF, but also offered an electrostatic repulsive mechanism to prevent adsorption of anionic bioanalytes on the capillary surface. 5.2 Experimental

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1385.2.1 Equipment On-line sample preconcentration and CE experiments were performed on a BioRad BioFocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA) equipped with a programmable, multi-wa velength UV/Vis detector. BioFocus 3000 operating software system (version 6.00) was used to collect and process the CE data. A Barnstead model 04741 Nanopure deionized water system (Barnstead/Thermodyne, Dubuque, IA) was used to prepare deionized water (~16M cm). A homemade gas pressure-operated capillary filling/purging device 36 was used for coating the fused-silica capillary. A Microcentaur model APO 5760 cent rifuge (Accurate Chemical and Scientific Corp., Westbury, NY) was used for centrifugation of the sol solutions. A Fisher model G560 Vortex Genie 2 system (Fisher Scientific, Pittsburgh, PA) was used for thorough mixing of the ingredients in sol solution. A Chemcadet model 5984-50 pH meter (ColePalmer Instrument Co., Chicago, IL) equipped with a TRIS-specific pH electrode (Sigma-Aldrich, St. Louis, MO) was used to measure the buffer pH. 5.2.2 Chemicals and materials Fused-silica tubing of 50-m i.d. was purchased from Polymicro Technologies (Phoenix, AZ) for the preparation of sol-gel coated columns. Sample vials (600L), HPLC grade methylene chloride and methanol were purchased from Fisher Scientific (Pittsburgh, PA). Mercaptoprop yltrimethoxysilane (MPTMS), n-octadecyltriethoxysilane (C18-TEOS) and phenyldimethylsilane (PheDMS) were purchased from United Chemical Technologies, Inc. (Bristol, PA). Tetramet hoxysilane (TMOS) (99+%), trifluoroacetic acid (TFA) (99%), dimethylsulfoxide (DMSO), and hydrogen peroxide 30 wt. % in water were purchased from Aldrich (Milwaukee, WI). Tris (hydroxymethyl)aminomethane hydrochloride (reagent grade) (Tris-HCl), tris(hydroxymethyl)aminomethane (reagent grade) (Tris-base) as well as myoglobin, conalbumin, arginine, lysine and asparagine standards were purchased from Sigma (St. Louis, MO). The background electrolyte (BGE) solutions were prepared by dissolving Trisbase and/or Tris-HCl in deionized water. The pH adjustments for the BGE solutions and samples were carried out by the addition of 1 M HCl, or 0.1 M NaOH.

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1395.2.3 Preparation of the sol-gel coating solutions The sol-gel solutions were prepared us ing the following ingredients: (a) three precursors (mercaptopropyltrimethoxysilane (MPTMS), tetramethoxysilane (TMOS) and n-octadecyltriethoxysilane (C18-TEOS)), (b) a deactivation re agent (phenyldimethylsilane (PheDMS)) and (c) a sol-gel catalyst (trifluo roacetic acid (TFA) containing 5% water)). The sol solution was vortexed to mix thoroughly and then centrifuged to remove the precipitates (if any). The clear sol solution in the top portion of the centrifuge tube was transferred to a clean vial and was further used to create a negatively charged sol-gel coating on the inner surface of a fused silica capillary. 5.2.4 Preparation of CE columns with a negatively charged sol-gel coating Preparation of the sol-gel open tubular CE columns involved the following two major steps: (1) hydrothermal treatment of the fuse d-silica capillary in ner surface and (2) creation of sol-gel coating on the hydrothermally treated surface. The purpose of the hydrothermal treatment was to clean the capillary inner surface and to enhance the surface silanol concentrations by hydrolysis of siloxane bridges, thereby increasing the bonding sites for chemical anchoring the sol-gel coating. Detailed coating procedure was described elsewhere by Hayes and Malik 42. Briefly, the capillary was coated by using an in-house built gas pressure-o perated filling/purging system 36. Under pressure, the capillary was filled with the sol-gel coating so lution. The sol solution was allowed to stay inside the capillary for a predetermined period (typically 15-30 min) to facilitate the hydrolytic polycondensation reactions responsible for the formation and growth of sol-gel network structures within the sol solution fi lling the capillary. At this point, the sol solution in the capillary was still a liquid and contained fragments of sol-gel network. In the course of sol-gel process, a portion of the sol-gel network fragments evolving in the vicinity of the fused silica capillary inner wall became chemically bonded to it via condensation reaction between the silanol gro ups on the capillary walls and the sol-gelactive functional groups on the sol-gel network. This bonded part of the sol-gel material formed the surface coating. The unbonded liquid portion of the partially gelled sol solution was then removed from the capillary using helium pressure (40 psi), and the coated capillary was conditioned at 150 C for 2 hours. To convert the mercaptopropyl

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140 groups into sulfonate moieties 64, a solution containing 30 wt % hydrogen peroxide was subsequently passed through the column at 40 psi for 5 hours. The coated capillary with negatively charged sulfonate groups in the surface-bonded sol-gel coating was further conditioned at 100 C for 10 hours. This step was followed by rinsing the sol-gel coated columns with 1 mL each of deionized water and the desired running buffer at 100 psi using the filling/purging system. After installation in the CE system, before the first run the column was further rinsed with deionized water and running buffer for 10 min each at 100 psi. Before each replicate run, the column was rinsed at 100 psi with DI water and running buffer but for a shorter time depending on the length of the column. 5.2.5 Preparation of samples Myoglobin, conalbumin, arginine, lysine and asparagines purchased from Sigma (St. Louis, MO) were dissolved in deio nized water to make the test samples. Concentrations of each sample are shown in section 5.3. 5.3 Results and discussion 5.3.1 Sol-gel reactions involved in the coating process Table 5.1 lists the names and chemical structures of sol-gel solution ingredients used in the present study. The ethoxysilyl groups in C18-TEOS and methoxysilyl groups in TMOS and MPTMS are sol-gel-active. They participate in the formation of the sol-gel polymeric network through hydrolytic polycondensation reactions. Scheme 5.1 illustrates complete hydrolysis of MPTMS, TMOS and C18-TEOS. This is a simplified scheme and illust rates only one possible outcome of the hydrolysis reactions. It is implied that part ial hydrolysis of the alkoxy groups is possible and the reaction mixture is likely to contain a variety of other products with varying numbers of hydrolyzed alkoxy groups. The hydrolysis products from sol-gel coprecursors can further enter into polycondensation reactions, which provide a simple and effective pathway for the formation of a sol-ge l network inside the capillary. A simplified scheme of polycondensation reactions likely to take place in the sol-gel process is presented in Scheme 5.2.

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141 Table 5.1 Names and chemical structures of the reagents used in the sol solution to fabricate negatively charged sol-gel sulfonated columns. Regent Function and Reagent Name Reagent Structure Sol-gel precursors: Tetramethoxysilane (TMOS) OCH3Si OCH3OCH3H3CO n-octadecyltriethoxysilane (C18-TEOS) Si(CH2)17H5C2O OC2H5OC2H5CH3 Mercaptopropyltrimethoxysilane (MPTMS) Si(CH2)3H3CO OCH3OCH3SH Deactivation reagent: Phenyldimethylsilane (PheDMS) Si H C H3CH3 Catalyst: Trifluoroacetic acid (TFA) HO O CF3

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142 Scheme 5.1 Illustration of the complete hydrolysis of sol-gel precursors a. Hydrolysis of MPTMS: 3H2O 3CH3OH Si(CH2)3 OCH3 H3CO OCH3 SH Si(CH2)3 OH HO OH SH b. Hydrolysis of TMOS: SiOCH3 OCH3 H3CO OCH3 SiOH OH HO OH 4H2O 4CH3OH c. Hydrolysis of C18-TEOS: Si(CH2)17 OC2H5 C2H5O OC2H5 CH3 3H2O Si(CH2)17 OH HO OH CH3 3C2H5OH

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143 Scheme 5.2 Condensations of hydrolysis products from TMSO, MPTMS, and C18-TEOS x Si(CH2)3HO OH O H O H Si OH OH HO Si(CH2)17CH3HO OH O H +y u SH -nH2O Si(CH2)3O O SH O Si O O O Si(CH2)17CH3O O O Si O O Si O O HO Si * + OH m

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144 The condensation process may further extend to chemically bind a portion of the evolving sol-gel network to th e silanol groups on the cap illary inner surf ace (shown in Scheme 5.3). The deactivating reagent, PheDMS, was a dded to the sol solution to derivatize residual silanol groups on the sol-gel coating or the capillary surface during thermal conditioning step that follows the coating process, and thereby provides deactivation of the sol-gel coated capillary (Scheme 5.4). MPTMS possesses the mercaptopropyl group, which is further oxidized into sulfonic acid moiety by passing hydrogen peroxide 64 through the coated capillary. Scheme 5.5 shows the oxidation of mercaptopropyl group into sulfonic acid.

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145 Scheme 5.3 Covalent bonding of the sol-gel coating to fused silica surface Si(CH2)3O O S H O Si O O O Si(CH2)17CH3O O O Si O O Si O O HO Si * Si O OH O -H2O Si(CH2)3O O SH O Si O O O Si(CH2)17CH3O O O Si O O Si O O Si Si O O O Silica surface Sol-gel coating bonded to the silica surface OHm m OH Silica surface

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146 Scheme 5.4 Deactivation of the sol-gel mediated fused-silica coated surface with PheDMS Si(CH2)3O O SH O Si O O O Si(CH2)17CH3O O O Si O O OH Si O O Si * Si O O O SiH H3C CH3 -H2 Si(CH2)3O O SH O Si O O O Si(CH2)17CH3O O O Si O O O Si O O Si Si O O O Si H3C CH3 Sol-gel coating bonded to silica surface Deactivated sol-gel coating bonded to silica surface Silica s u rface m Silica surface m

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147 Scheme 5.5 Oxidation of mercaptopropyl group into sulfonic acid moiety by H2O2Si(CH2)3O O SH O Si O O O Si(CH2)17CH3O O O Si O O O Si O O Si * Si O O O Si H3C CH3 3H2O2 Si(CH2)3O O SO3H O Si O O O Si(CH2)17CH3O O O Si O O O Si O O Si Si O O O Si H3C CH3 -3H2O Si(CH2)3O O SO3O Si O O O Si(CH2)17CH3O O O Si O O O Si O O Si Si O O O Si H3C CH3 Deprotonation Deactivated sol-gel coatingbondedto silica surface Deactivated sulfonated solgelcoatingbondedto silica surface Negatively charged solgelcoatingbondedto silica surface Silica surface Silica surface Silica surface m m m

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1485.3.2 Characterization of the sol-gel coating A series of electroosmotic flow measurements were performed to investigate the magnitude of the EOF under different pH conditions using dimethylsulfoxide (DMSO) as an EOF marker. Since the sulfonic acid moie ty is primarily responsible for the negative charge on the prepared sol-gel columns, the relative concentration of MPTMS in the sol solution used to prepare these columns is impor tant. To show the effect of the sol solution MPTMS content on the EOF at different pH co nditions, sol solutions with three different MPTMS-to-total precursor molar ratios were used to coat the capillaries. Table 5.2 lists the relative compositions of precursors in the used sol solutions. Figure 5.1 depicts the results from EOF evaluations on two sol-gel sulfonated columns and one uncoated fused silica capillary. As can be seen in Figure 5.1, the buffer pH has less influence on the EOF of sulfonated sol-gel columns. Also, the effect of buffer pH on the EOF decreases with the increase of MPTMS content in the sol solutio n which translates into an increase of sulfonate groups on the surface of the capilla ry. The silanol groups on the fused silica capillary surface is estimated have a pKa value of about 7.5 25. Unlike the silanol group on the fused silica, sulfonic acid has a pKa of about 2 63, and can stay dissociated at low pH conditions. However, the point of zero net charge generally recommended for fused silica in the field of capillary electrophoresis is pH 2.0 21, 22, 65. The measured EOF values for sulfonated sol-gel coated columns under acidic conditions are higher than that for uncoated fused silica cap illary. As shown in Figure 1, when pH was 3.75, the EOF obtained from the sol-gel coated column ( 74% MPTMS in the sol solution) was 1.57 x 10-4 cm2/V.s, which increased 162% compared with the value of 5.99 x 10-5 cm2/V.s obtained from an uncoated fused silica capillary. This significant difference in EOF between sulfonate coated and uncoated fused silica columns can be explained by the fact that sulfonic acid represents a much stronger acid than silanol. Under basic buffer conditions, the measured EOF values for the sol-gel sulfonated columns were lower than that of bare silica columns. For example, at pH 8.88, the EOF measured from the sol-gel coated column (74% MPTMS in the sol solution) was 3.84 x 10-5 cm2/V.s, which was 34% lower than the EOF value obtained on an uncoated column at same pH (5.81 x 10-5 cm2/V.s). Under such conditions, both silanol and sulfonic acid groups dissociate. The

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149 Table 5.2 MPTMS – tototal sol-gel precurso r molar ratio in the coating sol solutions used in the fabrication of negatively charged sol-gel sulfonated columns Column I Column II Column III Sol-gel precursors: mol. % mol. % mol. % Tetramethoxysilane (TMOS) 46% 26% 14% n-Octadecyltriethoxysilane (C18-TEOS) 14% 14% 12% Mercaptopropyltrimethoxysilane (MPTMS) 40% 60% 74%

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150 0.00E+00 2.00E-04 4.00E-04 6.00E-04 8.00E-04 01234567891011pHEOF (cm2/V.s)uncoated 40% MPTMS 74% MPTMS Figure 5.1 Electroosmotic flow vs. buffer pH values. 30 cm x 50 m i.d. columns, running buffer 50 mM tris-base, DMSO as the neutral marker, V = + 9 kV, injection 10 psi s, UV detection at 214 nm.

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151 lower EOF infers that the density of negative charge on the column surface has been reduced. The overall profile of the curves shows that pH has less prominent influence on EOF of the sol-gel sulfonated columns. However, the different EOF values obtained under basic and acidic conditions indicate that some silanols, either on the fused silica capillary surface or the sol-gel coating, are not shielded or deactivated. The effect of sol-gel coating on the repeatability of EOF was investigated by comparing relative standard deviations (RSD) for replicate EOF measurements carried out on a sulfonated sol-gel columns and an uncoated fused silica capillary of same dimensions (75 cm x 50 m i.d.) at pH 2.2 and 8.0, respectively. The sulfonated sol-gel column provided more consistent EOF than th at for the bare fused silica capillary as reflected by the smaller RSD values for the sol-gel sulfonated column. For example, the RSD value obtained on a sol-gel sulfonated column (MPTMS 40%) for 5 replicate runs at pH = 2.2 was 4.8%; while the RSD value obtained on an uncoated fused silica capillary was 24% under the same pH conditions. Figure 5.2 shows the run-to-run EOF repeatability data obtained on a sol-gel sulfonated column in four replicate experiments performed at pH 8.8 (10 mM buffer solution), using DMSO as the EOF marker. The migration times measured was (4.56 0.03) min. In order to investigate the reproducibility of the described coating procedure, the electroosmotic mobility has been measured on 5 columns prepared by the developed method. Dimethylsulfoxide was used as the EOF marker, and 50 mM Tris buffer (pH = 8.8) as the background electrolyte. An RSD value of 6.8% was obtained, which is indicative of good column-to-column reprod ucibility for the described sol-gel coating procedure.

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152 Table 5.3 The effect of sol-gel coating on the repeatability of migration time of a neutral EOF marker, DMSO pH = 8.0 pH = 2.2 Uncoated1 tR (min) Coated2 tR (min) Uncoated1 tR (min) Coated2 tR (min) Run #1 11.18 14.38 31.77 24.86 Run #2 12.08 14.45 46.66 22.05 Run #3 11.04 14.46 41.04 22.83 Run #4 10.97 14.42 51.94 22.49 Run #5 10.88 14.37 56.97 23.67 RSD (%) 4.3 0.3 21 4.8 1: Uncoated fused silica capillary column 75 cm x 50 m i.d. 2: Fused silica capillary column 75 cm x 50 m i.d. with a sol-gel sulfonated coating (MPTMS 40%). Operation conditions: sample DMSO, buffer 50 mM Tris HCl/Base for pH 8.0, and 50 mM Tris HCl for pH 2.2, applied voltage +15.00 kV, injection 10 psis, UV detection at 214 nm.

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153 0 2 4 6 8 10 12 14 16 18 05101520 min.mAU 4.54min 4.52min 4.57min 4.61min Figure 5.2 Migration time repeatability for a sol-gel sulfonated column. Operation conditions: column 50 cm x 50 m i.d., sample DMSO, buffer 10 mM 50/50 Tris HCl / Base pH 8.8, applied voltage + 15.00 kV, injection 10 psisec, UV detection at 214 nm.

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154 5.3.3 The zeitterionic solutes preconcent ration mechanism operated in the negatively charged sol-gel columns The attractive electrostatic interaction of the zwitterionic solutes (protein or amino acid) and the negative charge on the inner walls of the sol-gel coated fused silica capillary forms the basis for solute preconcentration on the described sol-gel columns. When such a zwitterionic test analyte is passing through the sulfonated sol-gel column with the opposite charge on the surface co ating, the positively charged analytes will electrostatically interact with the sol-gel coating leading to their extraction. Therefore, microextraction is accomplished by passin g the dilute sample solution through the column. In the presented method, the sample volume that can be used for analyte enrichment is not limited by one column volume as imposed by some other preconcentration techniques 18, 66. The sol-gel approach allows for the injection of multiple column volumes of the sample. By continuously passing the zwitterionic sample solution for an extended period through the sol-gel coated capillary with enhanced surface area and appropriate surface charge, the extraction can be accomplished from a large volume of the sample. With this desc ribed preconcentration method, sample was introduced into the column at 100 psi for an extended period. For example, sample was injected at 100 psi for 30 sec into a 30 cm long column. This approximately corresponded to more than 7 capillary volumes of the sample passed through the column for on-line extraction. This is an important advantage of the described on-line sample preconcentration method compared with other methods 18, 19, 66, 67, in which the possible maximum volume of the sample that could be injected into the CE system is limited by the volume of the column itself. It should be pointed out that in the case of very dilute samples, even one column volume of the samp le may not be enough to provide detectable amount of the analyte. The extracted analytes are then desorbed from the sol-gel coating by using a running buffer with a pH value above the pI of the extracted analytes. Under such pH conditions, the extracted zwitterions assume a net negative charge and desorb from the

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155 negatively charged sol-gel coating due to mu tual repulsion. The de sorbed zwitterions can be further focused and analyzed. The focusing of desorbed analytes was achieved by taking advantage of the dynamic pH junction between the sample solution and the background electrolyte zone 68. A schematic diagram (Figure 5.3) is presented below to illustrate the whole process including sample injection, extraction, desorption, and focusing of myoglobin or asparagine used as representative zwitterionic test biomolecules. As shown in Figure 5.3, after an extended period of sample injection (e.g., 30 s at 100 psi), facilitates the extraction of positively charged analytes on the negatively charged sol-gel coating (2a). A high electric field (e.g., E = +300 V/cm) is then applied using a running buffer (pH = 8.8), whose pH is greater than the pI of extracted analyte (for myoglobin pI ~ 7.0 69, for asparagine pI ~ 5.4 70) (2b). Under the effect of high electric field, a cathodic EOF is generated which drives the buffer (e.g. pH = 8.8) towards the cathode. Once the basic buffer enters the capillary filled with sample solution, a dynamic pH junction is formed between the running buffer and the sample solu tion. By coming in contact with the basic buffer, the extracted zwitterionic analyte molecules in the junction area acquire a net negative charge, and desorb from the sol-gel coating because of the repulsive forces between same sign of the charge on the anal yte and the sol-gel surface. The direction and migration rate of the desorbed analytes are under control of two opposite forces: (a) EOF and (b) electrophoretic mobility of the desorbed analyte molecules that have now turned into anions. Under the applied electric field, the negatively charged analytes residing in the buffer would tend to migrate toward the anode. Meanwhile, the EOF would tend to drive them toward the cathode. Since EOF is generally stronger than electrophoretic mobility, overall the analytes will move towa rd the cathode, pass through the on-column UV detector window and get detected.

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156a. Filling the capillary with the sample and analytes will be extracted on the capillary surface. b. Applying a high electric field V= +9 kV to generate EOF Anode Cathode c. Buffer solution is introduced into the capillary by EOF and a dynamic pH junction is formed to desorb extracted analytes out of the capillary. Anode Cathode EOF Cathode EOF anion cation neutral analyte Anode EOF window window dynamic pH junctiond. Desorbed analytes are focused at the border of pH junction and are carried to pass through the detector window. Figure 5.3 Illustration of on-line preconcentration using a negatively charged sol-gel column

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157 5.3.4 On-line preconcentration of zwitterionic molecules 5.3.4.1 On-line preconcentration of amino acids using sulfonated sol-gel columns A sol-gel coated column (74% MPTMS) was used to preconcentrate some test amino acids: argnine, lysine, asparagine and histidine using preconcentration method 1 described in Chapter Four. Table 5.4 lists the chemical structures and some physical properties of these test amino acids. Briefly, a sample was introduced into sol-gel column for an extended period (180 s.) which approximately corresponded to 25 capillary volumes. Then, sample matrix was removed from the column by a flow of deionized water while most of the extracted amino acid molecules remained attached to the capillary surface due to electrostatic attractive forces between the positively charged solute ions and the negatively charged capillary surface. After this, a high voltage was applied and electroosmotic flow was generated in a basic buffer solution, which formed a dynamic pH junction (the buffer between deioinized water) facilitating local pH change. The local pH change reversed the electrical charge on the extracted cations and led to the desorption from the surface due to electrical repulsive forces. The high voltage focused the desorbed analytes into narrow zones and detected when passing through the on-column optical window. Figure 5.4 presents an electropherogram representing the preconcentration of arginie on a sol-gel sulfonated column. As shown in Figure 5.4, remarkably high sample concentration sensitivity was achieved with the described method using the sulfnoated sol-gel column. It should be pointed out that the sample concentration was 5000 times higher for the used uncoated fused silica capillary column for which conventional injection was used. Except the injection protocol, all other operation conditions were the same. The electropherogram of arginine obtained with the preconcentration method shows 1382 mAU peak height, 54607 arbitrary units fo r peak area and 4472 arbitrary units for corrected peak area. While the electrophorogram obtained on an uncoated fused silica capillary column shows a peak with 3014 mAu peak height, 304799 arbitrary units for peak area and 97692 arbitrary units for corrected peak area.

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158 Table 5.4 Chemical structures and some physical properties of the test amino acids in this work Sample name Chemical structure Molecular weight (daltons) pI* Arginine H2NCH C CH2 OH O CH2 CH2 NH C NH 2 NH 174.20 10.8 Asparagine H2NCH C CH2 OH O C NH2 O 132.12 5.4 Lysine H2NCH C CH2 OH O CH2 CH2 CH2 NH 2 146.19 9.5 *: Data were calculated according to the procedure described in literature 70

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159 -2 -1 0 1 2 3 4 05101520 min.mAU A B Figure 5.4 Illustration of zwitterionic samp le preconcentration on a negatively charged sol-gel sulfonated column using arginine as a test sample. Oper ation conditions for electropherogram A: sample 50 mM arginine, uncoated column 50 cm x 50 um, buffer 10 mM Tris-base pH 8.86, injection 10 psi sec, V = +15.00 kV, polarity + to -, wavelength 214 nm. Conditions for electropherogram B: sample 10 M arginine, coated column 50 cm x 50 m, buffer 10 mM Tris-base pH 8.86, injection 100 psi for 180 s, UV detection at 214 nm. sample matrix was removed by deionized water, V = + 15.00 kV, polarity + to -.

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160 Preconcentrations of lysine and asparagines on a sol-gel sulfonated column are demonstrated in Figure 5.5 and 5.6, respectively. The repeatability of the sample preconcentration method was examined by a series of experiments and shown in terms of relative standard deviation of the migration time, peak height, peak area as well as corrected peak area. Table 5.5 shows the re peatability of the preconcentration method in five replicate experiments. As shown in Ta ble 5.5, quite good repeatability was obtained considering that these RSD values pertain to a sample preparation technique. The RSD values of migration times are in the range of 2.2% 5.9%. The RSD values in terms of peak height are no more than 7.5%. The RSD values of peak area and corrected peak area are in the range of 6.2% to 8.7%. For the presented method, the sensitiv ity enhancement factors (SEF) were calculated according to the method described by Quirino and Terabe 67, using the test amino acid samples. The equation is as follows. factor dilution method ration preconcent without obtained parameter peak method ration preconcent with obtained parameter peak SEF The SEFs calculated for the test amino acid analytes are presented in Table 5.6. The SEF values were calculated by peak height and peak area, obtained from preconcentration and traditional method. As can be seen in this table, remarkably high sample enrichment factors were achieved using the described sample preconcentration method. For example, the SEF value for analyte lysine reaches 7381 in terms of peak height.

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161 Figure 5.5 Illustration of zwitterionic samp le preconcentration on a negatively charged sol-gel sulfonated column using lysine as a test sample. Operation conditions for electropherogram A: sample 50 mM lysine, uncoated column 50 cm x 50 m, buffer 10 mM Tris-base pH 8.86, injection 10 psi sec, V = +15.00 kV, polarity + to -, UV detection wavelength 214 nm. Conditions for electropherogram B: sample 10 M lysine, sol-gel sulfonated column 50 cm x 50 m, buffer 10 mM Tris-base pH 8.86, injection 100 psi for 180 s, UV detection 214 nm, sample matrix was removed by deionized water, V = + 15.00 kV, polarity + to -.

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162 -3 -1 1 3 5 7 9 051015 min.mAU A B Figure 5.6 Illustration of zwitterionic samp le preconcentration on a negatively charged sol-gel sulfonated column using asparagine as a test sample. Operation conditions for electropherogram A: sample 50 mM lysine, uncoated column 50 cm x 50 m, buffer 10 mM Tris-base pH 8.86, injection 10 psi sec, V = +15.00 kV, polarity + to -, UV detection wavelength 214 nm. Conditions for electropherogram B: sample 10 M asparagine, sol-gel sulfonated column 50 cm x 50 m, buffer 10 mM Tris-base pH 8.86, injection 100 psi for 180 s, UV detection 214 nm, sample matrix was removed by deionized water, V = + 15.00 kV, polarity + to -.

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163 Table 5.5 Repeatability of sample preconcentration on a sulfonated sol-gel column using amino acids as test solutes Arginine Lysine Asparagine tR Peak Height Peak Area Corrected Area tR Peak Height Peak Area Corrected Area tR Peak Height Peak Area Corrected Area (min) (mAU) (arbitrary unit) (arbitrary unit) (min)(mAU)(arbitrary unit) (arbitrary unit) (min) (mAU)(arbitrary unit) (arbitrary unit) Run #1 12.71 1141 62093 4885 10.031848 71277 7106 9.63 2102 68133 7075 Run #2 12.33 1273 52539 4261 9.96 1935 69586 6987 9.72 2197 70607 7264 Run #3 12.07 1285 51592 4274 9.61 1965 62053 6457 8.94 2222 76197 8523 Run #4 11.85 1368 50200 4236 9.56 1995 66662 6973 8.85 2325 63798 7209 Run #5 12.21 1382 54607 4472 9.92 2138 68470 6902 10.13 1977 70850 6994 RSD(%)2.6 7.5 8.7 6.2 2.2 7.0 4.7 3.6 5.9 5.9 6.5 8.3 Operation conditions: 50 cm x 50 m i.d. sulfonated sol-gel column, 10 mM Tris buffer pH 8.8, 10 M arginine, lysine, and asparagines samples. Samples were injected at 100 psi for 180 s. Sample matrix was removed by DI water at 100 psi for 30 sec. V = + 15.00 kV, polarity + to -, UV detection wavelength 214 nm

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164 Table 5.6 Sensitivity enhancement factors obta ined on a sulfonated sol-gel columns using amino acids as test solutes SEF Sample By height By area By corrected area Arginine 5310 659 145 Asparagine 2010 1343 422 Lysine 7381 1518 364 Operation conditions for preconcentration experiment: 50 cm x 50 m i.d. sulfonated solgel column, 10 mM buffer pH 8.8, 10 M arginine, lysine, and asparagine. Samples were injected at 100 psi for 180 s. Sample matrix was removed by DI water at 100 psi for 30 sec. V = + 15.00 kV, polarity + to -, wa velength 214 nm. Conditions for traditional injection method: 50 cm x 50 m i.d. uncoated fused silica capillary column, 10 mM buffer pH 8.8, 50 mM arginine, lysine, and asparagine. Samples were injected at 10 psi sec. V = + 15.00 kV, polarity + to -, wavelength 214 nm.

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165 In the foregoing experiments, deionized water was used to remove the sample matrix after extraction was accomplished. In this process, a portion of the analytes extracted on the sol-gel surface of the column could have been rinsed out with sample matrix. To prevent this loss, a second preconcentration method was designed without the removal of matrix. The method was illustrated in section 5.3.3 of this chapter. Figure 5.7 illustrates two electropherograms of asparagine obtained on (a) a solgel sulfonated column using the preconcentration method and (b) an uncoated column with traditional injection. The asparagine sample enriched on the sol-gel coated column had a concentration 1000 times smaller than that used on the uncoated column. The peak height obtained on the sol-gel column was 8.9 mAU, and the corrected peak area was 2.01 x 106 arbitrary units. The electropherogram shown in Figure 6b was obtained on an uncoated column with traditional injection. The peak height and corrected peak area were 2.0 mAu and 2.74 x 105 arbitrary units, respectively. The sensitively was greatly increased by using the presented preconcentration method. SEF based on peak height was 4450, and SEF based on corrected peak area was 7335. Compared with the SEF of asparagine by preconcentration method with th e removal of sample matrix (2010 by peak area and 422 by corrected peak area), it cl early shows that the preconcentration method without the removal of sample matrix is more effective in sensitivity enhancement.

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166 -5 0 5 10 05101520min.mAU. a b Figure 5.7 Preconcentration of asparagine on negatively charged sol-gel coated column. Running buffer 50 mM Tris-Base (pH = 8.8), V = +9 kV, UV detector wavelength 214 nm. (a) injection for 30 sec at 100 psi, sample concentration 10 M (pH = 7.1); 60% MPTMS sol-gel coated 30 cm x 50 m ID colum; (b) injection at 10 psisec, 30 cm x 50 m ID uncoated column, sample concentration 10 mM.

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1675.3.4.2 On-line preconcentration of proteins using sulfonated sol-gel columns The 74% MPTMS sol-gel coated column was used to preconcentrate protein test samples. Figure 5.8 and 5.9 illu strate the comparison of tw o electropherograms obtained on a sol-gel coated column by preconcentration method without the removal of sample matrix and an uncoated fused silica capillary column with traditional sample injection. Figure 5.8 shows the results obtained from a protein test sample conalbumin, while Figure 5.9 demonstrates the results from myoglobin. Both figures indicate significant sensitivity enhancement using the preconc entration method on sulfonated sol-gel columns. For instance, the sol-gel column preconcentrated a 3.6 ppm conalbumin sample and gave a peak height of more than 3.5 mAU in Figure 5.8 (B). While with an uncoated column and conventional mode of sample injection, a 1797 ppm conalbumin sample gave an absorbance signal with peak height of around 1.5 mAu (shown in Figure 5.8 A). In Figure 5.9, the peak height obtained from a 2195 ppm (125 M) myoglobin sample was only 4.1 mAU (Figure 5.9 A). This value increased to more than 39 mAU (Figure 5.9 B) on sulfonated sol-gel column with a 21.95 ppm (1.25 M) myoblobin sample preconcentrated by the described method. Based on the results shown in Figure 5.8 and Figure 5.9, sensitivity enhancement factors (SEFs) were calculated. For conalbumin, the SEFs were 1143, 1012 and 2436 in terms of peak height, peak area and corrected peak area, respectively. For myoglobin, SEFs were 973, 3769, 3104 in terms of peak height, peak area and corrected peak area, respectively.

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168 -1 0 1 2 3 4 5 6 05101520min.mAU A B Figure 5.8 Preconcentration of conalbumin on a negatively charged sol-gel coated column. Running buffer 50 mM Tris-base (pH = 8.8), UV detector: 214 nm. A. uncoated fused silica capillary column 30 cm x 50 m i.d., sample 1797 ppm conalbumin solution (pH = 7.0), sample injection 10 psisec. V = +9 kV. B. Sulfonated sol-gel column 30 cm x 50 m i.d., sample 3.6 ppm conalbumin solution (pH = 7.0), sample injection 100psi for 30 sec. V = +9 kV.

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169 -10 -5 0 5 10 15 20 25 30 35 40 45 05101520min.mAU AB Figure 5.9 Preconcentration of myoglobin on a negatively charged sol-gel coated column. Running buffer 50 mM Tris-base (pH = 8.8), UV detection at 214 nm. A. uncoated fused silica capillary column 30 cm x 50 m i.d., sample 2195 ppm (125 M) myoglobin solution (pH = 7.1), sample injection 10 psisec. V = +9 kV. B. Sulfonated sol-gel column 30 cm x 50 m i.d., sample 21.95 ppm (1.25 M) myoglobin solution (pH = 7.1), sample injection 100psi for 60 sec. V = +9 kV.

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170 In order to investigate the influence of MPTMS content in the sol-gel coating solution preconcentration effect, three sulfonated sol-gel columns with different MPTMS composition were used to preconcentrate a protein sample (myoglobin) under the identical operation conditions. Figure 5.10 presents the electropherograms of myoglobin obtained after preconcentration on three different sulfonated sol-gel columns prepared by the described method using sol solutions containing (a) 40%, (b) 60%, and (c) 74% of MPTMS. For comparison, under identical conditions a myoglobin sample of 100-fold higher concentration was injected into an uncoated fused silica capillary using a conventional electromigration injection (Figure 5.10 d). In the obtained electropherogram (shown in Figure 5.10 d), the peak height and corrected peak area were 4.1 mAU and 3.32 x 104 arbitrary units, respectively. With the increase of the MPTMS content in the sol solution, the active extraction sites on the sol-gel coating also increased. Therefore, the preconcentration effect was enhanced. It should be pointed out that the sample concentration applied to the sol-gel coated columns was 100 times less than that of applied on the uncoated column. The peak height obtained on column “a” (prepared by using 40% MPTMS in the sol solution) was 4.0 mAU. This value increased to 39.9 mAU on column “c” (prepared by using 74% MPTMS in the sol solution). For these two columns, the corrected peak area (in arbitrary unit) for myoglobin increased from 7.68 x 104 (Figure 5.10a) to 1.03 x 106 (Figure 5.10c).

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171 Figure 5.10 Influence of sol solution MPTMS composition on the preconcentration of myoglobin on a negatively charged sol-gel column. Running buffer: 50 mM tris-base (pH = 8.8), UV detection at 214 nm. a: 40% MPTMS 30 cm x 50 m ID column. b: 60% MPTMS 30 cm x 50 m ID column. c: 74% MPTMS 30 cm x 50 m ID column.. a, b and c used sol-gel columns and the described preconcentration method. Sample: 1.25M myoglobin d: uncoated 30 cm x 50 m ID column, conventional injection (10 psi s). sample: 125 M myoglobin.

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172 Based on the results shown in Figure 5.10 sensitivity enhancement factors (SEFs) were calculated relative to an uncoated co lumn operated using traditional injection. Calculation based on peak heights provided SEF values of 97, 549, and 973 for column prepared by using sol solution containing 40%, 60% and 74% MPTMS, respectively. Analogous calculation made on the basis of corrected peak areas gave SEF values of 232, 1739, and 3104, respectively. The preconcentration effect of the negatively charged solgel coating on the test protein samples is summarized in Table 5.7. It is noticed that larger SEFs were obtained for the amino acid sample asparagine compared with those for the protein analyte. This may be explained by the differences in molecular sizes of these analytes. Being smaller in size, more amino acid molecules can be extracted on the surface of sol-gel coating than the much larger protein molecules. The run-to-run repeatability of the extraction process for myoglobin was investigated in terms of migration time, peak area, corrected peak area (peak area divided by migration time), and peak height RSDs. The RSD values in terms of migration time, peak heights, peak areas, and corrected peak areas were 5.1%, 6.0%, 3.4% and 7.8%, respectively. Figure 5.11 illustrates extraction repeatability using electropherograms for myoglobin obtained in replicate extraction experiments. The repeatability of preconcentration effect for protein test sample conalbumin is listed in Table 5.8. The presented RSD values (4.1% ~ 8.3%) indicate that the repeatability of the preconcentration method is quite good, considering that these RSD values pertain to a sample preparation technique and that the used solute is a biological macromolecule.

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173 Table 5.7 Sensitivity enhancement factors (SEFs) obtained on sol-gel coated columns using proteins as test solutes SEF sample column by peak height By peak area by corrected peak area myoglobina 40%MPTMS sol-gel column 97 215 232 myoglobina 60%MPTMS sol-gel column 549 1539 1739 myoglobina 74%MPTMS sol-gel column 973 3769 3104 Conalbuminb 74%MPTMS sol-gel column 1143 1012 2436 a Operation conditions: Preconcentration of myoglobin on negatively charged sol-gel columns 30 cm x 50 m i.d. Running buffer 50 mM tris-base ( pH = 8.80 ), V = + 9.0 kV, injection for 30 s at 100 psi, UV detection at 214 nm, sample concentration 21.95 ppm (1.25 M) ( pH = 7.1 ). bOperation conditions: Preconcentration of conalbumin on a negatively charged sol-gel column 30 cm x 50 m i.d. Running buffer 50 mM tris-base ( pH = 8.80 ), V = + 9.0 kV, injection for 30 s at 100 psi, UV detection at 214 nm, sample concentration 3.6 ppm ( pH = 7.0 ).

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174 Figure 5.11 Repeatability of myoglobin precon centration on a negatively charged sol-gel sulfonated column. Operation conditions: 40% MPTMS sol-gel sulfonated 30 cm x 50 m i.d. column, buffer 50 mM tris-base (pH = 8.8), applied voltage + 9 kV, injection for 30 s at 100 psi, UV detection at 214 nm, sample myoglobin, concentration 1.25 M (pH = 7.1).

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175 Table 5.8 Illustration of the preconcentration repeatability on a sol-gel sulfonated column using conalbumin as a test sample tR (min) Peak Height (mAU) Peak Area (arbitrary units) Corrected Area (arbitrary units) Run #1 1.99 3593 361531 189323 Run #2 2.01 3889 440466 206512 Run #3 2.01 3772 415090 219137 Run #4 1.99 3548 376752 181674 Run #5 2.01 3554 377366 187744 RSD(%) 0.55 4.1 8.3 7.9

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176 Further experiments were carried out to investigate the sorption/desorption of the ionic analytes on sol-gel sulfonated surface. Sample of asparagine was injected hydrodynamically for 180 seconds at 100 psi. This means that more than 25 column volumes of sample were passed through the column. This was followed by removing the sample matrix with over 4 column volumes of either deionized water (pH = 7.0) or 10 mM neutral buffer (pH = 7.0) or 10 mM running buffer (pH = 8.8) or dilute NaOH solution in water (pH = 8.8). Finally, an electric field was applied using basic running buffer (pH = 8.8) to desorb, focus, and carry the preconcentrated analyte to the detector. Figure 5.12 shows the results. As shown in Figure 5.12, a peak for asparagine was obtained in the electropherograms only wh en the sample matrix was removed by deionized water (Figure 5.12d). On the other hand, no sample peaks could be detected when a basic running buffer (pH = 8.8) was used to remove the sample matrix (Figure 5.12a). An incomplete desorption of the extracted asparagine was observed when a neutral buffer (pH = 7.0) or an aqueous NaOH solution (pH = 8.8) was used (Figure 5.12b and 5.12c).

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177 Figure 5.12 Effect of matrix removing media on preconcentration effectiveness using a sol-gel coated column. Operation conditions: column 50 cm x 50 m i.d. sol-gel sulfonated column prepared using 74% MPTM S in the coating solu tion; running buffer 10 mM tris-base (pH = 8.8); V = +15kV; sample 10 M asparagine; UV detection at 214 nm; sample injection 180 s at 100 psi; sample matrix was removed for 30 s at 100 psi by (A) 10 mM basic buffer (pH 8.8), (B) 10 mM neutral buffer (pH = 7.0), (C) a dilute NaOH solution (pH = 8.8), and (D) deionized water (pH = 7.0).

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1785.4 Conclusions A method was developed for the preparation of capillary columns with a sol-gel coating intrinsically carrying negatively charged sulfonic acid groups. Microextreaction has been successfully combined with dy namic pH junction to perform on-line preconcentration of zwitterionic biomolecules using proteins and amino acids as test samples. The sorption/desorption of the ex tracted analytes may be attributed to electrostatic interaction between the zwitterionic analytes and the negatively charged surface of the capillary as well as the effects arising from the dynamic pH junction and ion-exchange of the buffer. The presented on-line preconcentration method provided a significant enhancement in detection capability of a standard CE system equipped with a UV detector. On-line preconcentration and analysis results obtained on sulfonated sol-gel columns were compared with those obtained on an uncoated fused silica capillary of identical dimensions using conventional sample injections. Using UV detection, the sample preconcentration technique demonstrated here provided a sensitivity enhancement factor of more than 3,000 for myoglobin and more than 7,000 for asparagine. On-line preconcentration of zwitterionic molecules by using a negatively charged sol-gel coated column may prove to be a prom ising approach in trace analys is by CE. In addition, the sulfonated sol-gel column was successfully used to separate a protein mixture. The results indicate the preconcentration method developed here and coating procedure possess quite high run-to-run and co lumn-to-column reproducibilities. 5.5 Reference for Chapter Five (1) Hutchinson, J. P.; Zakaria, P.; Bowie, A. R.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2005, 77, 407-416. (2) Yeung, K.-C.; Lucy, C. A. J. Chromatogr. A 1998, 804, 319-325. (3) Foret, F.; Fanali, S.; Narid, A.; Bocek, P. Electrophoresis 1990, 11, 780-783. (4) Lin, C.-H.; Kaneta, T. Electrophoresis 2004, 25, 4058-4073. (5) Kist, T. B. L.; Mandaji, M. Electrophoresis 2004, 25, 3492-3497. (6) Britz-McKibbin, P.; Terabe, S. Chem. Rec. 2002, 2, 397-404.

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179 (7) Veuthey, J.-L. Anal. Bioanal. Chem. 2005, 381, 93-95. (8) Bosserhoff, A.-K.; Hellerbrand, C.; Buettner, R. Comb. Chem. High Throughput Screening 2000, 3, 455-466. (9) Wyss, R. J. Chromatogr. B 1995, 671, 381-425. (10) Prado, M. S. A.; Steppe, M.; Tarare s, J. F. M.; Kedor-Hackmann, E. R. M.; Santoro, M. I. R. M. J. Pharm. Biomed. Anal. 2005, 37, 273-279. (11) Bowman, J.; Tang, L.; Silverman, C. E. J. Pharm. Biomed. Anal. 2000, 23, 663669. (12) Chadwick, R. R.; Hsieh, J. C.; Resham, K. S.; Nelson, R. B. J. Chromatogr. A 1994, 671, 403-410. (13) McDonald, S.; Bishop, A. G.; Prenzler, P. D.; Robards, K. Anal. Chim. Acta 2004, 527, 105-124. (14) Mahnik, S. N.; Rizovski, B. ; Fuerhacker, M.; Maker, R. M. Anal. Bioanal. Chem. 2004, 380, 31-35. (15) Brueggemann, O.; Freitag, R. J. Chromatogr. A 1995, 717, 309-324. (16) Albin, M.; Grossman, P. D.; Moring, S. E. Anal. Chem. 1993, 63, 489A-497A. (17) Bigham, S.; Medlar, J.; Kabir, A.; Shende, C.; Alli, A.; Malik, A. Anal. Chem. 2002, 74, 752-761. (18) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (19) Chien, R. L.; Burgi, D. D. Anal. Chem. 1992, 64, 489A-496A. (20) Weinberger, R. Anal. Chem. 1991, 63, 823-827. (21) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons, Inc.: New York, N.Y., 1995. (22) Poole, C. F. The Essence of Chromatography; Elsevier Science: Amsterdam, The Netherlands, 2003. (23) Landers, J. P. Handbook of Capillary Electrophoresis; CRC Press: Boca Raton, FL, 1994. (24) Weinberger, R. Practical Capillary Electrophoresis, 2nd ed.; Academic Press: London, UK, 2000. (25) Behrens, S. H.; Grier, D. G. J. Chem. Phys. 2001, 115, 6716-6721.

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180 (26) F. Macritchie J. Colloid Interface Sci. 1972, 38, 484-488. (27) Millot, M. C.; Vidal-Madjar, C. Adv. Chromatogr. 2000, 40, 427-466. (28) Norde, W.; Fraaye, J.; Lykema, J. Protein Adsorption at Solid-Liquid Interfaces: a Colloid-Chemical Approach chap2; ACS: Washington, DC, 1987. (29) Holt, P. F.; Bowcott, J. E. L. AMA Arch. Ind. Hygiene Occupational Med. 1954, 9, 503-506. (30) Horbett, T. A.; Brash, J. L. Protein at Interfaces: Current Issues and Future Prospects Chap.1; ACS: Washington, D.C., 1987. (31) Gilges, M.; Kleemiss, M. H.; Schomburg, G. Anal. Chem. 1994, 66, 2038-2046. (32) Hjerten, S. Chromatogr. Rev. 1967, 9, 122-219. (33) Bentrop, D.; Kohr, J.; Engelhaardt, H. Chromatographia 1991, 32, 171-178. (34) Nilsson, J.; Speg el, P.; Nilsson, S. J. Chromatogr. B 2004, 804, 3-12. (35) Wang, T.; Hartwick, R. A. J. Chromatogr. 1992, 594, 325-340. (36) Hayes, J. D.; Malik, A. J. Chromatogr. B 1997, 695, 3-13. (37) Malik, A.; Zhao, Z.; Lee, M. L. J. Microcol. Sep. 1993, 5, 199-205. (38) Tran, N. T.; Taverna, M.; Miccoli, L.; Angulo, J. F. Electrophoresis 2005, 26, 3105-3122. (39) Minnoor, E.; Liu, Y.; Pietrzyk, D. J. J. Chromatogr. A 2000, 884, 297-309. (40) Huang, M.; Yi, G.; Bradshaw, J. S.; Lee, M. L. J. Microcol. Sep. 1993, 5, 199-205. (41) Hsieh, Y.-Y.; Lin, Y.-H.; Yang, J.-S.; Wei, G.-T.; Tien, P.; Chau, L.-K. J. Chromatogr. A 2002, 952, 255-266. (42) Hayes, J. D.; Malik, A. Anal. Chem. 2001, 73, 987-996. (43) Hayes, J. D.; Malik, A. Anal. Chem. 2000, 72, 4090-4099. (44) Zhang, M.; Yang, C.; Rassi, E. E. Anal. Chem. 1999, 71, 3277-3282. (45) Liu, Q.; Lin, F.; Hartwick, R. A. J. Chromatogr. Sci. 1997, 35, 126-130. (46) Smith, J. T.; Rassi, Z. E. J. High Resol. Chromatogr. 1992, 15, 573-578. (47) Sun, P.; Landman, A.; Barker, G. E.; Hartwick, R. A. J. Chromatogr. A 1994, 685, 303-312.

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181 (48) Hautala, J. T.; Linden, M. V.; Wiedmer, S. K.; Ryhanen, S. J.; Saily, M. J.; Kinnunen, P. K. J.; Riekkola, M.-L. J. Chromatogr.A 2003, 1004, 81-90. (49) Wiedmer, S. K.; Jussila, M.; Hakala, R. M. S.; Pystynen, K.-H.; Riekkola, M.-L. Electrophoresis 2005, 26, 1920-1927. (50) Allen, D.; El Rassi, Z. Analyst 2003, 128, 1249-1256. (51) Guo, Y.; Imahori, G. A.; Colon, L. A. J. Chromatogr.A 1996, 744, 17-29. (52) Malik, A. Electrophoresis 2002, 23, 3973-3992. (53) Allen, D.; Rassi, Z. E. J. chromatogr.A 2004, 1029, 239-247. (54) Brinker, C.; Scherer, G. Sol-Gel Science; Academic Press: Boston, MA, 1990. (55) Iler, R. K. The Chemistry of Silica; John Wiley and Sons, Inc.: New York, 1979. (56) Wang, D.; Chong, S. L.; Malik, A. Anal. Chem. 1997, 69, 4566-4576. (57) Shende, C.; Kabir, A.; Townsend, E.; Malik, A. Anal. Chem. 2003, 75, 3186-3198. (58) Chen, Z.; Uchiyama, K.; Hobo, T. J. Chromatogr. A 2002, 942, 83-91. (59) Ishizuka, N.; Kobayashi, H.; Minakuchi, M. H.; Nakanishi, K.; Hirao, K.; Hosoya, K.; Ikegami, T.; Tanaka, N. J. Chromatogr. A 2002, 960, 85-96. (60) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511-2516. (61) Viberg, P.; Nilsson, S.; Skog, K. Anal. Bioanal. Chem. 2004, 378, 1729-1734. (62) Pedersen-Bjergaard, S.; Rasmussen, K. E.; Halvorsen, T. G. J. Chromatogr. A 2000, 902, 91-105. (63) The Columbia Encyclopedia, 6th ed.; Columbia University, New York, 2001. (64) Margolese, D.; Melero, J. A.; Christians en, S. C.; Chmelka, B. F.; Stucky, G. D. Chem. Mater. 2000, 12, 2448-2459. (65) Altria, K. D. Capillary Electrophoresis Guidbook Principles, Operation, and Applications; Humana Press: Totowa, NJ, 1996. (66) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-1644. (67) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023-1030. (68) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1242-1252. (69) Righetti, P. G.; Caravaggio, T. J. Chromatogr. 1976, 127, 1-25.

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182 (70) Mathews, C. K.; Holde, K. E. v.; Ahern, K. G. Biochemistry 3rd edition, 3rd ed.; Benjamin Cummings: San Francisco, CA, 2000.

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ABOUT THE AUTHOR Wen Li was born in Dalian, Liaoning Province, China. She obtained her Bachelor’s degree in Chemical Engineeri ng from Beijing Institute of Technology. In the Fall of 2000, she enrolled at the Univer sity of South Florida and joined to Dr. Abdul Malik’s research group in the Spring of 2001. The work presented here was conducted under the guidance of Dr. Abdul Malik. During her study at USF, she published several works re lating to her research.