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Use of model compounds to study potential removal of pharmaceuticals using octolig®

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Title:
Use of model compounds to study potential removal of pharmaceuticals using octolig®
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English
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Chang, Wen-shan
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University of South Florida
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Environment
Chromatography
Antibiotic
Amoxicillin
LGB
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: The existence of pharmaceuticals in the environment has some adverse effects, and may pose threat to the organisms in the environment. The possibility of removing certain pharmaceuticals from wastewater was tested using Octolig(R), a commercially available material with polyethyldiamine moieties covalently attached to high-surface area silica gel. Selected drug models were subjected to column chromatography in efforts to effect removal by means of ion encapsulation, the effectiveness of which would depend upon having appropriate anionic functional groups. The experimental results suggested that the model compounds, Rose Bengal, Eosin Y, Erythrosine , ZPS, and Lissamine Green B were successfully encapsulated by Octolig(R), while Methylene Blue with quaternary ammonium groups was (statistically) not. In contrast, complete success was attained for removing of each of three xanthenylbenzenes (Rose Bengal, Eosin Y, Erythrosine) that have both phenolic and carboxylic acid groups. In addition complete success was attained for ZPS (zinc phthalocyaninetetrasulfonate) with sulfonate groups present. A test of a real pharmaceutical compound, Amoxicillin, indicated that Octolig(R) can be used to remove this compound from aqueous media.
Thesis:
Thesis (M.S.)--University of South Florida, 2010.
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Includes bibliographical references.
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by Wen-shan Chang.
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Use of Model Compounds to Study Potential Removal of Pharmaceuticals Using Octolig¨ by Wen shan Chang A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Art and Science University of South Florida Co Major Professor: Kirpal S. Bisht, Ph.D. Co Major Professor : Dean F. Martin, Ph.D. Xiao Li, Ph.D. Date of Approval: April 5, 2010 Keywords: environment, chromatography, antibiotic, Amoxicillin, LGB Copyr ight 2010, Wen shan Chang

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Acknowledgements I would like to acknowledge Dr. Kirpal S. Bisht for his and wisdom guidance and warming advises. I am heartily thankful to his understanding and encouragement when I meet difficulties in my graduated career. I would like to express my sincere gratitude Dr. Dean F. Martin for his support throughout my graduate career. This thesis would not have been possible without his inspiration. I would like to thank Dr. Xiao Li for her excellent teaching that deepe ned my thinking in this research. I thank Ms. Meagan Small for her technical assistance and relevant discussions of my research. I thank to Mr. John Seals for ordering reagents for this project.

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Note to Reader The original of this document contains color that is necessary for understanding the data. The original dissertation is on file with the USF library in Tampa, Florida.

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i Table of Contents List of Tables i ii List of Figures i v Abstract v Intro duction 1 Background 1 Definition and the Use of Pharmaceuticals 2 Occurrence in the En vironment 3 Pathways to the Environment 4 Effects 5 Sources of Pharmaceuticals 7 Assessment 9 Solutions to the Problem 1 0 Statement of the Problem 12 Plan of Attack 13 Experimental Methods 15

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ii Materials and Methods 16 Source of Reagents and Materials 16 Analytical Methods 17 Chromatography Experiment 18 Molar Extinction Coefficient Measurement 18 Test for Fluorescence 20 Application to Amoxicillin 20 Results and Discussion 21 Compounds Selection 21 Medical Uses 25 Aggregation 26 Experiment Results 26 Conclusions 3 3 Literature Cited 39 Appendices 47 Appendices A: Experimental data for mo del compounds 48 Appendices B: Spectra of model compounds 88 About the Author End Page

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iii List of Tables Table 1. Molar extinction coefficients and measured wavelength of model compounds. 19 Table 2. The compound names, CAS numbers, therapeutic uses and chemical formulas of the top five most prescribed pharmaceuticals in 2008 24 Table 3. Passage of aqueous sample over a 2 cm id chromatographic column packed with 22cm of Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). 29 Table 4. Passage of aqueous Amoxicillin sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). 32

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iv List of Figures Figure 1. Possible structure of encapsulation of anions (A n ) by Octolig¨ showing a one arm model. 14 Figure 2. Chemical structure of Octolig¨. 17 Figure 3. Struc tures of the top 5 most prescribed pharmaceuticals in 2008. 22 Figure 4. S tructures of model compounds. 27 Figure 5. pH effect on percentage removal of Rose Bengal. 35 Figure 6. pH effect on percentage removal of Eosin Y. 35 Figure 7. pH eff ect on percentage removal of Erythrosine. 36 Figure 8. pH effect on percentage removal of ZPS. 37 Figure 9. pH effect on percentage removal of Lissamine Green B. 37

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v Use of Model Compounds to Study Potential Removal of Pharmaceuticals Usi ng Octolig¨ Wen shan Chang ABSTRACT The existence of pharmaceuticals in the environment has some adverse effects, and may pose threat to the organisms in the environment. The possibility of removing certain pharmaceuticals from wastewater was tested u sing Octolig¨, a commercially available material with polyethyldiamine moieties covalently attached to high surface area silica gel. Selected drug models were subjected to column chromatography in efforts to effect removal by means of ion encapsulation, th e effectiveness of which would depend upon having appropriate anionic functional groups. The experimental results suggested that the model compounds, Rose Bengal, Eosin Y, Erythrosine ZPS, and Lissamine Green B were successfully encapsulated by Octolig ¨, while Methylene Blue with quaternary ammonium groups was (statistically) not. In contrast, complete success was attained for

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vi removing of each of three xanthenylbenzenes (Rose Bengal, Eosin Y, Erythrosine) that have both phenolic and carboxylic acid grou ps. In addition complete success was attained for ZPS (zinc phthalocyaninetetrasulfonate) with sulfonate groups present. A test of a real pharmaceutical compound, Amoxicillin, indicated that Octolig¨ can be used to remove this compound from aqueous media.

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1 Introduction Background With the rapid developing of human population, the demanding of pharmaceuticals also has increased exponentially. However, without a proper disposal, drugs may contaminate our environment. In recen t years, the occurrence and fate of pharmaceutical substances in the environment have become an emerging issue in the world. As a result, recent decades have seen increase attention being given to potential adverse effects from residues of pharmaceuticals There has been a proliferation of research concerned with pharmaceuticals in the environment ( K Ÿ mmerer, 2008 ). Regarding the potential adverse influences on wildlife and organisms, regulations associated with pharmaceuticals are relatively few in number.

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2 Defini tion and the us e of pharmaceuticals Pharmaceutical drugs are defined as those organic or inorganic compounds used in the diagnosis, cure, mitigation, treatment, or prevention of disease (Buser et al, 1999). Pharmaceuticals are not onl y applied to humans' usage, but the definition of pharmaceuticals can be extended to veterinary and plant pharmaceuticals, or even illicit drugs. What is more, pharmaceuticals are used in aquaculture, livestock farming, horticulture and bee keeping ( KŸmmer er, 2003 ). Addition of pharmaceuticals to water directly can be done through usage in aquaculture, e.g., feeding and injecting (Stuart, 1983). There are more than 100 aquatic species cultured in the United State, but the development of the new pha rmaceuticals which are used for aquatic diseases are scarce. There are only five FDA approved drugs available for aquaculture treatments (Gloyd, 1992) In general, the use of aquaculture pharmaceuticals for treating fish diseases or modulating fish growth is not sophisticated.

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3 Occurrence in the environment Many individual pharmaceuticals and their metabolites are found in the environment and the sewage plants. Many investigations show that pharmaceutically active compounds cannot be totall y eliminated during wastewater treatment, and also can only be partially biodegraded in the environment (Daughton and Ternes, 1999; Heberer, 2002) The issue of persistent pharmaceutical compounds and residues is associated with the occurre nce of pharmaceutical compounds in the environment. Many individual pharmaceutical compounds and metabolites have been found in the environment and the occurrence of pharmaceuticals has been investigated in many countries, including the E.U and the U.S. Th e improvements in analytical techniques are beneficial to the quantitative tracking of pharmaceuticals in queous environments. Several studies (Halling Sorensen et al., 1998; Ku mmerer, 2001; Heberer, 2002; Kolpin et al., 2002; Boyd et al., 2003) have noted that pharmaceuticals are present in wastewater treatment plant effluents, hospital wastewater effluents, surface water, ground water, and this will likely result in indirect h uman exposure to pharmaceuticals via drinking water supplies. A national survey released in 2002 reported that pharmaceuticals, hormones and other

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4 organic pollutants were present in more than 80% of surface water streams tested (Kolpin et al., 2002). In addition, as Heberer (2002) noted in his review, more than 80 pharmaceutical active compounds have been detected in the g/L range in the aquatic environment Because of the obvious effect on the environment, more data exist for antibiotics than ot her pharmaceuticals; what's more, antibiotics are widely used for human medicine, veterinary medicine and agriculture. As a result, there are more obvious environmental issues involving antibiotics ( K Ÿ mmerer, 2003 ). Pathways to the environment T here are many possible routes for pharmaceuticals to enter the environment. Most pharmaceuticals enter the environment through sewage treatment plants, agriculture run off, landfill leaching, or from direct application in the environment, as for example, t he pharmaceuticals used in the aquaculture. Some pharmaceuticals pathways to environment had been summarized by Ku mmerer (2008 ) who also described a similar pathway for antibiotics ( Ku mmerer, 2003 ). Not only through direct disposal of unused ph armaceutical can chemicals enter our environment, but also the medications that a re only partially biodegradable will go into

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5 the environment through organism's metabolicsystem. There are also large amounts of active pharmaceutical ingredients (APIs) that enter our environment by going down to the drains directly (Bound and Voulvoulis, 2005). Effects Since pharmaceutical compounds are designed for medical usage, the lifetime of the chemicals should be long enough in order to do the therapeutic w ork. As a result, pharmaceutical compounds may persist in the environment ( Jones et al., 2002). Although some drugs can be eliminated by human and animal's metabolism or sewage systems, many researchers have pointed out that some pharmaceuticals cannot be completely eliminated by sewage treatment plants (Daughton and Ternes, 1999; Zwiener et al., 2000). Therefore, pharmaceuticals may remain in our environment, and pose threat to the organisms in our environment. As the result, pharmaceuticals are potentiall y toxic substances and may be defined as a new class of pollutants in the environment (Ku mmerer, 2001). Although the discharge of pharmaceutical could be present in low concentration, it may still cause significant effects, a hypothesis had been mentioned previously ( Daughton and Ternes, 1999 ). It is that combination of low concentration pharmaceuticals and their transformation products through long term accumulation may

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6 have some ecologically significant consequences. The others hypothesis is th at the pharmaceuticals remaining in domestic water may have risks for human after lifetime ingestion. One concern is that antimicrobials may affect, qualitatively or quantitatively, the resident microbial community in sediments (Nygaard et al, 19 92). A second concern is that a certain class of pharmaceutical compounds like antibiotics may cause long term change to bacteria, and make bacteria resistant. The resistant bacteria and, perhaps, multiple resistant bacteria, may be involved in sewage, soi l, or other environmental components. Such bacterial resistance has been detected in wastewater and in sewage treatment plants (Guardabassi et al, 1998; Witte, 1998). A third concern is that the pharmaceutical compounds, especially endocrine disrupting che micals (EDCs), are suspected of causing harmful influences to the endocrine systems of human and animals ( Ghijsen and Hoogenboezem, 2000 ). In addition to active pharmaceuticals, their metabolites also need to be considered for their harmful effec ts. Pharmaceuticals may change during the digestion process by organisms and the additional molecules that are formed in the transformation process can cause pharmaceutical contamination, i.e., drug molecules may be altered by human and animals' metabolism (Golan et al. 2007). Some drugs structures are largely changed by the microorganisms in the guts or by the enzymes in human's metabolism before they are excreted. Therefore, their pharmaceutical properties are different to their parent drugs.

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7 Eve n though it is very possible that pharmaceuticals in the environments have adverse impacts, the risk of this issue is very hard to evaluate. For many pharmaceutical compounds, their potential effects on organisms are not completely understood, especially when the pharmaceutical compounds co exist in mixtures and form the so called chemical "cocktails ( Halling Sorensen et al., 1998). Sources of pharmaceuticals Even though there are several thousands APIs used as drugs in the world, there is ina dequate data for usage for total pharmaceuticals. Drug production plants may also be making significant contribution to the total pharmaceutical concentration in the environment (Larsson et al, 2007). Amounts of pharmaceutical products are not manufactured evenly throughout the world. In fact, production of pharmaceuticals is concentrated in five countries USA, Japan, Germany, France and the UK, which are the most industrialized countries. Production in these five countries, represents two thirds of all medicine produced. In addition, 15% of the world's population lives in high income countries and use about 90% of total medicine, the use in USA accounts for over 52% of total medicinal consumption ( World Health Organization, 2004 ). Antibiotics a re also used on a worldwide basis. In 2000, 16,200 tons of antibiotics were produced in the United States, of which 70% was used for livestock (Union of

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8 Concerned Scientists, 2001). European Federation of Animal Health (1999) presented data that 13,288 t ons of antibiotics were reportedly used in the EU (European Union) and Switzerland, of which 65% was for human medicine, 29% was used in the veterinary field and 6% was used as growth promoters. Besides, it was estimated the total antibiotic consumption wa s between 100,000 and 200,000 tons world wide (Wise, 2002). What's more, 10 most prescribed medicines account for 12% of total usage of all medicine ( World Health Organization, 2004 ). Pharmaceutical manufacturing processes involve in many series of steps, which can be taken in many different sites. Therefore, there is a risk that pharmaceuticals may enter the environment from many places during the production of APIs. However, according to Williams and Cook (2007), no studies have documented whether drug manufacturers could be main sources for pharmaceuticals in the environment. Hospitals are one of the major contributors for the pharmaceuticals in the environments. In hospitals, pharmaceuticals are used for surgeries and other medical purpo ses, so hospitals could also be a concentrated source of waste pharmaceuticals, either through disposal of expired drugs, or through the metabolism of patients. For example, Ciprofloxcin was found in a German hospital effluent with a concentration of 0.7 1 25 g/L (Hartmann et al, 1999). Amoxicillin was found in another German hospital effluent with a concentration of 920 980 g/L ( KŸmmerer, 2001 ). Considering that there were 203 hospitals in Florida alone in 2006 with around 2373 thousand patients served

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9 pe r day ( Bureau of the Census, 2008 ), there is an obvious need to develop methods to effect the removal of these agents. Assessment Although pharmaceutical compounds in the environment have been considered a possible risk, the scientific investiga tions of the problems have not received much attention. In order to determine concentration of pharmaceuticals in aqueous sample, the assessments should be sensitive enough to reach as low as nanogram per liter level. Several assessments have been made by different analytical methods to determine the concentration of pharmaceuticals in biological samples, ex. blood and urine, and the detection limit can be lower to !g per liter (Ternes, 2001). According to published research, high performance liq uid chromatography (HPLC), gas chromatography with mass spectrometry (GC/MS) gas chromatography with tandem mass spectrometry (GC/MS 2 ), liquid chromatography with mass spectrometry (LC/MS), liquid chromatography with tandem mass spectrometry (LC/MS 2 ) (H irsch, 1998) have been used in pharmaceutical analyses. Liquid chromatography with tandem mass spectrometry (LC/MS 2 ) has become very popular and is commonly used in assessment methods used in pharmaceutical analysis

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10 because of its high sensitivit y and its ability to confirm compounds. This method can provide detailed information of drug's chemical structure, and it is possible to separate and detect the pharmaceuticals that have the same molecular mass but different product ions ( D’az Cruz and Bar cel—, 2005 ). Solutions to the problem The possible solutions for reducing the adverse effects due to the presenting of pharmaceuticals in the environments could be differentiated into two categories or approaches. One is before the pharmaceutic al compounds are discharged into the environment, preventing the unwanted pharmaceutical compounds enter the environment improperly. The other is reducing of the amounts of pharmaceuticals compounds already existing in the environment. In order to prevent pharmaceuticals from entering the environment improperly, an acceptable solution could be reduction of the amounts of pharmaceuticals discharged into the environment. Proper drug disposal is an emerging environmental issue. The previously recommen ded disposal methods for unused drugs were dumping in the toilet or sink. By doing this, children are protected from accidental poisoning in the home. However, improper disposals may have adverse impacts on our environment and human health. Since the moder n sewage systems are not designed to deal with unused

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11 pharmaceuticals, it is not proper to flush them down the toilets or pour them down to the sinks. Throwing unwanted medicines to the trash is also unacceptable. Pharmaceuticals also have the potential to leach out from the landfill. Therefore, returning unwanted medicines to pharmacies or hospitals may be good environmental friendly options, but since they may not want them, then approved source of incineration could be a better option. Another p otential method for preventing waste pharmaceuticals enter the environment is creating additional waste stream for health care organizations (Smith, 2009). Since health care organizations, such as hospitals, are one of the main sources of pharmaceutical pa ssage to the environment, developing safe and effective pharmaceutical waste management streams can reduce the amount of pharmaceuticals that go into the environments. Recycling can also reduce the pharmaceutical waste amount in the environment. T he unwanted but potentially usable drugs can be returned to the manufacturer through hospitals or pharmacies. By doing this, unwanted pharmaceuticals are not treated as wastes, and disposed to our environment. In addition, recycling unwanted drugs can ease the burden of waste disposal, and unwanted drugs have the potential to be reused. Besides the aforementioned three solutions for preventing the pharmaceutical compounds from entering the environment improperly, a lot of researchers are putting

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12 e fforts on removing the pharmaceutical compounds already in the environment. For examples, there is an increasing interest on the use of advanced oxidation processes (AOPs) for the removal of pharmaceuticals residues from municipal wastewater treatment plan t effluents in recent year (Comninellis et al, 2008). APOs are a set of treatments that can remove organic and inorganic compounds by oxidation. This technique is based on the intermediacy of high reactive chemicals such as hydroxyl radicals to destruct th e target compounds. Different reagents oxidize the compounds intend to be removed. The most common used reagent is Fenton's reagent because it is simple and high efficient in organic compounds' removal (Faouzi et al, 2006). Statement of the problem More than 80 active pharmaceutical ingredients from various therapeutic usages, such as antibiotics, analgesics and hormones have been detected in the aquatic environment ( Heberer, 2002; Daughton and Ternes, 1999 ) and these ingredients affect human he alth through drinking water or aquatic recreational activities. There are numerous negative effects due to environmental existence of pharmaceutical compounds. Most of active pharmaceutical ingredients enter organisms through the pathways in the a quatic environment. In order to eliminate the pharmaceuticals compounds, finding

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13 an effective way to probe entry of the pharmaceuticals into aquatic environment could be useful. Plan of attack While considerable attention has been paid to resear ch issues related to removing the pharmaceuticals in the environment, simple and cost efficient methods still need to be developed for removing pharmaceuticals. A previous study indicated that Octolig¨, a polyethylenediamine covalently attached to a high s urface area silica gel, is capable of removing such anions as phosphate, sulfate, nitrate, and nitrite in a aquatic environment, presumably through a process of encapsulation by the protonated polyethylenediamine groups (Stull and Martin, 2009). A possibl e mechanism of anion encapsulation by Octolig is shown in Fig. 1. The anions were presumed to be associated with "arms" of polyethylenediamine where there are positive charges on the surface of stationary phase ( silica gel of Octolig¨). The purpose of thi s research was to ascertain the potential possibility of using Octolig¨ to remove pharmaceuticals before or after reaching an aquatic environment.

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14 Fig. 1. Possible structure of encapsulation of anions (A n ) by Octolig¨ showing a one arm model (Stu ll and Martin, 2009 ). Octolig¨ has the advantages of simplicities, stabilities, re generation and comparative low cost ($40/kg wholesale). Using Octolig¨ to remove the pharmaceutical compounds may be simple, and easy to operate than other treatme nt processes. Octolig¨ has enormous stability coefficients, and is also stable in pH ranges from 0.5 to 10.5 and at temperatures from 0¡C to 80¡C. Besides, Octolig¨ can be regenerated by a small volume of dilute acid hundreds of times without losing its ca pacity (Metre General, Inc, 2009). It is proposed that certain pharmaceuticals could be removed through chromatography using a commercially available product called Octolig¨. The purpose of

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15 this research is to investigate if Octolig¨ has the poten tial to remove pharmaceutical compounds from the aquatic environment. To evaluate this possibility, this research will focus on testing model compounds in terms of available functional groups. Experiments with model compounds could serve as the basis for a study of real pharmaceuticals, and the practicality of the proposed method of removing pharmaceutical compounds could be demonstrated through a real pharmaceutical compound study. Experimental methods In order to deepen our understanding of h ow pharmaceuticals can be removed by Octolig¨, the present study examines whether Octolig¨ might be suitable for removing pharmaceuticals from different aqueous matrix by using model compounds. The working hypothesis is that certain pharmaceuticals might have appropriate functional groups to enable appropriate encapsulation by Octolig¨. A chromatography procedure was developed for the examination, and a series of model compounds were chosen. The primary criterion for selecting model compounds was that they have the functional group(s) and they are easy to observe visually during chromatography. After testing model compounds, a pharmaceutical compound, Amoxicillin, was chosen to test the practical application to a real pharmaceutical compound.

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16 Ma terials and Methods Source of reagents and materials Octolig¨ (CAS Registry Number 404899 06 5), a polyethylenediamine covalently attached to a high surface area silica gel with a density of 0.455 g/cm 3 was a gift from Metre General, Inc., Fre derick, Co. The structure of Octolig¨ is presented in Fig. 2. Methylene blue was obtained from EMD Chemicals Inc. Rose Bengal was obtained from J.T.Baker Chemical Co. Eosin Y was obtained from Sigma Aldrich Chemical Co., and Erythrosine was acquired from J Preston LTD. ZPS was a gift from Procter and Gamble. Lissamine Green B was purchased from Acros Organics Co and Amoxicillin was from Sigma Aldrich Chemical Co. Well water samples were obtained from a private well at 3402 Valencia Road in Original Carrol lwood, Tampa, FL. Prior to use; in order to remove undissolved solids, the water was filtered through a 3 Millipore membrane filter using an all glass apparatus.

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17 Fig. 2. Chemical structure of Octolig¨. Analytical methods Measurements o f total dissolved solids (TDS) of aqueous samples were done by a Fisher Scientific digital conductivity meter, and the pH values were obtained by an Orion model 290A pH/ISE meter connected with an Orion pH triode electrode (modal 9107BN). The concentration s of solutions were acquired from the absorbance measurements using a Shimadzu UV 2401 PC UV/Vis recording spectrophotometer. Data were collected, and spectra were saved to a disk using OriginPro 8.0 for further use.

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18 Chromatography experiments The chromatography process was similar as Octolig used before (Martin et al, 2008). The chromatography procedure was as follows. The Octolig was used as received with a pretreatment by suspending the solid in DI water then decanting the water to rem ove the fines. A CHEMGLASS column, 2cm (id) equipped with a glass frit and a Teflon stopcock, was packed with Octolig and washed with about 1L of appropriate solvent, i.e. DI (deionized) water, tap water and well water were used as different matrices. A queous samples were chromatographed using a rate of 10mL/min using a Spectra/chron peristaltic pump. A series of 50 mL fractions were collected, and measured by conductivity meter, pH meter and UV VIS absorbance meter. The average concentrations of the ef fluents were compared with the input concentrations, and the percentage removal was calculated and recorded. Molar extinction coefficient measurements Serial dilutions for each model compound were prepared from a known stock solution. Absorbance values were recorded for a wavelength near the max for each dilution using a Shimadzu UV 2401 PC spectrophotometer. Concentrations were

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19 prepared to ensure that absorbance values did not exceed OD=1.5. Molar extinction coefficients were determined using the Beer Lambert law, in which the slope of the absorbance versus concentration plot is equal to the extinction coefficient times the path length, in which the path length was 1.0 cm. The data analysis used EXCEL software and the molar extinction coeffi cients could be obtained from the linear equations. Measured wavelengths and absorbance for each model compounds are shown in Table 1. Molar extinction coefficient of Rose Bengal, which is 57,886M 1 cm 1 at 544nm, was acquired as the slope value i n the trend line. The molar extinction coefficients of Eosin Y, Erythrosine and other dyes were acquired by the same procedures (Table 1). Table 1. Molar extinction coefficients and measured wavelength of model compounds. Dye max nm max Methylene b lue 662 73004 Rose Bengal 544 57886 Eosin Y 516 71976 Erythrosine 526 71235 ZPS 668 23279 Lissamine Green B 635 91891

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20 Test for fluorescence A sample of Amoxicillin and all model compounds were tested using light from a Model UVL 56 Blak Ray¨ Lamp, i.e., long wave UV 366nm obtained from Ultraviolet Products, Inc San Gabriel, CA. No compound showed fluorescence by using Blak Ray¨ Lamp in a darken laboratory. Application to Amoxicillin After testing model compounds, Amoxicillin wa s chosen to be tested for the possibility of this technique to be applied on real pharmaceutical compounds. The extinction coefficient of Amoxicillin was measured, and acquired as 1040cm 1 M 1 at 275 nm. The procedure was the same as previous procedure for model compounds.

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21 Results and Discussion Compound selection The number of criteria can characterize a pharmaceutical popularity is one. The top five most prescribed drugs in 2008(Towner, 2009) were Hydrocodone Lisinopril, Simvastati n, Levothyroxine, and Amoxicillin. CAS numbers, therapeutic usages, and chemical formula are shown in Table 2. Structural features may also characterize pharmaceuticals and the simplest way is by functional groups. Accordingly, Lisinopril, Levothyroxine an d Amoxicillin have either carboxyl (all three) or phenolic groups (last two) or both (last two). Amino groups could also be of interest, and these are found in Lisinopril, Levothyroxine and Amoxicillin. The structures of these pharmaceuticals are shown in Fig. 3.

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22

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23 Fig. 3. Structures of the top 5 most prescribed pharmaceuticals in 2008 (Towner, 2009)

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24 Table 2. The compound names, CAS numbers, therapeutic uses and chemical formulas of the top five most prescribed pharmaceuticals in 2008 (Jones et al, 2002; Towner, 2009) Compound Name CAS Number Therapeutic Use Chemical Formula Hydrocodone 125 29 1 Analgesic C 18 H 21 N O 3 Lisinopril 83915 83 7 Analgesic C 21 H 31 N 3 O 5 Simvastatin 79902 63 9 lipid lo wering drug C 25 H 38 O 5 Levothyroxine 51 48 9 Hormone replacement C 15 H 11 I 4 N O 4 Amoxicillin 26787 78 0 Ant ibiotic C 16 H 19 N 3 O 5 S In considering the functional groups, the model compounds were selected for the evaluation of the potential of pharmaceuticals to be encapsulated by Octolig A series of model compounds, which are Methylene blue, Rose Bengal, Eosin Y, Erythrosine, ZPS, and Lissamine Green B were selected for testing. There experimental results may lead to a better understanding of the possibility of removing the pharmaceuticals i n aquatic environment by Octolig Lissamine Green B is one of the common dyes and had been reported to be removed by one of the APOs process Electro Fenton oxidation (Rosales, 2009). Amoxicillin is a widely used antibiotic, and had been document ed to be present in Sewage Treatment

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25 plants (STPs) effluents. Concentrations of Amoxicillin up to 120 ngL 1 have been reported in STP effluents in Italy ( Andreozzi, 2005 ). Because of having the functional groups, carboxyl and amino, that have the potential to be encapsulated by Octolig¨, Amoxicillin was chosen for experimental test. Presumably carboxyl groups would be favorable for removal; amino groups would not be. Also it was commercially available for a reasonable price. Lisinopril was commercially avai lable, but not at a reasonable price. Medical uses Methylene blue, a guanylate cyclase inhibitor is used to treat vasoplegia which is a frequent complication after cardiopulmonary bypass ( Leyh, 2003) Rose Bengal (4, 5, 6, 7 tetrachloro 20, 40, 50, 70 tetraiodofluorescein disodium, or RB) is a water soluble photo sensitizer with a high molar extinction coefficient in the red region of the spectrum, and Provecuts Pharmaceuticals discovered about a decade ago that Rose Bengal can kill cancer cells, but not normal cells; besides, it was found that Rose Bengal can be used as a treatment for metastatic melanoma (Thompson et al, 2008). PV 10 is ten percent (w/v) Rose Bengal in saline, and PV 10 treatment for melanoma is safe and tolerated for patients. Unfortunately, Food & Drug Administration has not approved it as a medical treatment. Since Rose Bengal is not patentable, no pharmaceutical company can make much profit by producing it (McDuffie, 2009).

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26 Aggregation While doing the experiment, it was found that there are undissolved solids in Rose Bengal solutions when concentration is high. When Rose Bengal is in aqueous solution at a concentration of 2.5# 10 5 M Rose Bengal is present as its monomer (Fini et al, 2007). The aggregation of Rose B engal impacts on the photochemical response, such as absorbance at high concentration (Daraio and San Romˆn, 2001). In order to prevent the aggregation of Rose Bengal that impacts the absorbance when measuring the sample by UV VIS, the sample containers we re covered by aluminum foil, and the samples were prepared with concentrations lower than 2.5# 10 5 M. Experiment results A series of model compounds were selected for testing their potential for encapsulation by Octolig¨. Methylene blue, for exam ple, has a pair of tertiary amino groups. Fluorescein and the halogenated derivatives are substituted xanthenylbenzenes that have both phenolic groups and carboxylic acid groups. The series includes Rose Bengal, Eosin Y, Erythrosine, and sodium fluorescei n. The structures of model compounds are shown in Fig. 4. After chromatography process, the percentage removed

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27 of each model compounds were obtained, and experimental results are show in Table 3, and more detailed data are presented in the Appendices.

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28 Fig. 4. Structures of model compounds.

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29 Table 3. Passage of aqueous sample over a chromatographic column packed with Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Dye, (Matrix) Batch ¤ Fraction TDS(ppm) p H Concentration* %Removed Methylene blue   (DI water) No. 1 Stock 2 7.34 3.321 --4 15 1.90.2 6.090.24 3.3180.11 1.922.57 No. 1 Stock 3 7.61 0.876 --4 9 4.00 6.330.22 0.8440.02 3.722.71 No. 1 Stock 7 5.56 2.429 --4 10 5.20 .4 5.820.26 2.5500.06 0.000.00 Rose Bengal   (DI water) No. 1 Stock 3 6.09 4.170 --4 9 6.50.55 5.630.35 0.9490.266 77.36.4 No. 1 Stock 4 6.58 1.042 --4 9 5.00 5.780.10 0.0080.071 99.20.7 No. 1 Stock 4 7.05 9.600 --4 10 5.00 6.650.24 0.0450.022 99.50.2 No. 2 Stock 4 7.18 9.647 --4 10 5.00 6.340.35 0.0700.010 99.30.1 (Well water) No. 2 Stock 159 7.93 21.921 --6 10 220.33.87 6.740.07 0.0120.011 99.90.0 Eosin Y   (DI water) No. 1 Stock 9 6.27 55.185 --

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30 4 10 14.00.0 6.240.08 0.7130.009 87.10.2 No. 1 Stock 10 6.68 56.213 --4 10 20.00.0 5.990.08 0.0020.005 100.00.2 No. 1 Stock 10 6.72 86.290 --4 10 13.00.5 5.980.08 0.0350.031 100.00.0 No. 2 Stock 10 6.83 90.044 --4 10 15.20.3 5.880.04 0.9270.065 99.00.1 (Well water) No. 2 Stock 164 7.97 85.167 --6 10 205.31.3 6.700.09 0.0540.037 99.90.0 Erythrosine   (DI water) No. 1 Stock 30 7.48 59.662 --4 10 31.10.8 6.710.06 0.6420.027 98.90.0 No. 1 Stock 32 9.54 73.14 --4 10 34.90.3 6.220.13 0.0940.020 99.90.0 No. 2 Stock 28 8.66 78.753 --4 10 31.90.3 6.130.10 0.0460.027 99.90.0 (Tap water) No. 2 Stock 293 7.76 94.897 --4 10 352.77.5 7.450.08 0.1220.019 99.90.0 (W ell water) No. 2 Stock 203 8.33 105.566 --4 10 199.91.2 8.420.06 1.5040.070 98.60.1 ZPS   (DI water) No. 1 Stock 16 6.99 126.29 --4 10 23.33.7 6.790.16 3.8600.159 96.90.1 No. 2 Stock 17 7.80 22.77 --

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31 4 10 19.40.9 6.450.30 0 .2640.034 98.80.2 (Well water) No. 2 Stock 183 7.54 4.081 --4 10 234.13.9 7.090.16 0.0000.000 100.00.0 No. 2 Stock 175 8.34 67.335 --4 10 342.63.1 7.170.05 2.7300.090 95.90.4 (DI water) No. 2 Stock 16 6.48 57.219 --4 10 18.30. 8 6.430.05 1.4240.145 97.50.0 Lissamine Green B (DI water) No. 1 Stock 9 6.27 9.087 --4 10 8.10.3 6.200.10 0.0170.005 99.80.1 No. 1 Stock 10 6.24 9.294 --4 10 6.90.4 6.100.13 0.0080.005 99.90.1 (Well water) No. 2 Stock 169 8.09 7 .636 --4 10 2116 6.640.14 0.0050.001 99.90.0 No. 2 Stock 174 7.92 7.542 --4 10 2135 6.640.10 0.0020.002 1000.0 No. 2 Stock 175 7.88 0.74 --4 10 2164 6.920.01 0.0020.001 99.70.1 Concentration unit is 10 6 M   A 2.0 cm id id chromatographic column was used, and the total volume of Octolig¨ used was 69mL 3 A 3.0 cm id chromatographic column was used, and the total volume of Octolig¨ used was 127mL 3 ¤ Octolig¨ was obtained in two different batches from Metre General, Inc.

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32 In order to test the possibility of application on real pharmaceutical compound, a real pharmaceutical compound, Amoxicillin, was chosen, and tested by the same chromatography procedure. After chromatography process, the percentage removed o f Amoxicillin was obtained, and experimental results are shown in Table 4, and more detailed data are presented in the Appendices. Table 4. Passage of aqueous Amoxicillin sample over a 3.0 cm id chromatographic column packed with 18cm of Octolig¨ at a fl ow rate of 10 mL/min (50 mL aliquots were collected). Matrix Fraction TDS, ppm pH Concentration, 10 6 M %Removed DI water Stock 4 6.17 839.269 --4 10 60 7.010.18 4.9453.653 99.40.4 Stock 3 6.19 1229.808 --4 10 61 6.830.07 2.8853.092 99.80. 3 Stock 5 5.97 741.346 --4 10 21 5.590.10 17.1709.594 98.80.6 Well water Stock 119 6.56 750.000 --4 10 17614 6.480.06 11.6760.012 99.20.4 Stock 153 7.12 912.500 --4 10 19324 6.720.06 57.41822.258 96.91.2

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33 Conclusions The experimental results show Octolig¨ could effectively remove model compounds with proper anionic functional groups. For example, removal of Methylene blue with quaternary ammonium groups and one phenolic group was less than 2% averaged over three tria ls (Table 3) and within experimental error was a complete failure. In contrast, model compounds, Rose Bengal, Eosin Y, Erythrosine ZPS and Lissamine Green B, were successfully encapsulated by Octolig¨. Rose Bengal, Eosin Y, Erythrosine, which have both ph enolic and carboxylic acid groups, were removed quantitatively by Octolig¨ in aquatic environment In addition, complete success was attained for ZPS and Lissamine Green B with sulfonate anions present Matrix effects were studied using t hree different water sources DI water, tap water, and well water from Floridian Aquifer. For example, the Trial 3 and Trial 4 of Erythrosine with different matrixes (DI water and tap water) showed the same percentage removals (99.9%). Besides, the data indicate the removal ability of Octolig¨ did not depend on different batches. For example, as the data shows (Table 3), the stock concentrations were similar for Trial 3 (9.600# 10 6 M ) and Trial 4 (9.647# 10 6 M ) of Rose Bengal test s and the percentage remov als were also similar (99.2% and 99.5%) while using two different batches of Octolig¨. The result s of Student's T test showed no

PAGE 43

34 significant different between the two data groups from different batches of Octolig¨ (P=0.146). While encapsulating compounds with phenolic and carboxylic acid groups, no significant correlation was discerned between pH values and removal ability of Octolig¨ for Erythrosine for a pH value above 7, just as was observed for Rose Bengal and Eosin Y. For example, the Trial 3, 4, and 5 of Erythrosine with different pH values (9.54, 8.66 and 7.76) showed the same percentage removals (99.9%). Figure 7 also shows no obvious pH effects on removal of Erythrosine, presumably because the pH value was above that need for removal of a carboxylic proton. These three fluorescein dyes have pKa values around 5 (Levitanf, 1977;Mchedlov Petrosyan et al, 2006 ) and when the pH value above 7, the ionization of the proton on carboxylic acid functional group is more the 99%. However, when pH was lower than about 6.5, the removal ability of Octolig¨ for Rose Bengal and Eosin Y was not good enough. By increasing the pH value, Octolig¨ removed a greater percentage of xanthenylbenzene. Figure 5 shows the fact that percentage removal can be increased from 77.3% to 99.2% when pH over 6.58. There is also obvious pH effect on removal of Eosin Y (Fig. 6). For example, the percentage removal of Eosin Y sample with the pH of 6.27 was 87.1%, but with a pH value greater than 6.68, the percentage rem oval could be increased to over 99%.

PAGE 44

35 Fig. 5. pH effect on percentage removal of Rose Bengal. Fig. 6. pH effect on percentage removal of Eosin Y.

PAGE 45

36 Fig. 7. pH effect on percentage removal of Erythrosine. In adddtion, while encapsulat ing compounds with sulfonate anions groups, there was no significant relationship between pH values and removal ability of Octolig¨, e.g., for ZPS and Lissamine Green B. For example, the percentage removals did not increase with higher pH values in Trial 2 and 3of ZPS. The pH values are 7.54 in Trial 3 and 7.80 in Trial 2, but the percentage removals are 100% for Trial 3 and 98.8% for Trial 2. Figures 8 and 9 also show there is no obvious relationship between pH and percentage removal of ZPS and Lissamine Green B.

PAGE 46

37 Fig. 8. pH effect on percentage removal of ZPS. Fig. 9. pH effect on percentage removal of Lissamine Green B.

PAGE 47

38 Octolig¨ not only has the potential to remove simple anions, such as phosphate, sulfate, nitrate, and nitrite from wa ter, but also has the potential to remove dyes and those pharmaceutical compounds with suitable functional groups in an aquatic environment at low concentration. Selected drug models compounds were subjected to column chromatography in efforts to effect re moval by means of ion encapsulation, the effectiveness of which would depend upon having appropriate anionic functional groups. The experimental results demonstrated that Methylene Blue with quaternary ammonium groups could not be removed succes sfully. In contrast, complete success was attained for removal of each of three xanthenylbenzenes (Rose Bengal, Eosin Y, and Erythrosine ) that have both phenolic and carboxylic acid groups. In addition complete success was attained for ZPS (zinc phthalocy aninetetrasulfonate) and Lissamine Green B with sulfonate anions present. The real pharmaceutical compound, Amoxicillin, with carboxyl group can be removed by Octolog¨ successfully. Overall, The pharmaceutical compounds which have phenol, carboxylic acid a nd sulfonate functional groups have the potential to be removed by Octolig¨.

PAGE 48

39 Literature Cited Andreozzi, R.; Caprio, V.; Ciniglia, C.; de Champdor`e, M.; Lo Giudice, R.; Marotta, R.; Zuccato, E. Antibiotics in the environment: Occurrence in Ita lian STPs, fate and preliminary assessment on algal toxicity of Amoxicillin. Environ. Sci. Eng, 2005 38, 6832 6838. ! Bound, J.P. and Voulvoulis, N. Household disposal of pharmaceuticals as a pathway for aquatic contamination in the United Kingdom, 2005 Environ. Health Persp, 113(12), 1705 1711. Bureau of the Census. (2008). Community Hospitals States: 2000 and 2006. Statistical Abstract of the United States, 2009 128th ed., United States Department of Commerce: Washington, DC: 2009; Table 165.

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40 Buser H.R.; Poiger, T.; Muller, M.D. Occurrence and environmental behavior of the chiral pharmaceutical drug Ibuprofen in surface water and wastewater. Environ. Sci. Technol, 1999 33, 2529 2535. Comninellis, C.; Kapalka, A.; Malato, S.; Parsons, S.A.; Poulios, I. and Mantzavinos, D. Advanced oxidation processes for water treatment: advances and trends for R&D. J. Chem. Technol. Biotechnol, 2008 83 769 776. Daraio, M.E.; San Romˆn, E. Aggregation and photophysics of Rose Bengal in Alumina coated colloidal sus pensions. Helv. Chim. Acta, 2001 84, 2601 2614. Daughton C.G.; Ternes T.A. Pharmaceuticals and personal products in the environment: Agents of subtle change? Environ. Health Persp, 1999 107(6), 907 938 D’az Cruz M.S. and Barcel— D. LC MS 2 trace anal ysis of antimicrobials in water, sediment and soil. Trends Analy, Chem, 2005 24(7), 645 657. Rosales E. ; Pazos M. ; Longo M.A. ; Sanroman M.A. Electro Fenton decoloration of dyes in a continuous reactor: A promising technology in colored wastewater treatmen t. Chem. Eng. J 2009 155(1 2), 62 67.

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41 Faouzi, M.; Ca nizares, P.; Gadri, A.; Lobato, J.; Nasr, B.; Paz, R.; Rodrigo, M.A.; Saez, C. Advanced oxidation processes for the treatment of wastes polluted with azoic dyes. Electrochim. Acta, 2006 52 (1), 32 5 331. Fini, P.; Loseto, R.; Catucci, L.; Cosma, P.; Agostiano, A. Study on the aggregation and electrochemical properties of Rose bengal in aqueous solution of Cyclodextrins. Bioelectrochemistry, 2007 70, 44 49. Ghijsen, R. T.; Hoogenboezem, W. Endocrin e disrupting compounds in the Rhine and Meuse Basin occurrence in surface, process, and drinking water, sub project of the National Research Project on the occurrence of endocrine disrupting compounds. Association of river waterworks RIWA, De Eendracht, Sc hiedam, Netherlands, 2000 p 96. Gloyd, J.S. Aquatic Species Drugs -new field for CVM; challenge for veterinary profession. J. Am. Vet. Medi. Assoc, 1992 201, 25 26. Golan, D.; Tashjian, A.H.; Ar mstrong, E. J.; Armstrong, A.W. Principles of Pharmacology The Pathophysiologic Basis of Drug Therapy Williams & Wilkins:Philadelphia, PA, 2007.

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42 Guardabassi, L.; Petersen, A.; Olsen, J.E.; Dalsgaard, A. Antibiotic resistance in Acinetobacter spp. Iso lated from sewers receiving waste effluent from a hospital and a pharmaceutical plant. Appl. Environ. Microbiol, 1998 64, 3499 3502. Hartmann, A.; Golet, E. M.; Gartiser, S.; Alder, A. C.; Koller, T.; Widmer, R. M. Primary DNA damage but not mutagenicit y correlated with ciprofloxacin concentrates in German hospital waste waters. Arch. Environ. Contam. Toxicol, 1999 36, 115 119. Heberer, T. Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicol. Letters, 2002 131, 5 17. Halling Sorensen, B.; Nielsen, S.N.; Lanzky, P.F.; Ingerslev, F.; Lutzhoft, H.C.; Jorgensen, S. E. Occurrence, fate and effects of pharmaceutical substances in the environment a review. Chemosphere, 1998 36, 357 3 93. Jones, O.A.; Voulvoulis, H. N.; Lester, J. N. Aquatic environmental assessment of the top 25 English prescription pharmaceuticals. Wat. Res, 2002 36, 5013 5022. Kolpin, D.W.; Furlong, E.T.; Meyer, M.T.; Thurman, E.M.; Zaugg, S. D.; Barber, L. B.; H. T. Buxton, H. T. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999 2000: a National Reconnaissance. Environ. Sci. Technol, 2002 36, 1202 1211.

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43 KŸmmerer, K. Drugs in the environment: Emission of drugs, diagnostic aid s ad disinfectants into wastewater by hospitals in relation to other sources --a review. Chemosphere, 2001 45 957 169. KŸmmerer, K. Significance of antibiotics in the environment. J. Antimicrob. Chemotherapy, 2003 52 : 5 7. K Ÿ mmerer K. Ed. Pharmaceuti cals in the Environment. Sources, Fate, Effects and Risk, (3 rd ed.) Springer: Berlin, Heidelberg, 2008. Larsson, D.G.J.; de Pedro, C and Paxeus, N. Effluent from drug manufactures contains extremely high levels of pharmaceuticals. J. Haz. Materials, 2007 148:3, 751 755. Levitanf, H. Food, drug, and cosmetic dyes: Biological effects related to lipid solubility. Proc. Nati. Acad. Sci, 1977 74(7), 2914 2918. Leyh, R.G.; Kofidis, T.; StrŸber, M.; Fischer, S.; Knobloch, K.; Hagl. C. Methylene blue: the dru g of choice for catecholamine refractory vasoplegia following cardiopulmonary bypass? J. Thorac. Cardiovasc. Surg 2003 6, 1426 31.

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44 Martin, D.F.; Aguinaldo,J.S.; Kondis, N.P.; Stull, F.W.; O'Donnell, L.F.; Martin, B.B. Alldredge, R.L. Comparison of effe ctiveness of removal of nuisance anions by metalloligs, metal derivatives of Octolig¨. J. Environ. Sci. Health Part A, 2008, 43, 1296 1302. McDuffie, H.F. Why Rose bengal isn't available latter. Chem. Engn. News, 2009 87(46), 4. Mchedlov Petrosyan, N.O .;Kukhtik, V.I.; Egorova, S.I. Protolytic equilibria of fluorescein halo derivatives in aqueous organic systems. Russian J. of Gene. Chem. 2006 76(10), 1607 1617. Metre General, Inc 2009 The Chemistry of Octolig¨. http://www.octolig.com/chemistry.html. Accessed March 12, 2010. Nygaard, K; Lunestad, B. T.; Hektoern, H. Resistance to oxytetracycline, oxolinic acid and furazolidine in bacteria from marine sediments. Aquaculture, 1992 104, 21 36. Smith, C.A. 2009. Risk Management of Pharmaceuticals Enter ing POTWs and Municipal Landfills from Routine Hospital Waste Management Practices. From the Keep Antibiotics Working (update 2004). Accessed November 3, 2009.

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45 Stuart, N.C. Treatment of fish disease. Vet. Record, 1983 112, 173 177. Stull, F.W.; Martin, D.F. Comparative ease of separation of mixtures of selected nuisance anions (nitrate, nitrite, sulfate, phosphate) using Octolig¨. J. Environ. Sci. Health, Part A, 2009 44, 1551 1556. Ternes, T.A. Analytical methods for the determination of pharmaceutic als in aqueous environmental samples. Trends Anal. Chem, 2001 20(8), 419 434. Thompson, J.F.; Hersey, P.; Wachter, E. chemoablation of metastatic melanoma using intralesional Rose bengal. Melanoma Res, 2008 18(6), 405 411. Towner, B. The Fifty most pr escribed drugs. AARP Bull. 2009. October. AARP, Washington, D.C. Union of Concerned Scientist. 70 Percent of all antibiotics given to healthy livestock. Press release, 2001 January 8. Cambridge, MA, USA. Wise, R. Antimicrobial resistance: priorities fo r action. J. Antimicrob. Chemotherapy, 2002 49, 585 586.

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46 Witte, W. Medical; consequences of antibiotic use in agriculture. Science, 1998 279, 996 997. Williams, R. T.; Cook, J. C. Exposure to pharmaceuticals present in the environment. Drug Inf. J, 20 07 41 133 141. World Health Organization. 2004. The World Medicines Situation http://www.ops.org.bo/textocompleto/ime23901.pdf. Accessed March 12, 2010. Zwiener, C.; Frimmel, F.H. Oxidative treatment of pharma ceuticals in water. Water Res, 2000 34, 1881 1885. ! ! ! !

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47 ! ! ! ! !""#$%&'#( ) ! ! ! !

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48 Appendices A: Experimental data for model compounds Table A. Passage of Methylene blue solution sample with pH adjustment over a 2 cm id chromatographic co lumn packed with 25cm of Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Trial Matrix Batch Fraction TDS, ppm pH Concentration, 10 6 M %Removal Stock 2 7.34 3.321 --1 DI water 1 4 15 1.90.2 6.090.24 3.3180.11 1.922.57 Stock 3 7.61 0.876 --2 DI w ater 1 4 9 4.00 6.330.22 0.8440.02 3.722.71 Stock 7 5.56 2.429 --3 DI water 1 4 10 5.20.4 5.820.26 2.5500.06 0.000.00

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49 Table A 1. Passage of Methylene blue aqueous sample with pH adjustment over a 2 c m id chromatographic column packed with 25cm of Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 2 7.45 0.558 0.041 2 2 7.13 0.844 0.062 3 2 6.77 2.498 0.182 4 2 6.70 2.874 0.210 5 2 6.54 3.109 0.227 6 2 6.51 3.249 0.237 7 2 6.44 3.287 0.240 8 2 6.07 3.343 0.244 9 2 5.73 3.360 0.245 10 2 5.75 3.362 0.245 11 2 6.18 3.421 0.250 12 2 5.97 3.443 0.251 13 2 5.87 3.462 0.253 14 2 5.82 3.457 0.252 15 1 5.54 3.454 0.252 S ample 2 7.34 3.321 0.242 Average Concentration= 3.318 #10 6 M Standard Deviation= 0.175 %Removal= 1.922.57

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50 Table A 2. Passage of Methylene blue aqueous sample with pH adjustment over a 2 cm id chromatographic column packed with 25cm of Octolig¨ with NaOH pre rinsed at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 3 6.61 0.511 0.037 2 2 6.45 0.535 0.039 3 4 6.67 0.740 0.054 4 4 6.45 0.806 0.059 5 4 6.27 0.829 0.061 6 4 6.66 0. 841 0.061 7 4 6.35 0.853 0.062 8 4 6.21 0.864 0.063 9 4 6.03 0.870 0.064 Sample 3 7.61 0.876 0.064 Average Concentration= 0.844 #10 6 M Standard Deviation= 0.237 %Removal= 3.722.71

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51 Table A 3. Passage of Methylene blue aqueous sample over a 2 cm id chromatographic column packed with 25cm of Cuprilig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 8 6.35 0.604 0.044 2 7 5.87 1.780 0.130 3 8 5.70 2.319 0.169 4 6 6.12 2 .453 0.179 5 5 6.18 2.513 0.183 6 5 5.73 2.583 0.189 7 5 5.66 2.558 0.187 8 5 5.53 2.608 0.190 9 5 5.71 2.593 0.189 Sample 7 5.56 2.429 0.177 Average Concentration= 2.551 #10 6 M Standard Deviation= 0.059 %Removal= 0.000.00

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52 Table B. Passa ge of aqueous Rose Bengal sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Trial Matrix Batch Fraction TDS, ppm pH Concentration, 10 6 M %Removal Stock 3 6.09 4.170 --1 DI water 1 4 9 6.50.55 5.630.35 0.9490.266 77.36.4 Stock 4 6.58 1.042 --2 DI water 1 4 9 5.00 5.780.10 0.0080.071 99.20.7 Stock 4 7.05 9.600 --3 DI water 2 4 10 5.00 6.650.24 0.0450.022 99.50.2 Stock 4 7.18 9.647 --4 DI water 2 4 10 5.00 6.340.35 0.0700.010 99.30.1 Stock 159 7.93 21.921 --5 Well water 2 6 10 220.33.87 6.740.07 0.0120.011 99.90.0

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53 Table B 1. Passage of Rose Bengal aqueous sample over a 2 cm id chromatographic colu mn packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 14 6.84 0.567 0.033 2 12 5.88 0.608 0.035 3 10 6.41 0.709 0.041 4 7 6.12 0.799 0.046 5 7 5.58 1.305 0.076 6 7 5.11 1.278 0.074 7 6 5.59 0.754 0.044 8 6 5.51 0.761 0.044 9 6 5.89 0.794 0.046 Sample 3 6.09 4.170 0.241 Average Concentration= 0.949 #10 6 M Standard Deviation= 0.266 %Removal= 77.36.4

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54 Table B 2. Passage of Rose B engal aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 8 5.34 0.003 0.000 2 6 5.82 0.016 0.001 3 6 5.79 0.023 0.001 4 5 5.90 0.011 0.001 5 5 5.88 0.021 0.001 6 5 5.80 0.008 0.000 7 5 5.69 0.001 0.000 8 5 5.66 0.003 0.000 9 5 5.74 0.005 0.000 Sample 4 6.58 1.042 0.060 Average Concentration= 0.008 #10 6 M Sta ndard Deviation= 0.007 %Removal= 99.20.7

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55 Table B 3. Passage of Rose Bengal aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 9 7.78 0.002 0.000 2 8 7.56 0.050 0.003 3 6 7.34 0.022 0.001 4 5 7.14 0.065 0.004 5 5 6.80 0.035 0.002 6 5 6.71 0.065 0.004 7 5 6.59 0.043 0.002 8 5 6.55 0.070 0.004 9 5 6.46 0 .035 0.002 10 5 6.31 0.000 0.000 Sample 4 7.05 9.600 0.556 Average Concentration= 0.045 #10 6 M Standard Deviation= 0.025 %Removal= 99.50.0

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56 Table B 4. Passage of Rose Bengal aqueous sample with pH adjustion with NaOH over a 2 cm id chromatogra phic column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 8 7.49 0.000 0.000 2 7 7.34 0.000 0.000 3 6 7.16 0.064 0.004 4 5 6.82 0.076 0.00 4 5 5 6.67 0.073 0.004 6 5 6.65 0.091 0.005 7 5 6.34 0.067 0.004 8 5 6.06 0.061 0.004 9 5 5.98 0.062 0.004 10 5 5.83 0.060 0.003 Sample 4 7.18 9.647 0.558 Average Concentration= 0.070 #10 6 M Standard Deviation= 0.011 %Removal= 99.30.1

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57 Tabl e B 5. Passage of Rose Bengal aqueous sample in well water with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50mL aliquots were collected). Fraction No. TDS, ppm pH Con centration, 10 6 M Absorbance 1 5 6.76 0.000 0.000 2 35 6.25 0.023 0.001 3 176 6.56 0.033 0.002 4 213 6.63 0.035 0.002 5 215 6.65 0.006 0.000 6 222 6.72 0.017 0.001 7 223 6.75 0.000 0.000 8 223 6.78 0.016 0.001 9 223 6.81 0.004 0.000 10 223 6.83 0. 006 0.000 Sample 159 7.93 21.921 1.269 Average Concentration= 0.012 #10 6 M Standard Deviation= 0.012 %Removal= 99.90.0

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58 Table C. Passage of aqueous Eosin Y sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Trial Matrix Batch Fraction TDS, ppm pH Concentration, 10 6 M %Removal Stock 9 6.27 55.185 --1 DI water 1 4 10 14.00.0 6.240.08 0.7130.009 87.10.2 Stock 10 6.68 56.213 --2 DI water 1 4 10 20.0 0.0 5.990.08 0.0020.005 100.00.2 Stock 10 6.72 86.290 --3 DI water 1 4 10 13.00.5 5.980.08 0.0350.031 100.00.0 Stock 10 6.83 90.044 --4 DI water 2 4 10 15.20.3 5.880.04 0.9270.065 99.00.1 Stock 164 7.97 85.167 --5 Well water 2 6 10 205.31.3 6.700.09 0.0540.037 99.90.0

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59 Table C 1. Passage of Eosin Y aqueous sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fracti on No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 3 7.78 0.681 0.049 2 4 7.14 0.709 0.051 3 12 6.46 0.695 0.05 4 14 6.26 0.722 0.052 5 14 6.13 0.695 0.05 6 14 6.11 0.722 0.052 7 14 6.26 0.709 0.051 8 14 6.33 0.709 0.051 9 14 6.31 0.709 0.051 10 14 6.3 0.722 0.052 Sample 9 6.27 11.031* 0.794* Average Concentration= 0.071 #10 6 M Standard Deviation= 0.001 %Removal= 87.10.2 Sample was diluted 1:5.

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60 Table C 2. Passage of Eosin Y aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 2 5.72 0.000 0.000 2 4 5.65 0.000 0.000 3 15 5.84 0.000 0.000 4 20 5 .87 0.000 0.000 5 20 5.93 0.003 0.002 6 20 5.95 0.000 0.000 7 20 5.98 0.000 0.000 8 20 6.07 0.000 0.000 9 20 6.05 0.014 0.001 10 20 6.11 0.000 0.000 Sample 10 6.68 11.240* 0.809* Average Concentration= 0.001 #10 6 M Standard Deviation= 0.001 %Remo val= 100.00.2 Sample was diluted 1:5.

PAGE 70

61 Table C 3. Passage of Eosin Y aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collect ed). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 2 5.73 0.102 0.007 2 3 5.50 0.023 0.002 3 10 5.78 0.085 0.006 4 12 5.86 0.014 0.001 5 13 5.92 0.086 0.006 6 14 5.89 0.015 0.001 7 13 6.10 0.085 0.006 8 13 6.07 0.013 0.001 9 13 6.02 0 .015 0.001 10 13 5.97 0.015 0.001 Sample 10 6.72 17.256* 1.242* Average Concentration= 0.035 #10 6 M Standard Deviation= 0.035 %Removal= 100.00.0 Sample was diluted 1:5.

PAGE 71

62 Table C 4. Passage of Eosin Y aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 3 6.52 1.039 0.075 2 4 6.28 0.852 0.061 3 12 5.99 1.05 3 0.076 4 15 5.98 0.852 0.061 5 15 5.94 1.000 0.072 6 15 5.89 0.856 0.062 7 15 5.86 0.992 0.071 8 15 5.89 0.863 0.062 9 16 5.85 0.989 0.071 10 15 5.87 0.860 0.062 Sample 10 6.83 18.006* 1.296* Average Concentration= 0.927 #10 6 M Standard Deviatio n= 0.073 %Removal= 99.00.1 Sample was diluted 1:5.

PAGE 72

63 Table C 5. Passage of Eosin Y aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance 1 3 5.57 0.056 0.004 2 26 5.09 0.014 0.001 3 172 6.02 0.097 0.007 4 202 6.51 0.014 0.001 5 205 6.65 0.097 0.007 6 206 6.69 0.014 0.001 7 206 6.73 0.097 0.007 8 206 6.75 0.02 8 0.002 9 206 6.79 0.097 0.007 10 206 6.81 0.028 0.002 Sample 164 7.97 17.003* 1.226* Average Concentration= 0.054 #10 6 M Standard Deviation= 0.041 %Removal= 99.90.0 Sample was diluted 1:5.

PAGE 73

64 Table D. Passage of aqueous Erythrosine sample over a 2 cm id chromatographic column packed with Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Trial Matrix Batch Fraction TDS, ppm pH Concentration, 10 6 M %Removal Stock 30 7.48 59.662 --1 DI water 1 4 10 31.10.8 6.710.06 0.6 420.027 98.90.0 Stock 32 9.54 73.14 --2 DI water 1 4 10 34.90.3 6.220.13 0.0940.020 99.90.0 Stock 28 8.66 78.753 --3 DI water 2 4 10 31.90.3 6.130.10 0.0460.027 99.90.0 Stock 293 7.76 94.897 --4 Tap water 2 4 10 352.77.5 7.4 50.08 0.1220.019 99.90.0 Stock 203 8.33 105.566 --5 Well water 2 4 10 199.91.2 8.420.06 1.5040.070 98.60.1

PAGE 74

65 Table D 1. Passage of Erythrosine aqueous sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in B atch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 12 6.74 0.014 0.001 100.0 2 17 6.57 0.197 0.014 99.7 3 31 6.70 0.604 0.043 99.0 4 32 6.77 0.660 0.047 98.9 5 32 6.7 9 0.632 0.045 98.9 6 32 6.74 0.632 0.045 98.9 7 31 6.69 0.632 0.045 98.9 8 31 6.70 0.702 0.050 98.8 9 30 6.65 0.618 0.044 99.0 10 30 6.62 0.618 0.044 99.0 Sample 30 7.48 5.9662* 0.425* Average Concentration= 0.642 #10 6 M Standard Deviation= 0.027 %Removal= 98.90.0 *Sample was diluted 1:10.

PAGE 75

66 Table D 2. Passage of Erythrosine aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots we re collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 3 6.84 0.056 0.004 99.9 2 6 6.45 0.042 0.003 99.9 3 27 5.97 0.126 0.009 99.8 4 34 5.90 0.070 0.005 99.9 5 35 6.23 0.112 0.008 99.8 6 35 6.24 0.070 0.005 99.9 7 35 6.2 7 0.112 0.008 99.8 8 35 6.28 0.070 0.005 99.9 9 35 6.30 0.112 0.008 99.8 10 35 6.29 0.112 0.008 99.8 Sample 32 9.54 7.314* 0.521* Average Concentration= 0.094 #10 6 M Standard Deviation= 0.023 %Removal= 99.90.0 *Sample was diluted 1:10.

PAGE 76

67 Table D 3. Passage of Erythrosine aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 2 5.84 0.028 0.002 100.0 2 5 5.53 0.014 0.001 100.0 3 25 5.73 0.084 0.006 99.9 4 31 5.93 0.042 0.003 99.9 5 32 6.05 0.084 0.006 99.9 6 32 6.14 0.042 0.003 99.9 7 32 6.08 0.084 0.006 99.9 8 32 6.21 0.042 0.003 99.9 9 32 6.2 5 0.028 0.002 100.0 10 32 6.22 0.000 0.000 100.0 Sample 28 8.66 7.873* 0.56* Average Concentration= 0.046 #10 6 M Standard Deviation= 0.030 %Removal= 99.90.0 *Sample was diluted 1:10.

PAGE 77

68 Table D 4. Passage of Erythrosine aqueous sample with pH a djustion with NaOH over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 4 7.15 0.042 0.003 100.0 2 4 7 7.35 0.070 0.005 99.9 3 265 7.38 0.168 0.012 99.8 4 337 7.31 0.140 0.010 99.9 5 345 7.32 0.154 0.011 99.8 6 355 7.47 0.112 0.008 99.9 7 359 7.49 0.126 0.009 99.9 8 358 7.49 0.098 0.007 99.9 9 358 7.5 0.126 0.009 99.9 10 357 7.55 0.098 0.007 99.9 Sample 293 7.76 9.490* 0.676* Average Concentration= 0.122 #10 6 M Standard Deviation= 0.021 %Removal= 99.90.0 *Sample was diluted 1:10.

PAGE 78

69 Table D 5. Passage of Erythrosine aqueous sample with pH adjustion with NaOH over a 2 cm id chromatographi c column packed with 25cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 202 8.1 0.000 0.000 100.0 2 195 8.27 0.281 0.020 99.7 3 198 8.27 1.376 0.09 8 98.7 4 198 8.31 1.600 0.114 98.5 5 200 8.35 1.572 0.112 98.5 6 200 8.42 1.572 0.112 98.5 7 201 8.42 1.502 0.107 98.6 8 201 8.47 1.446 0.103 98.6 9 201 8.49 1.432 0.102 98.6 10 198 8.49 1.404 0.100 98.7 Sample 203 8.33 10.557* 0.752* Average C oncentration= 1.504 #10 6 M Standard Deviation= 0.079 %Removal= 98.60.1 *Sample was diluted 1:10.

PAGE 79

70 Table E. Passage of aqueous ZPS sample over a 2 cm id chromatographic column packed with Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were colle cted). Trial Matrix Batch Fraction TDS, ppm pH Concentration, 10 6 M %Removal Stock 16 6.99 126.294 --1 DI water 1 4 10 23.33.7 6.790.16 3.8600.159 96.90.1 Stock 17 7.80 22.767 --2 DI water 2 4 10 19.40.9 6.450.30 0.2640.034 98.80.2 Stock 183 7.54 4.081 --3 Well water 2 4 10 234.13.9 7.090.16 0.0000.000 100.00.0 Stock 175 8.34 67.335 --4 Well water 2 4 10 342.63.1 7.170.05 2.7300.090 95.90.4 Stock 16 6.48 57.219 --5 DI water 2 4 10 18.30.8 6.430.05 1.4240 .145 97.50.0

PAGE 80

71 Table E 1. Passage of ZPS aqueous sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M A bsorbance %Removed 1 2 7.57 0.086 0.002 99.9 2 9 7.36 0.601 0.014 99.5 3 13 7.29 3.265 0.076 97.4 4 15 7.16 4.038 0.094 96.8 5 28 6.86 3.909 0.091 96.9 6 26 6.80 4.081 0.095 96.8 7 25 6.75 3.952 0.092 96.9 8 24 6.70 3.694 0.086 97.1 9 23 6.67 3.65 1 0.085 97.1 10 22 6.62 3.694 0.086 97.1 Sample 16 6.99 12.629* 0.294* Average Concentration= 3.860#10 6 M Standard Deviation= 0.178 %Removal= 96.90.1 *Sample was diluted 1:10.

PAGE 81

72 Table E 2. Passage of ZPS aqueous sample over a 2 cm id chromato graphic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 5 7.19 0.000 0.000 100.0 2 8 7.19 0.000 0.000 100.0 3 17 7.13 0.129 0 .003 99.4 4 21 7.07 0.215 0.005 99.1 5 20 6.76 0.258 0.006 98.9 6 20 6.27 0.258 0.006 98.9 7 19 6.29 0.258 0.006 98.9 8 19 6.26 0.258 0.006 98.9 9 19 6.24 0.258 0.006 98.9 10 18 6.23 0.344 0.008 98.5 Sample 17 7.80 277* 0.053* Average Concentra tion= 0.264#10 6 M Standard Deviation= 0.039 %Removal= 98.80.2 *Sample was diluted 1:10.

PAGE 82

73 Table E 3. Passage of ZPS aqueous sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliq uots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 92 6.53 0.000 0.000 100.0 2 103 6.58 0.000 0.000 100.0 3 178 6.68 0.000 0.000 100.0 4 225 6.90 0.000 0.000 100.0 5 235 6.96 0.000 0.000 100.0 6 236 6.99 0.000 0 .000 100.0 7 237 7.02 0.000 0.000 100.0 8 238 7.11 0.000 0.000 100.0 9 235 7.27 0.000 0.000 100.0 10 233 7.41 0.000 0.000 100.0 Sample 183 7.54 4.081* 0.095* Average Concentration= 0.000#10 6 M Standard Deviation= 0.000 %Removal= 100.00.0 *Sample was diluted 1:10

PAGE 83

74 Table E 4. Passage of ZPS aqueous sample over a 2 cm id chromatographic column packed with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Abso rbance %Removed 1 20 6.31 0.025 0.001 100.0 2 134 6.00 1.812 0.042 97.3 3 299 6.94 2.119 0.049 96.9 4 335 7.06 2.636 0.061 96.1 5 344 7.14 3.029 0.071 95.5 6 345 7.16 2.948 0.069 95.6 7 344 7.19 2.918 0.068 95.7 8 344 7.20 2.711 0.063 96.0 9 342 7 .20 2.649 0.062 96.1 10 344 7.21 2.223 0.052 96.7 Sample 175 8.34 67.335 1.567 Average Concentration= 2.730#10 6 M Standard Deviation= 0.272 %Removal= 95.9 0.4

PAGE 84

75 Table E 5. Passage of ZPS aqueous sample over a 2 cm id chromatographic column p acked with 25cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 11 6.22 1.418 0.033 97.5 2 11 6.21 1.418 0.033 97.5 3 15 6.30 1.461 0.034 97.4 4 20 6.41 1.461 0.034 97.4 5 19 6.45 1.418 0.033 97.5 6 18 6.51 1.418 0.033 97.5 7 18 6.48 1.418 0.033 97.5 8 18 6.45 1.418 0.033 97.5 9 18 6.36 1.418 0.033 97.5 10 17 6.35 1.418 0.033 97.5 Sample 16 6.48 57.219 1.332 Average Concentration= 1.424#10 6 M Standard Deviation= 0.015 %Removal= 97.50.0

PAGE 85

76 Table F. Passage of aqueous Lissamine Green B sample over a 3 cm id chromatographic column packed with Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Trial Matrix Batch Fracti on TDS, ppm pH Concentration, 10 6 M %Removal Stock 9 6.27 9.087 --1 DI water 1 4 10 8.10.3 6.200.10 0.0170.005 99.80.1 Stock 10 6.24 9.294 --2 DI water 1 4 10 6.90.4 6.100.13 0.0080.005 99.90.1 Stock 169 8.09 7.636 --3 Well water 2 4 10 2116 6.640.14 0.0050.001 99.90.0 Stock 174 7.92 7.542 --4 Well water 2 4 10 2135 6.640.10 0.0020.002 1000.0 Stock 175 7.88 0.74 --5 Well water 2 4 10 2164 6.920.01 0.0020.001 99.70.1

PAGE 86

77 Table F 1. Passage of L issamine Green B aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 13 6.77 0.033 0. 003 99.6 2 10 6.52 0.000 0.000 100.0 3 8 6.42 0.011 0.001 99.9 4 9 6.38 0.011 0.001 99.9 5 8 6.30 0.022 0.002 99.8 6 8 6.23 0.022 0.002 99.8 7 8 6.16 0.011 0.001 99.9 8 8 6.13 0.022 0.002 99.8 9 8 6.11 0.022 0.002 99.8 10 8 6.08 0.011 0.001 99.9 Sample 9 6.27 9.087 0.835 Average Concentration= 0.017#10 6 M Standard Deviation= 0.006 %Removal= 99.80.1

PAGE 87

78 Table F 2. Passage of Lissamine Green B aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fration No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 9 6.49 0.011 0.001 99.9 2 8 6.38 0.000 0.000 100.0 3 7 6.35 0.000 0.000 100.0 4 7 6.31 0.011 0.001 99.9 5 7 6.30 0.000 0.000 1 00.0 6 7 6.14 0.011 0.001 99.9 7 7 6.04 0.011 0.001 99.9 8 7 6.00 0.011 0.001 99.9 9 7 5.97 0.000 0.000 100.0 10 6 5.96 0.011 0.001 99.9 Sample 10 6.24 9.294 0.854 Average Concentration= 0.008#10 6 M Standard Deviation= 0.005 %Removal= 99.90.1

PAGE 88

79 Table F 3. Passage of Lissamine Green B aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fration No. TDS, ppm pH Concentration, 10 6 M Absorbanc e %Removed 1 4 7.79 0.005 0.000 99.9 2 21 6.43 0.005 0.000 99.9 3 145 6.15 0.004 0.000 99.9 4 197 6.32 0.003 0.000 100.0 5 210 6.57 0.003 0.000 100.0 6 213 6.67 0.005 0.000 99.9 7 213 6.71 0.005 0.000 99.9 8 214 6.74 0.006 0.001 99.9 9 213 6.67 0. 004 0.000 100.0 10 215 6.79 0.006 0.001 99.9 Sample 169 8.09 7.636 0.702 Average Concentration= 0.005#10 6 M Standard Deviation= 0.001 %Removal= 99.90.0

PAGE 89

80 Table F 4. Passage of Lissamine Green B aqueous sample over a 3 cm id chromatographic c olumn packed with 18cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fration No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 15 7.08 0.000 0.000 100.0 2 32 6.51 0.000 0.000 100.0 3 155 6.38 0.004 0.000 99 .9 4 201 6.45 0.001 0.000 100.0 5 213 6.54 0.002 0.000 100.0 6 215 6.63 0.000 0.000 100.0 7 215 6.67 0.000 0.000 100.0 8 216 6.70 0.005 0.000 99.9 9 216 6.73 0.000 0.000 100.0 10 216 6.75 0.004 0.000 100.0 Sample 174 7.542 0.693 Average Concen tration= 0.002#10 6 M Standard Deviation= 0.002 %Removal= 100.00.0

PAGE 90

81 Table F 5. Passage of Lissamine Green B aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots we re collected). Fration No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 11 6.36 0.002 0.00221 99.7 2 24 6.28 0.003 0.00278 99.6 3 151 6.41 0.002 0.00143 99.8 4 207 6.91 0.002 0.00218 99.7 5 216 6.90 0.001 0.00125 99.8 6 218 6.91 0.003 0.002 33 99.7 7 218 6.92 0.001 0.00099 99.9 8 218 6.92 0.003 0.00233 99.7 9 218 6.92 0.001 0.00099 99.9 10 219 6.95 0.002 0.00221 99.7 Sample 175 7.88 0.074 0.06805 Average Concentration= 0.002#10 6 M Standard Deviation= 0.001 %Removal= 99.70.1

PAGE 91

82 Table G. Passage of aqueous Amoxicillin sample over a 3 cm id chromatographic column packed with Octolig¨ at a flow rate of 10 mL/min (50 mL aliquots were collected). Trial Matrix Batch Fraction TDS, ppm pH Concentration, 10 6 M %Removal Stoc k 4 6.17 839.269 --1 DI water 1 4 10 60 7.010.18 4.9453.653 99.40.4 Stock 3 6.19 1229.808 --2 DI water 1 4 10 61 6.830.07 2.8853.092 99.80.3 Stock 5 5.97 741.346 --3 DI water 2 4 10 21 5.590.10 17.1709.594 98.80.6 Stock 1 19 6.56 750.000 --4 Well water 2 4 10 17614 6.480.06 11.6760.012 99.20.4 Stock 153 7.12 912.500 --5 Well water 2 4 10 19324 6.720.06 57.41822.258 96.91.2

PAGE 92

83 Table G 1. Passage of Amoxicillin aqueous sample over a 3 cm id chromatographic c olumn packed with 18cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 5 7.73 32.692 0.034 96.3 2 5 7.59 13.462 0.014 98.5 3 6 7.43 14.423 0.015 98.4 4 7 7.32 1.923 0.002 99.8 5 7 7.21 9.615 0.010 98.9 6 6 7.08 1.923 0.002 99.8 7 6 6.90 8.654 0.009 99.0 8 6 6.95 1.923 0.002 99.8 9 6 6.85 9.615 0.010 98.9 10 6 6.74 0.962 0.001 99.9 Sample 4 6.17 893.269 0.929 Average Concentration= 4.945#10 6 M Standard Deviation= 4.096 %Removal= 99.40.5

PAGE 93

84 Table G 2. Passage of Amoxicillin aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 1 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 7 7.29 0.000 0.000 100.0 2 6 7.22 0.000 0.000 100.0 3 6 7.04 0.000 0.000 100.0 4 7 6.94 0.000 0.000 100.0 5 6 6.89 2.885 0.003 99.8 6 6 6.84 0.000 0.000 100.0 7 6 6.79 6.731 0.007 99.5 8 6 6 .87 0.962 0.001 99.9 9 5 6.78 8.654 0.009 99.3 10 5 6.73 0.962 0.001 99.9 Sample 3 6.19 1229.808 1.279 Average Concentration= 2.885#10 6 M Standard Deviation= 3.467 %Removal= 99.80.3

PAGE 94

85 Table G 3. Passage of Amoxicillin aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 2 5.99 0.000 0.000 100.0 2 2 6.15 0.962 0.001 99.9 3 2 5.93 10.577 0.011 99.3 4 2 5.76 9.615 0.010 99.4 5 2 5.69 9.615 0.010 99.4 6 2 5.60 6.731 0.007 99.5 7 2 5.58 16.346 0.017 98.9 8 3 5.57 18.269 0.019 98.8 9 3 5.48 21.154 0.022 98.6 10 3 5.45 38.462 0.040 97.4 Sample 5 5.97 741.346 0.771 Aver age Concentration= 17.170#10 6 M Standard Deviation= 10.756 %Removal= 98.80.7

PAGE 95

86 Table G 4. Passage of Amoxicillin aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquo ts were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 8 7.09 0.000 0.000 100.0 2 8 7.41 0.000 0.000 100.0 3 59 6.68 0.000 0.000 100.0 4 143 6.34 0.962 0.001 99.9 5 171 6.45 17.308 0.018 98.8 6 179 6.50 13.462 0.014 9 9.1 7 181 6.50 10.577 0.011 99.3 8 182 6.50 9.615 0.010 99.4 9 184 6.51 13.462 0.014 99.1 10 189 6.54 16.346 0.017 98.9 Sample 119 6.56 750.000 0.780 Average Concentration= 11.676#10 6 M Standard Deviation= 4.887 %Removal= 99.20.4

PAGE 96

87 Table G 5. Passage of Amoxicillin aqueous sample over a 3 cm id chromatographic column packed with 18cm of Octolig¨ in Batch 2 at a flow rate of 10 mL/min (50 mL aliquots were collected). Fraction No. TDS, ppm pH Concentration, 10 6 M Absorbance %Removed 1 2 6.5 2 19.231 0.020 98.9 2 4 7.07 37.500 0.039 97.9 3 71 6.87 47.115 0.049 97.4 4 133 6.72 44.231 0.046 97.6 5 188 6.71 41.346 0.043 97.7 6 203 6.70 36.538 0.038 98.0 7 206 6.61 44.231 0.046 97.6 8 206 6.74 53.846 0.056 97.0 9 206 6.77 75.962 0.079 95.8 10 207 6.81 105.769 0.110 94.2 Sample 153 7.12 912.500 0.949 Average Concentration= 57.418#10 6 M Standard Deviation= 22.258 %Removal= 96.91.4

PAGE 97

88 Appendices B: Spectra for model compounds Fig A 1. Spectra for Methylene blue Trial 1 aqueous fractions and sample. Fig A 2 Spectra for Methylene blue Trial 2 aqueous fractions and sample.

PAGE 98

89 Fig A 3 Spectra for Methylene blue Trial 3 aqueous fractions and sample. Fig B 1 Spectra for Rose Bengal Trial 1 aqueous fractions and sample

PAGE 99

90 Fig B 2 Spectra for Rose Bengal Trial 2 aqueous fractions and sample Fig B 3 Spectra for Rose Bengal Trial 3 aqu eous fractions and sample

PAGE 100

91 Fig B 4 Spectra for Rose Bengal Trial 4 aqueous fractions and sample Fig B 5 Spectra for Rose Bengal Trial 5 aqueous fractions and sample !

PAGE 101

92 ! Fig C 1 Spectra for Eosin Y Trial 1 aqueous fractions and sample ! Fig C 2. Spectra for Eosin Y Trial 2 aqueous fractions and sample

PAGE 102

93 Fig C 3 Spectra for Eosin Y Trial 3 aqueous fractions and sample Fig C 4 Spectra for Eosin Y Trial 4 aqueous fractions and sample

PAGE 103

94 ! Fig C 5 Spectra for Eosin Y Trial 5 aqueous fractions and sample ! Fig D 1 Spectra for Erythrosin e Trial 1 aq ueous fractions and sample

PAGE 104

95 ! Fig D 2 Spectra for Erythrosin e Trial 2 aqueous fractions and sample Fig D 3 Spectra for Erythrosin e Trial 3 aqueous fractions and sample

PAGE 105

96 ! F ig D 4 Spectra for Erythrosin e Trial 4 aqueous fractions and sample ! Fig D 5 Spectra for Erythrosin e Trial 5 aqueous fractions and sample !

PAGE 106

97 ! Fig E 1 Spectra for ZPS Trial 1 aqueous fractions and sample ! Fig E 2 Spectra for ZPS Trial 2 aqueous fractions and sample

PAGE 107

98 ! Fig E 3 Spectra for ZPS Trial 3 aqueous fractions and sample Fig E 4. Spectra for ZPS Trial 4 aqueous fractions and sample

PAGE 108

99 Fig E 5. Spectra for ZPS Trial 5 aqueous fractions and sample FigF 1. Spectra for Lissamine Green B Trial 1 aqueous fractions and sample.

PAGE 109

100 FigF 2. Spectra for Lissamine Green B Trial 2 aqueous fractions and sample. FigF 3. Spectra for Lissamine Green B Trial 3 aqueous fractions and sample.

PAGE 110

101 FigF 4. Spectra for Lissamine Green B Trial 4 aqueous fractions and sample. FigF 5. Spectra for Lissamine Green B Trial 5 aqueous fractions and sample.

PAGE 111

102 FigG 1. Spectra for Amoxicillin Trial 1 aqueous fractions and sample. FigG 2. Spectra for Amoxicillin Trial 2 aqueous fractions and sample.

PAGE 112

103 FigG 3. Spectra for Amoxicillin Trial 3 aqueous fractions and sample. FigG 4. Spectra for Amoxicillin Trial 4 aqueou s fractions and sample.

PAGE 113

104 FigG 5. Spectra for Amoxicillin Trial 5 aqueous fractions and sample.

PAGE 114

105 About the Author Wen Shan Chang received her Bachelor's Degree in Chemistry from the T unghai University in Taiwan After complete undergraduate program, she went to America, and entered the Master program at the University of South Florida in 2008. During the time at the University of South Florida, Ms. Chang worked with Dr. Dean F. Martin, and focused her research on Environmental Analytical Chemistry about the pharmaceutical compounds. She started working as Teach Assistant while in the Master's program, and teaching General Chemistry Laboratory courses as an instructor.


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(OCoLC)
040
FHM
c FHM
049
FHMM
090
XX9999 (Online)
1 100
Chang, Wen-shan.
0 245
Use of model compounds to study potential removal of pharmaceuticals using octolig
h [electronic resource] /
by Wen-shan Chang.
260
[Tampa, Fla] :
b University of South Florida,
2010.
500
Title from PDF of title page.
Document formatted into pages; contains X pages.
502
Thesis (M.S.)--University of South Florida, 2010.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
3 520
ABSTRACT: The existence of pharmaceuticals in the environment has some adverse effects, and may pose threat to the organisms in the environment. The possibility of removing certain pharmaceuticals from wastewater was tested using Octolig(R), a commercially available material with polyethyldiamine moieties covalently attached to high-surface area silica gel. Selected drug models were subjected to column chromatography in efforts to effect removal by means of ion encapsulation, the effectiveness of which would depend upon having appropriate anionic functional groups. The experimental results suggested that the model compounds, Rose Bengal, Eosin Y, Erythrosine ZPS, and Lissamine Green B were successfully encapsulated by Octolig(R), while Methylene Blue with quaternary ammonium groups was (statistically) not. In contrast, complete success was attained for removing of each of three xanthenylbenzenes (Rose Bengal, Eosin Y, Erythrosine) that have both phenolic and carboxylic acid groups. In addition complete success was attained for ZPS (zinc phthalocyaninetetrasulfonate) with sulfonate groups present. A test of a real pharmaceutical compound, Amoxicillin, indicated that Octolig(R) can be used to remove this compound from aqueous media.
590
Advisor: Kirpal S. Bisht, Ph.D.
653
Environment
Chromatography
Antibiotic
Amoxicillin
LGB
690
Dissertations, Academic
z USF
x Chemistry
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.3355