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1 United Chemical Technologies, Inc. 2731 Bartram Road, Bristol, Pennsylvania 19007 800-541-0559www.unitedchem.com "Using Mechanisms of Solid Phase Extraction.

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Presentation on theme: "1 United Chemical Technologies, Inc. 2731 Bartram Road, Bristol, Pennsylvania 19007 800-541-0559www.unitedchem.com "Using Mechanisms of Solid Phase Extraction."— Presentation transcript:

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2 1 United Chemical Technologies, Inc Bartram Road, Bristol, Pennsylvania www.unitedchem.com "Using Mechanisms of Solid Phase Extraction to Improve Your Bioanalytical Results"

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4 3 The force known as solid phase extraction is unusually strong.

5 4 Types of Base Materials for SPE Packings Silica – NA and K Silicates Silica – NA and K Silicates Fluorosil ® Mg Silicates Fluorosil ® Mg Silicates Alumina Alumina Carbon Carbon Polystyrene Polystyrene Polystyrene – Divinyl benzene Polystyrene – Divinyl benzene Polystyrene – N-Vinylpyrrolidone Polystyrene – N-Vinylpyrrolidone Cellulose Cellulose Hydroxyapatite Hydroxyapatite Fullerenes Fullerenes Cyclodextrin Cyclodextrin Agarose Agarose

6 5 MSMS EIC 20 ng/mL Salbutamol – 1 mL Urine 78:20:2 Elution Solvent (Dichloromethane:Isopropanol:Ammonium Hydroxide) CLEAN SCREEN ®

7 6 Abundance Time--> Ion ( to ): 3788.D Time--> Abundance Ion ( to ): 3790.D PC: CONDOA BEG-TMS CleanScreen Time--> Abundance Ion ( to ): 3793.D Time--> Abundance Ion ( to ): 3795.D PC: CONDOA BEG-TMS Cerex 10 ng/mL Equine Urine 1 mL BEG-TMS CleanScreen 10 ng/mL Equine Urine 1 mL BEG-TMS Cerex Benzoylecgonine Recovery Comparisons from Clean Screen and Cerex Solid Phase Columns Sample is 1 mL equine urine, Positive Control is commercially obtained CONDOA multiconstituent control

8 7 SPE Steps Condition sorbents Condition sorbents Apply sample Apply sample Wash interferences Wash interferences Dry sorbent Dry sorbent Elute analyte Elute analyte

9 8 Types of Sorbent-Analyte Interactions Polar Polar Non-polar Non-polar Ion-exchange Ion-exchange Covalent Covalent Copolymeric Copolymeric

10 9 The Real Mechanism of Solid Phase Extraction

11 10 Polar Extractions Also called hydrophilic or normal phase Also called hydrophilic or normal phase Unequal distribution of electrons Unequal distribution of electrons Involves hydrogen bonding, pi-pi and dipole/ Involves hydrogen bonding, pi-pi and dipole/ dipole interactions dipole interactions Sorbents - silica, diol, diethylamino, Sorbents - silica, diol, diethylamino, cyanopropyl cyanopropyl Applications - lipids, oil additives, Applications - lipids, oil additives, carbohydrates, phenols, oil soluble vitamins carbohydrates, phenols, oil soluble vitamins Analytes - amines, hydroxyls, carbonyls, Analytes - amines, hydroxyls, carbonyls, aromatic rings, heteroatoms (O, S, N, P) aromatic rings, heteroatoms (O, S, N, P) Matrix - non-polar, organic Matrix - non-polar, organic Elution solvents - medium to high polarity Elution solvents - medium to high polarity

12 11 Non-Polar Extractions Also called hydrophobic or reverse phase Also called hydrophobic or reverse phase Interactions between sorbent C-H bonds and Interactions between sorbent C-H bonds and analyte C-H bonds analyte C-H bonds Involves van der Waals / dispersion forces Involves van der Waals / dispersion forces Sorbents - C2, C3,C4, iC4, tC4, C5, C6, C7, C8, Sorbents - C2, C3,C4, iC4, tC4, C5, C6, C7, C8, C10, C12, C18, C20, C30 phenyl and cyclohexyl C10, C12, C18, C20, C30 phenyl and cyclohexyl Applications - drugs of abuse, TDM, pesticides Applications - drugs of abuse, TDM, pesticides Analytes - protonated / neutral state, aromatics Analytes - protonated / neutral state, aromatics & alkyl chains & alkyl chains Matrix - biologicals, water, aqueous buffers Matrix - biologicals, water, aqueous buffers Elution solvents - typically non-polar to Elution solvents - typically non-polar to moderately polar moderately polar

13 12 Sample pH vs. Recovery Copolymeric DAU Column A/N Extraction Hydrophobic Retention (CH 3 ) 2 CHCH 2 CC H CH 3 OH O Ibuprofen

14 13 Sample pH vs. Recovery of Ibuprofen Sample added at pH Sample added at pH 5 Ibuprofen Meprobamate Glutethimide Phenobarbital Phenytoin

15 14 Chain Length Effect (Recovery & Extract Cleanliness) Butabarbital64% Amobarbital87% Pentobarbital88% Secobarbital89% Glutethimide78% 96% 94% 96% 98% C18C e 4 2.0e 4 1.0e e 4 2.0e 4 1.0e endogenous peaks: area = 71,628 endogenous peaks: area = 11,257

16 15 Chain Length Effect (Recovery & Extract Cleanliness) Butabarbital64% Amobarbital87% Pentobarbital88% Secobarbital89% Glutethimide78% 96% 94% 96% 98% C18C e 4 2.0e 4 1.0e e 4 2.0e 4 1.0e endogenous peaks: area = 71,628 endogenous peaks: area = 11,257

17 16 Chain Length Effect (Recovery & Extract Cleanliness) Butabarbital93% Amobarbital97% Pentobarbital98% Secobarbital98% Glutethimide96% % 73% 84% 98% 70% Ct4Cn IST D e 4 2.0e 4 1.0e IST D e 4 2.0e 4 1.0e 4 0 endogenous peaks: area = 18,271 endogenous peaks: area = 1,336

18 17 Yoda teaches Luke the ways of the tube.

19 18 Ion Exchange Mechanisms Ionic interactions occur between charged sorbent & analyte of opposite charge Ionic interactions occur between charged sorbent & analyte of opposite charge pH is manipulated to ionize analytes functional group pH is manipulated to ionize analytes functional group Ionic bonds are strong & retain analyte Ionic bonds are strong & retain analyte Hydrophobic interferences washed away with organic solvents Hydrophobic interferences washed away with organic solvents Polar interferences removed with aqueous or weak aqueous / organic washes Polar interferences removed with aqueous or weak aqueous / organic washes Elute solvents containing stronger counterions or by changing pH Elute solvents containing stronger counterions or by changing pH For ionic/hydrophobic analytes, elute by simultaneously disrupting both interactions For ionic/hydrophobic analytes, elute by simultaneously disrupting both interactions

20 19 Cation Exchange Extractions Cation exchange sorbents negatively charged Cation exchange sorbents negatively charged Basic analytes manipulated to carry positive charge Basic analytes manipulated to carry positive charge Opposites attract forming strong bonds Opposites attract forming strong bonds Sorbents Sorbents –Benzenesulfonic acid (strong) –Propylsulfonic acid (strong) –Carboxylic acid (weak) Applications include basic drugs, catecholamines, pharmaceuticals, herbicides Applications include basic drugs, catecholamines, pharmaceuticals, herbicides Analytes Analytes –Amines –Pyrimidines (cations) Matrix - aqueous Matrix - aqueous Basic elution solvents to neutralize analyte Basic elution solvents to neutralize analyte

21 20 Anion Exchange Extractions Anion exchange sorbents positively charged Anion exchange sorbents positively charged Acidic analytes manipulated to carry negative charge Acidic analytes manipulated to carry negative charge Opposites attract forming strong bonds Opposites attract forming strong bonds Sorbents Sorbents –1 , 2  amine –Aminopropyl (weak) –Quaternary amine (strong) –Diethylamino (weak) Applications include phosphates, acidic drugs, organic acids, fatty acids, vitamins Applications include phosphates, acidic drugs, organic acids, fatty acids, vitamins Analytes Analytes –Phosphates –Carboxylic acids –Sulfonic acids (cations) Matrix - aqueous Matrix - aqueous Acidic elution solvents to neutralize analyte Acidic elution solvents to neutralize analyte

22 21 Copolymeric Extractions Hydrophobic & ionic retention mechanisms Hydrophobic & ionic retention mechanisms Reverse phase sorbent with cation OR anion exchange Reverse phase sorbent with cation OR anion exchange Acidic, basic & neutral analyte applications Acidic, basic & neutral analyte applications Matrix - aqueous Matrix - aqueous Selective washes Selective washes Elution solvents mixture of organics with acid or base Elution solvents mixture of organics with acid or base Superior sample clean up Superior sample clean up

23 22 C8 vs. Copolymeric Extraction Time (minutes) Time (minutes) C8ColumnCopolymericColumn

24 23

25 24 pKa, pH & Ionization % of Compound in Ionic State FunctionalityIonization State pH units away from pKa FunctionalityIonization State pH units away from pKa

26 25 Ion exchange columns possess charged functional groups which allow analytes to bind upon sample application. Prior to column use, these groups require counter ions at these charged sites. The standard counter ion for cation exchangers is the hydronium ion and for anion exchangers is the chloride ion. From time to time during sample application, a charged analyte is not strong enough to displace the counter ion & therefore does not bind to the column. In cases such as these, a weaker counter ion is required. Two such columns with weaker counter ions (Quaternary amine with acetate counter ion) & (Quaternary amine with hydroxide counter ion) are commercially available. In terms of strength, the acetate ion is stronger than the hydroxide ion. Specialty Anion Exchange Columns CHQAX Silica Backbone Quaternary Amine anion exchanger Acetate counter ion (Standard anion exchanger carries Cl - ) CAQAX

27 26 Larger numbers reflect greater ability of the ion to displace other ionic materials from the bonded surfaces. Cations Anions Ba Ag Benzene Sulfonate500 Pb Citrate220 Hg I Cu + 5.3Phenate Sr HSO Ca CIO Ni NO Cd Br - 50 Cu CN - 28 CO HSO - 27 Zn BrO27 Cs NO 2 24 Rb + 2.6CI - 22 K + 2.5HCO Fe IO Mg Formate Mn Acetate NH Propionate Na + 1.5F H + 1.0OH Li Standard cation exchange counter ion Strong Cation Exchanger Benzenesulfonic Acid (BCX) Strong Anion Exchanger Quaternary Amine (QAX) - Si - (CH 2 ) 3 N + (CH 3 ) 3 Relative Counter ion Selectivity

28 27 ROBINUL (GLYCOPYRROLATE) FROM EQUINE URINE BY LCMSMS (CLEAN UP ® CUCCX-2) 500 mg / 14 mL I. CONDITION SPE COLUMN 1.Wash with 2 x 2.5 ml MeOH 2.Wash with 2 x 2.5 ml phosphate buffer (0.1m, pH 7.0) II. SAMPLE PREPARATION 1.Buffer 5 ml of urine to pH 7.0 by adding 3 mL of 0.1M phosphate buffer (pH 7.0) 2.Add (12.5 ng) of mepenzolate (internal standard) 3.Add 5 ml of water to the sample 4.Vortex or shake thoroughly 5.Centrifuge for 5 min at 800g 6.Apply supernatant to SPE column III. WASH COLUMN 1.Wash column with 5 ml of MeOH 2.Wash column with 5 ml of H 2 O IV. DRY COLUMN 1.5 min. with vacuum at 25 mm Hg V. ELUTE OF GLYCOPYRROLATE 1.Elute with 4 ml of methanol – 0.5M ammonium acetate buffer pH 3.00 VI. BLOWN DOWN 1.Blown down eluent at 60°C under nitrogen and reconstitute with 0.1 mL of MeOH

29 28 LCMSMS of Glycopyrrolate

30 29 Meprobamate NH 2 C O O CH 2 C CH 3 O C O Polar Drug Copolymeric DAU Column A/N Extraction Hydrophobic Retention CH 2 NH 2

31 30 A/N Drug Recovery vs. Changes in Elution Solvents Hexane/Ethyl AcetateMethylene Chloride Ibuprofen Meprobamate

32 31 Amphetamine Structures CH 2 CH NH 2 CH 3 CH 2 CHNH 2 CH 3 CH 3 CCN CH 3 OH HHHAmphetamine pKa = 9.9 Methamphetamine Ephedrine pKa = 9.6

33 32 Recovery vs. Different Elution Solvents 1d-Amphetamine 2d-Methamphetamine 3PPA 4Pseudoephedrine 5Meperidine 6Lidocaine 7PCP 8Methadone 9Propoxyphene 10Cocaine 11Codeine 12Diazepam 13Nordiazepam 14Chlordiazepoxide Elution: MeCl 2 / IPA / NH 4 OH (78/20/2)

34 33 Recovery vs. Different Elution Solvents 1d-Amphetamine 2d-Methamphetamine 3PPA 4Pseudoephedrine 5Meperidine 6Lidocaine 7PCP 8Methadone 9Propoxyphene 10Cocaine 11Codeine 12Diazepam 13Nordiazepam 14Chlordiazepoxide Elution: EA / NH 4 OH (98/2)

35 34 Solubility Best wash solvents are those in which the compound of interest is insoluble. Example:Vancomycin Insoluble in Methanol Wash:100% methanol Soluble in H 2 O Elution:80:20 methanol/H 2 O

36 35 Technical Document P-105 Purification of Small Molecule Libraries Desalting Samples Using Pharmasil™ Reverse Phase SPE Principle: The generation of small molecule libraries for screening against biological targets has emerged as an area of intense interest in the pharmaceutical industry. SPE has been demonstrated to expedite work up and purification of organic molecules synthesized in solution, and in the automated construction of small molecule libraries. Samples that have been synthesized in aqueous salt, buffer solutions, or low polarity organic solvents containing salts may require the removal of those salts prior to analysis. Pharmasil TM Reverse Phase SPE can be used to desalt these libraries. Application: This application details the use of Pharmasil™ CEC18, a highly loaded reverse phase sorbent, for desalting synthetic mixtures. In combinatorial chemistry and organic synthesis salts are sometimes present in the reaction mixtures. Once the reaction is complete, it is usually necessary to separate the products of the reaction from the salts. If the salt is not removed it can interfere with further testing as well as ruin expensive analytical equipment. This can be done using a highly loaded reverse phase SPE column to selectively remove the salt from the reaction mixture.

37 36 Advantages of Pharmasil™ Based Sorbents Complete removal of salts Clean background High recoveries High levels of purification of anaytes Applicable to a broad range of compounds Simple easy to develop methods Chemistry of Pharmasil™ CEC18 Sorbent

38 37 Purification Profile This profile is based on the use of a Pharmasil™ CEC mg column (columns are available with varying volumes). This column is capable of removal of salts. The method can be scaled up as necessary by using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately The following profile is meant to be a guideline for these types of purifications. Each drug class has its own specific requirements based on solubility, stability, and pKa and may require slight adjustments in methodology. Therefore think of the following profile as a beginning rather than a final method. Sample Pre-treatment Samples may or may not require pretreatment before addition. The primary concern using desalting columns is to adjust the pH of the compound of interest so that it is totally molecular. This may require the addition of an acid or base. Desalting can be done out of low polarity organic solvents such as hexane or methylene chloride as long as the compound of interest is protonated. Column Conditioning Condition the column 1 ml of Methanol followed by 1 ml of water. Column Equilibration Condition the column with buffer: If sample is a base, you want the pH to be >9 If sample is an acid, you want the pH to be<2.5 Apply the sample to the column under gravity. The salts will flow through the column and the sample will stick to the column. The volume of the sample is not important and will probably be dictated by the equipment you use. The critical factor is concentration and capacity of the sorbent. If the concentration of the compound of exceeds the capacity of the sorbent you will not get the highest recovery. If you think this is a problem use a larger bed mass. Product Purification Wash the column with 1ml of DI water or hexane. Product Elution Elute compound of interest with 1ml of methanol, ethyl acetate, or the organic solvent of your choice.

39 38 Technical Document P-102 Purification of Small Molecule Libraries TIN (Sn) Removal by Pharmasil™ Ion Exchange SPE Principle: The generation of small molecule libraries for screening against biological targets has emerged as an area of intense interest in the pharmaceutical industry. Ion exchange chromatography has been demonstrated to expedite work up and purification of organic molecules synthesized in solution, and in the automated construction of small molecule libraries. The advantage of ion exchange chromatography over more traditional small molecule purification modes such as flash chromatography or HPLC is that one can reliably predict the elution characteristics of a broad range of molecules solely by the presence or absence of an ionizable site on the molecule. Application: This application details the use of Pharmasil™ TAX, a highly loaded weak cation exchange sorbent, for the removal of tin catalysts from organic synthesis mixtures. In combinatorial chemistry and organic synthesis tin compounds are common catalysts. Once the reaction is complete, it is usually necessary to separate the products of the reaction from the catalysts. If the catalyst is not removed it can interfere with further testing as well as ruin expensive analytical equipment. This can be done using a highly loaded weak cation exchanger to selectively remove the tin catalyst from the reaction mixture.

40 39 Chemistry of Pharmasil™ TAX Sorbent Advantages of Pharmasil™ Based Sorbents Complete removal of tin catalyst Clean background High recoveries High levels of purification of anaytes Applicable to a broad range of compounds Simple easy to develop methods

41 40 Purification Profile This profile is based on the use of a Pharmasil™ TAX 500 mg column (columns are available with varying volumes). This column is capable of removal of up to50mg of tin. The method can be scaled up as necessary by using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately The following profile is meant to be a guideline for these types of purifications. Each drug class has its own specific requirements based on solubility, stability, and pKa and may require slight adjustments in methodology. Therefore think of the following profile as a beginning rather than a final method. Sample Pre-treatment Samples may or may not require pretreatment before addition. The primary concern using ion exchangers is to adjust the pH of the compound of interest so that it is totally ionized. This may require the addition of an acid or buffer. Ion exchange can be done out of organic solvents such as methanol or ethyl acetate as long as the compound of interest is ionized... Tin catalysts are strong cations and are charged across the complete pH range. Column Conditioning Condition the column 1 ml of Methanol followed by 1 ml of water. Column Equilibration Condition the column with buffer: If sample is a base, you want the pH at 7-8. If sample is an acid, you want the pH at 3-4. Sample Application Apply the sample to the column under gravity. The tin will stick to the column. The volume of the sample is not important and will probably be dictated by the equipment you use. The critical factor is concentration and capacity of the sorbent. If the concentration of the tin of exceeds the capacity of the sorbent you will not get the highest removal of tin. If you think this is a problem use a larger bed mass. Product Purification Wash the column with 1ml of buffer used in column equilibration. Product Elution Elute compound of interest with 1ml of methanol.

42 41 Purification of Small Molecule Libraries Palladium (Pd) Removal by Pharmasil™ Ion Exchange SPE Technical Document P-103 Purification of Small Molecule Libraries Palladium (Pd) Removal by Pharmasil™ Ion Exchange SPE Principle: The generation of small molecule libraries for screening against biological targets has emerged as an area of intense interest in the pharmaceutical industry. Ion exchange chromatography has been demonstrated to expedite work up and purification of organic molecules synthesized in solution, and in the automated construction of small molecule libraries. The advantage of ion exchange chromatography over more traditional small molecule purification modes such as flash chromatography or HPLC is that one can reliably predict the elution characteristics of a broad range of molecules solely by the presence or absence of an ionizable site on the molecule. Application: This application details the use of Pharmasil™ TAX, a highly loaded weak cation exchange sorbent, for the removal of palladium catalysts from organic synthesis mixtures. In combinatorial chemistry and organic synthesis palladium compounds are common catalysts. Once the reaction is complete, it is usually necessary to separate the products of the reaction from the catalysts. If the catalyst is not removed it can interfere with further testing as well as ruin expensive analytical equipment. This can be done using a highly loaded weak cation exchanger to selectively remove the tin catalyst from the reaction mixture.

43 42 Chemistry of Pharmasil™ TAX Sorbent Advantages of Pharmasil™ Based Sorbents Complete removal of palladium catalyst Complete removal of palladium catalyst Clean background Clean background High recoveries High recoveries High levels of purification of anaytes High levels of purification of anaytes Applicable to a broad range of compounds Applicable to a broad range of compounds Simple easy to develop methods Simple easy to develop methods

44 43 Purification Profile This profile is based on the use of a Pharmasil™ TAX 500 mg column (columns are available with varying volumes). This column is capable of removal of up to50mg of palladium. The method can be scaled up as necessary by using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately The following profile is meant to be a guideline for these types of purifications. Each drug class has its own specific requirements based on solubility, stability, and pKa and may require slight adjustments in methodology. Therefore think of the following profile as a beginning rather than a final method. Sample Pre-treatment Samples may or may not require pretreatment before addition. The primary concern using ion exchangers is to adjust the pH of the compound of interest so that it is totally ionized. This may require the addition of an acid or buffer. Ion exchange can be done out of organic solvents such as methanol or ethyl acetate as long as the compound of interest is ionized... Palladium catalysts are strong cations and are charged across the complete pH range. Adjust the sample to pH 9 with buffer or ammonium hydroxide. Column Conditioning Condition the column 1 ml of Methanol followed by 1 ml of water. Column Equilibration Condition the column with buffer of pH 9. Sample Application Apply the sample to the column under gravity. The palladium will stick to the column. The volume of the sample is not important and will probably be dictated by the equipment you use. The critical factor is concentration and capacity of the sorbent. If the concentration of the palladium exceeds the capacity of the sorbent you will not get the highest removal of palladium. If you think this is a problem use a larger bed mass. Product Purification Wash the column with 1ml of buffer used in column equilibration. Wash the column with 1ml of buffer used in column equilibration. Product Elution Elute compound of interest with 1ml of methanol.

45 44 Technical Document P-104 Purification of Small Molecule Libraries TFAA Removal by Pharmasil™ Ion Exchange SPE Principle: The generation of small molecule libraries for screening against biological targets has emerged as an area of intense interest in the pharmaceutical industry. Ion exchange chromatography has been demonstrated to expedite work up and purification of organic molecules synthesized in solution, and in the automated construction of small molecule libraries. The advantage of ion exchange chromatography over more traditional small molecule purification modes such as flash chromatography or HPLC is that one can reliably predict the elution characteristics of a broad range of molecules solely by the presence or absence of an ionizable site on the molecule. Application: This application details the use of Pharmasil™ CHQAX, a highly loaded quaternary amine exchange sorbent, for the removal of acid catalysts from organic synthesis mixtures. In combinatorial chemistry and organic synthesis TFAA is a common catalyst. Once the reaction is complete, it is usually necessary to separate the products of the reaction from the catalyst. If the catalyst is not removed it can interfere with further testing as well as ruin expensive analytical equipment. This can be done using a highly loaded quaternary amine exchanger to selectively remove the acid catalyst from the reaction mixture.

46 45 Chemistry of Pharmasil™ CHQAX Sorbent Advantages of Pharmasil™ Based Sorbents Complete removal of acid catalyst Complete removal of acid catalyst Clean background Clean background High recoveries High recoveries High levels of purification of anaytes High levels of purification of anaytes Applicable to a broad range of compounds Applicable to a broad range of compounds Simple easy to develop methods Simple easy to develop methods

47 46 Purification Profile This profile is based on the use of a Pharmasil™ CHQAX 500 mg column (columns are available with varying volumes). This column is capable of removal of up to 50mg of TFAA. The method can be scaled up as necessary by using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately. The following profile is meant to be a guideline for these types of purifications. Each drug class has its own specific requirements based on solubility, stability, and pKa and may require slight adjustments in methodology. Therefore think of the following profile as a beginning rather than a final method. Sample Pre-treatment Samples may or may not require pretreatment before addition. The primary concern using ion exchangers is to adjust the pH of the compound of interest so that it is totally ionized. This may require the addition of a pH 7 buffer. Ion exchange can be done out of organic solvents such as methanol or ethyl acetate as long as the compound of interest is ionized... acid catalysts are strong anions and are charged across the complete pH range. Column Conditioning Condition the column with 1 ml of methanol followed by 1 ml of DI water. Column Equilibration Condition the column with pH 7 buffer. Application Apply the sample to the column under gravity. The TFAA will stick to the column. The volume of the sample is not important and will probably be dictated by the equipment you use. The critical factor is concentration and capacity of the sorbent. If the concentration of the TFAA exceeds the capacity of the sorbent you will not get the highest removal of TFAA. If you think this is a problem use a larger bed mass. Product Purification Wash the column with 1ml of buffer used in column equilibration. Product Elution Elute compound of interest with 1ml of methanol.

48 47 The Determination of Trace Metal Concentrations in Acid Mine Drainage By Michael W. Beneteau Advisor: Dr. Carol M. Babyak

49 48 TAX Column % Recovery of Pb at Various Concentrations ppm0.050 ppm1.00 ppm2.00 ppm

50 49 Metal Recoveries on Various Phases Cu (ll) Zn (ll) As (V) Sn (Vl) Se (lV) Hg (ll) Cr (lll) PSA BCX-HL CCX TAX THX NAX

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