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United Chemical Technologies, Inc. 2731 Bartram Road, Bristol, Pennsylvania 19007 800-541-0559 www.unitedchem.com

"Using Mechanisms of Solid Phase Extraction to Improve Your Bioanalytical Results". United Chemical Technologies, Inc. 2731 Bartram Road, Bristol, Pennsylvania 19007 800-541-0559 www.unitedchem.com. mtelepchak@unitedchem.com 215-781-3850.

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United Chemical Technologies, Inc. 2731 Bartram Road, Bristol, Pennsylvania 19007 800-541-0559 www.unitedchem.com

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

  2. mtelepchak@unitedchem.com 215-781-3850

  3. The force known assolid phase extraction is unusually strong.

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

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

  6. Abundance Abundance Ion 240.00 (239.70 to 240.70): 3790.D Ion 240.00 (239.70 to 240.70): 3788.D 6000000 6000000 4000000 4000000 2000000 2000000 0 0 Time--> Time--> 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Abundance Abundance Ion 240.00 (239.70 to 240.70): 3793.D Ion 240.00 (239.70 to 240.70): 3795.D 6000000 6000000 4000000 4000000 2000000 2000000 0 0 Time--> Time--> 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 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 10 ng/mL Equine Urine 1 mL BEG-TMS CleanScreen PC: CONDOA BEG-TMS CleanScreen PC: CONDOA BEG-TMS Cerex 10 ng/mL Equine Urine 1 mL BEG-TMS Cerex

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

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

  9. The Real Mechanism of Solid Phase Extraction

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

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

  12. Copolymeric DAU Column A/N Extraction Hydrophobic Retention Sample pH vs. Recovery CH3 OH (CH3) 2CHCH2 C C O H Ibuprofen

  13. 3 4 3 4 5 5 1 2 2 1 Sample added at pH 6 Sample added at pH 5 1 2 3 4 5 Sample pH vs. Recovery of Ibuprofen Ibuprofen Meprobamate Glutethimide Phenobarbital Phenytoin

  14. 4 4 3.0e4 2.0e4 1.0e4 0 3.0e4 2.0e4 1.0e4 0 3 5 5 3 2 2 1 1 0 2 4 6 8 0 2 4 6 8 C18 C2 1 96% 94% 94% 96% 98% Butabarbital 64% Amobarbital 87% Pentobarbital 88% Secobarbital 89% Glutethimide 78% 2 3 4 5 Chain Length Effect(Recovery & Extract Cleanliness) endogenous peaks: area = 71,628 endogenous peaks: area = 11,257

  15. 4 4 3.0e4 2.0e4 1.0e4 0 3.0e4 2.0e4 1.0e4 0 3 5 5 3 2 2 1 1 0 2 4 6 8 0 2 4 6 8 C18 C2 1 96% 94% 94% 96% 98% Butabarbital 64% Amobarbital 87% Pentobarbital 88% Secobarbital 89% Glutethimide 78% 2 3 4 5 Chain Length Effect(Recovery & Extract Cleanliness) endogenous peaks: area = 71,628 endogenous peaks: area = 11,257

  16. 4 4 ISTD 3.0e4 2.0e4 1.0e4 0 3.0e4 2.0e4 1.0e4 0 ISTD 3 5 2 5 1 3 2 1 0 2 4 6 8 10 0 2 4 6 8 10 1 2 3 4 5 Chain Length Effect(Recovery & Extract Cleanliness) Ct4 Cn4 28% 73% 84% 98% 70% Butabarbital 93% Amobarbital 97% Pentobarbital 98% Secobarbital 98% Glutethimide 96% endogenous peaks: area = 1,336 endogenous peaks: area = 18,271

  17. Yoda teaches Luke the ways of the tube.

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

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

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

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

  22. 0 4 8 12 16 20 0 4 8 12 16 20 Time (minutes) Time (minutes) C8 Column Copolymeric Column C8 vs. CopolymericExtraction

  23. 2

  24. pKa, pH & Ionization % of Compound in Ionic State Functionality Ionization State pH units away from pKa 2< 1< at pKa 1> 2> Acid Anion (-) 1 9 50 91 99 Base Cation (+) 99 91 50 9 1

  25. CAQAX Specialty AnionExchange Columns 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. Silica BackboneQuaternary Amine anion exchangerAcetate counter ion(Standard anion exchanger carries Cl- ) CHQAX Silica BackboneQuaternary Amine anion exchangerAcetate counter ion(Standard anion exchanger carries Cl- )

  26. Relative Counter ion Selectivity Cations Anions Ba2+ 8.7 Ag2+ 7.6 Benzene Sulfonate 500 Pb2+ 7.5 Citrate 220 Hg2+ 7.2 I - 175 Cu+ 5.3 Phenate- 110 Sr2+ 4.9 HSO4- 85 Ca2+ 3.9 CIO3- 74 Ni2+ 3.0 NO3- 65 Cd2+ 2.9 Br- 50 Cu2+ 2.9 CN - 28 CO2+ 2.8 HSO - 27 Zn2+ 2.7 BrO 27 Cs2+ 2.7 NO2 24 Rb+ 2.6 CI - 22 K+ 2.5 HCO3- 6.0 Fe2+ 2.5 IO33- 5.5 Mg2+ 2.5 Formate - 4.6 Mn2+ 2.3 Acetate - 3.2 NH4+ 1.9 Propionate - 2.6 Na+ 1.5 F - 1.6 H+ 1.0 OH - 1.0 Li+ 0.8 Larger numbers reflect greater ability of the ion to displace other ionic materials from the bonded surfaces. Strong Cation Exchanger Benzenesulfonic Acid (BCX) Strong Anion Exchanger - Si - (CH2)3 N+ (CH3)3 Quaternary Amine (QAX) Standard cation exchange counter ion

  27. 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 H2O 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 ROBINUL (GLYCOPYRROLATE) FROM EQUINEURINE BY LCMSMS(CLEAN UP® CUCCX-2) 500 mg / 14 mL

  28. LCMSMS of Glycopyrrolate

  29. Polar Drug Copolymeric DAU Column A/N Extraction Hydrophobic Retention Meprobamate O O CH3 NH2 C CH2 O NH2 C O CH2 C CH3

  30. Hexane/Ethyl Acetate Methylene Chloride 1 2 1 2 1 2 1 2 Ibuprofen Meprobamate 1 1 2 2 A/N Drug Recovery vs. Changes in Elution Solvents

  31. CH2 CH NH2 CH 3 CH CH CH NH 3 2 2 CH 3 H H H C C N CH3 OH CH3 Amphetamine Structures Amphetamine pKa = 9.9 Methamphetamine pKa = 9.9 Ephedrine pKa = 9.6

  32. 4 2 3 1 Recovery vs. Different Elution Solvents 1 d-Amphetamine 2 d-Methamphetamine 3 PPA 4 Pseudoephedrine 5 Meperidine 6 Lidocaine 7 PCP 8 Methadone 9 Propoxyphene 10 Cocaine 11 Codeine 12 Diazepam 13 Nordiazepam 14 Chlordiazepoxide Elution: MeCl2 / IPA / NH4OH (78/20/2)

  33. 3 2 4 1 Recovery vs. Different Elution Solvents 1 d-Amphetamine 2 d-Methamphetamine 3 PPA 4 Pseudoephedrine 5 Meperidine 6 Lidocaine 7 PCP 8 Methadone 9 Propoxyphene 10 Cocaine 11 Codeine 12 Diazepam 13 Nordiazepam 14 Chlordiazepoxide Elution: EA / NH4OH (98/2)

  34. Best wash solvents are those in which the compound of interest is insoluble. Solubility • Example:Vancomycin • Insoluble in Methanol • Wash: 100% methanol • Soluble in H2O • Elution: 80:20 methanol/H2O

  35. 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. Technical Document P-105Purification of Small Molecule LibrariesDesalting Samples Using Pharmasil™ Reverse Phase SPE

  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

  37. Purification Profile This profile is based on the use of a Pharmasil™ CEC18 500 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.

  38. 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. Technical Document P-102Purification of Small Molecule LibrariesTIN (Sn) Removal by Pharmasil™ Ion Exchange SPE

  39. 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 Chemistry of Pharmasil™ TAX Sorbent

  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.

  41. 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. Technical Document P-103Purification of Small Molecule LibrariesPalladium (Pd) Removal by Pharmasil™ Ion Exchange SPE

  42. Advantages of Pharmasil™ Based Sorbents • Complete removal of palladium catalyst • 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™ TAX Sorbent

  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. Product Elution Elute compound of interest with 1ml of methanol.

  44. 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. Technical Document P-104Purification of Small Molecule LibrariesTFAA Removal by Pharmasil™ Ion Exchange SPE

  45. Advantages of Pharmasil™ Based Sorbents • Complete removal of acid catalyst • 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™ CHQAX Sorbent

  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.

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

  48. 120 100 80 60 40 20 0 0.250 ppm 0.050 ppm 1.00 ppm 2.00 ppm TAX Column % Recovery of Pb at Various Concentrations

  49. Metal Recoveries on Various Phases

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