Finding the best separation for enantiomeric mixtures Chiral Technologies Europe Illkirch Cedex – France Chiral Technologi...
Analytical Method Development Goals <ul><li>Resolution of the enantiomeric pair </li></ul><ul><li>Resolution from impuriti...
Method Development Overview  for Polysaccharide-derived CSPs Racemic mixture HPLC SFC Organic mobile phases Water compatib...
Choice of LC and SFC Technique <ul><li>Recognition behavior </li></ul><ul><li>Timing and productivity </li></ul><ul><li>So...
Choice of LC and SFC Technique <ul><li>SFC Advantages of Small Scale Separations: </li></ul><ul><li>Resolution of the enan...
HPLC method development  in organic mobile phases
Polysaccharide-derived Columns <ul><li>Amylose-based </li></ul><ul><li>CHIRALPAK AD-H  (Coated) </li></ul><ul><li>CHIRALPA...
Primary Screening <ul><li>Immobilised </li></ul><ul><li>CHIRALPAK IA </li></ul><ul><li>CHIRALPAK IB </li></ul><ul><li>CHIR...
Primary Screening
Primary Screening <ul><li>Immobilised </li></ul><ul><li>Alkane / EtOH </li></ul><ul><li>Alkane / 2-PrOH </li></ul><ul><li>...
Basic Molecule – Analytical Scale IA IB IC Hept/EtOH/DEA Hept/IPA/DEA Hept/THF/DEA Hept/DCM/DEA Separation Retention time ...
Basic Molecule – Analytical Scale CHIRALPAK  IB n -heptane/2-PrOH/DEA 70/30/0.1 12 min 7 min
Complementary Properties of CSPs <ul><li>CHIRALPAK IA </li></ul><ul><li>CHIRALPAK IB </li></ul><ul><li>CHIRALPAK IC </li><...
Complementary Properties of CSPs <ul><li>Advantages </li></ul><ul><li>Specific resolutions </li></ul><ul><li>Changes in en...
SFC method development  in organic mobile phases
Compatible Co-Solvents in SFC <ul><li>Coated CSPs </li></ul><ul><li>Ethanol </li></ul><ul><li>Methanol </li></ul><ul><li>2...
Compatible Co-Solvents in SFC <ul><li>Extended Solvent Range </li></ul><ul><li>MtBE </li></ul><ul><li>Ethyl acetate </li><...
CSPs in SFC: Analytical Scale CO 2  / MeOH 90/10 T=25°C Flow rate: 3.0ml/min. CHIRALPAK IA CO 2  / THF (+1%DEA) 70/30 T=30...
CSPs in SFC: Analytical Scale <ul><li>All the advantages of the SFC applications on polysaccharide-derived CSPs </li></ul>...
HPLC method development  in water compatible mobile phases
Water Compatible Mobile Phases Aqueous  Solution Organic modifier Identify the compound nature Acidic Neutral Basic HCOOH ...
Separations RP-Mode CHIRALPAK IA furoin MeOH/H 2 O 55:45 min 0 2 4 6 8
Separations RP-Mode CHIRALPAK IC 2,3-dibenzoyl- DL-tartaric  acid HCOOH aq. (pH2.0) ACN: 55% min 0 2 4 6 8
Faster screening and enhanced resolution: 3-µm CSPs
Fast Analysis: CHIRALPAK IA-3 (4.6 x 50 mm) Hexane-EtOH 80:20   (+0.1% AE) Flow rate:  5.0  ml/min DCM-MeOH 98:2 (+0.1% AE...
Looking for new separations: different chiral selectors
Different Substituents; New Enantiorecognition CHIRALCEL OD CHIRALPAK AD CHIRALCEL OZ CHIRALPAK AZ CHIRALPAK IC CHIRALPAK AY
Are New Selectors Needed? Metolachlor Hexane / EtOH 95:5 1ml/min, 25°C (4.6 x 250 mm) CHIRALPAK IA  CHIRALPAK IB  CHIRALPA...
Are New Selectors Needed? Metolachlor Hexane / EtOH 95:5 1ml/min, 25°C (4.6 x 250 mm) CHIRALPAK AS-H CHIRALPAK AD-H CHIRAL...
Are New Selectors Needed? Metolachlor Hexane / EtOH 95:5 1ml/min, 25°C (4.6 x 250 mm) CHIRALPAK AY-H
Chiral Technologies Worldwide <ul><li>Quality </li></ul><ul><li>Expertise </li></ul><ul><li>Solutions </li></ul>www.chiral...
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Finding the best separation for enantiomeric mixtures

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Finding the best separation for enantiomeric mixtures

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  • Speak the line, “Can we achieve this with a minimal number of columns and efficient screening strategies?”
  • Speak, “Analytical, but also preparative considerations.”
  • Speak, « Looking for a given elution order »
  • Speak that these are Polysacchasride-derived CSPs Also speak: « These will irreversibly destroy coated CSPs »
  • Speak « These will irreversibly destroy coated CSPs »
  • Speak, “An alternative possibility would be using polar organic mode to be water compatible.”
  • Different substituents New enantiorecognition
  • Finding the best separation for enantiomeric mixtures

    1. 1. Finding the best separation for enantiomeric mixtures Chiral Technologies Europe Illkirch Cedex – France Chiral Technologies, Inc. West Chester, PA
    2. 2. Analytical Method Development Goals <ul><li>Resolution of the enantiomeric pair </li></ul><ul><li>Resolution from impurities </li></ul><ul><li>Short analysis time </li></ul><ul><li>Suitable elution order </li></ul><ul><li>Compatibility of the sample media with mobile phase and column </li></ul><ul><li>Low LOD/LOQ </li></ul><ul><li>Reproducibility and robustness of the method </li></ul><ul><li>Stability of the sample under analytical conditions </li></ul>
    3. 3. Method Development Overview for Polysaccharide-derived CSPs Racemic mixture HPLC SFC Organic mobile phases Water compatible mobile phases CHOICE OF SEPARATION MODE CHOICE OF TECHNOLOGY
    4. 4. Choice of LC and SFC Technique <ul><li>Recognition behavior </li></ul><ul><li>Timing and productivity </li></ul><ul><li>Solubility considerations </li></ul><ul><li>Stability issues </li></ul><ul><li>Amount of mixture to be separated </li></ul><ul><li>Solvent volume to be evaporated </li></ul><ul><li>Equipment availability </li></ul>09/01/2007 00:05:36 09/01/2007 00:52:44 09/01/2007 00:29:10 -205.0 256.0 717.0 1178.0 1639.0 2100.0 Cyclique Cyclique signal UV (PIC04002:ao_F_mes_UV_DETECTOR) signal UV (PIC04002:ao_F_mes_UV_DETECTOR) (09/01/2007 00:05:36) -17.1 mv (09/01/2007 00:05:36) -17.1 mv -42.4 mv (0 jours, 00:47:09) (09/01/2007 00:52:44) -59.5 mv (09/01/2007 00:52:44) -59.5 mv -42.4 mv (0 jours, 00:47:09)
    5. 5. Choice of LC and SFC Technique <ul><li>SFC Advantages of Small Scale Separations: </li></ul><ul><li>Resolution of the enantiomeric pair </li></ul><ul><li>Less solvent to be evaporated </li></ul><ul><li>Single solvent </li></ul><ul><li>Short retention times </li></ul><ul><li>Enhanced solubility in certain cases </li></ul><ul><li>Less perturbance when injecting in different solvent </li></ul>
    6. 6. HPLC method development in organic mobile phases
    7. 7. Polysaccharide-derived Columns <ul><li>Amylose-based </li></ul><ul><li>CHIRALPAK AD-H (Coated) </li></ul><ul><li>CHIRALPAK AS-H (Coated) </li></ul><ul><li>CHIRALPAK AZ-H (Coated) </li></ul><ul><li>CHIRALPAK AY-H (Coated) </li></ul><ul><li>CHIRALPAK IA (Immobilised) </li></ul>Cellulose-based CHIRALCEL OD-H (Coated) CHIRALCEL OJ-H (Coated) CHIRALCEL OZ-H (Coated) CHIRALPAK IB (Immobilised) CHIRALPAK IC (Immobilised)
    8. 8. Primary Screening <ul><li>Immobilised </li></ul><ul><li>CHIRALPAK IA </li></ul><ul><li>CHIRALPAK IB </li></ul><ul><li>CHIRALPAK IC </li></ul><ul><li>Coated </li></ul><ul><li>CHIRALPAK AD-H </li></ul><ul><li>CHIRALCEL OD-H </li></ul><ul><li>CHIRALPAK AS-H </li></ul><ul><li>CHIRALCEL OJ-H </li></ul>
    9. 9. Primary Screening
    10. 10. Primary Screening <ul><li>Immobilised </li></ul><ul><li>Alkane / EtOH </li></ul><ul><li>Alkane / 2-PrOH </li></ul><ul><li>MtBE mixtures </li></ul><ul><li>Dichloromethane (or THF) mixtures </li></ul><ul><li>Coated </li></ul><ul><li>Alkane / EtOH </li></ul><ul><li>Alkane / 2-PrOH </li></ul><ul><li>EtOH / MeOH </li></ul><ul><li>Acetonitrile </li></ul>
    11. 11. Basic Molecule – Analytical Scale IA IB IC Hept/EtOH/DEA Hept/IPA/DEA Hept/THF/DEA Hept/DCM/DEA Separation Retention time > 30 min Separation Retention time > 30 min 70/30/0.1 70/30/0.1 90/10/0.1 80/20/0.1 Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -50 0 50 100 150 200 250 300 350 400 450 mAU -50 0 50 100 150 200 250 300 350 400 450 1: 290 nm, 4 nm CE070128 - IA - Heptane-EtOH - 70-30-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU 0 50 100 150 200 250 300 mAU 0 50 100 150 200 250 300 1: 290 nm, 4 nm CE070128 - IA - Heptane-IPA - 70-30-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -2,5 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 mAU -2,5 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 1: 290 nm, 4 nm CE07128 - IA - Hept-THF 90-10-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -5 0 5 10 15 20 25 30 mAU -5 0 5 10 15 20 25 30 1: 290 nm, 4 nm CE07128 - IA - Hept-DCM 80-20-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -50 0 50 100 150 200 250 300 350 400 450 mAU -50 0 50 100 150 200 250 300 350 400 450 1: 290 nm, 4 nm CE070128 - IB - Heptane-EtOH - 70-30-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -25 0 25 50 75 100 125 150 175 200 225 250 275 mAU -25 0 25 50 75 100 125 150 175 200 225 250 275 1: 290 nm, 4 nm CE070128 - IB - Heptane-IPA - 70-30-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -50 0 50 100 150 200 250 300 350 400 450 mAU -50 0 50 100 150 200 250 300 350 400 450 1: 290 nm, 4 nm CE070128 - IC - Heptane-EtOH - 70-30-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -25 0 25 50 75 100 125 150 175 200 225 250 mAU -25 0 25 50 75 100 125 150 175 200 225 250 1: 290 nm, 4 nm CE070128 - IC - Heptane-IPA - 70-30-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -2,5 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 mAU -2,5 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 1: 290 nm, 4 nm CE07128 - IC - Hept-THF 90-10-Rep2 Retention Time Minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 mAU -10 0 10 20 30 40 50 mAU -10 0 10 20 30 40 50 1: 290 nm, 4 nm CE07128 - IC - Hept-DCM 80-20-Rep2 Retention Time
    12. 12. Basic Molecule – Analytical Scale CHIRALPAK IB n -heptane/2-PrOH/DEA 70/30/0.1 12 min 7 min
    13. 13. Complementary Properties of CSPs <ul><li>CHIRALPAK IA </li></ul><ul><li>CHIRALPAK IB </li></ul><ul><li>CHIRALPAK IC </li></ul>(D) (L) min 0 2.5 5 7.5 10 12.5 15 17.5 (D) (L) FMOC-D,L-Leucine n -hexane/2-PrOH/TFA 90/10/0.1 (D) min 0 2.5 5 7.5 10 12.5 15 17.5 (L) min 0 2.5 5 7.5 10 12.5 15 17.5
    14. 14. Complementary Properties of CSPs <ul><li>Advantages </li></ul><ul><li>Specific resolutions </li></ul><ul><li>Changes in enantiomeric elution order </li></ul><ul><li>Different elution of impurities </li></ul>
    15. 15. SFC method development in organic mobile phases
    16. 16. Compatible Co-Solvents in SFC <ul><li>Coated CSPs </li></ul><ul><li>Ethanol </li></ul><ul><li>Methanol </li></ul><ul><li>2-Propanol </li></ul><ul><li>Acetonitrile </li></ul><ul><li>Other alcohols </li></ul><ul><li>Immobilised CSPs </li></ul><ul><li>Ethanol </li></ul><ul><li>Methanol </li></ul><ul><li>2-Propanol </li></ul><ul><li>Acetonitrile </li></ul><ul><li>Other alcohols </li></ul>
    17. 17. Compatible Co-Solvents in SFC <ul><li>Extended Solvent Range </li></ul><ul><li>MtBE </li></ul><ul><li>Ethyl acetate </li></ul><ul><li>THF </li></ul><ul><li>Dichloromethane </li></ul><ul><li>Chloroform </li></ul><ul><li>1,4-dioxane </li></ul><ul><li>Acetone </li></ul><ul><li>DMSO or DMF (as injection solvents) </li></ul>
    18. 18. CSPs in SFC: Analytical Scale CO 2 / MeOH 90/10 T=25°C Flow rate: 3.0ml/min. CHIRALPAK IA CO 2 / THF (+1%DEA) 70/30 T=30°C Flow rate: 3.0ml/min. CHIRALPAK IC 0 200 400 600 800 1000 1200 (sec)
    19. 19. CSPs in SFC: Analytical Scale <ul><li>All the advantages of the SFC applications on polysaccharide-derived CSPs </li></ul><ul><li>Possibility of eluting and/or injecting in different solvents (DCM, THF, …) when using the immobilised CSPs </li></ul>
    20. 20. HPLC method development in water compatible mobile phases
    21. 21. Water Compatible Mobile Phases Aqueous Solution Organic modifier Identify the compound nature Acidic Neutral Basic HCOOH pH 2.0 40% ACN 60% MeOH H 2 O 40% ACN 60% MeOH 20mM NH 4 HCO 3 pH 9.0 40% ACN 60% MeOH
    22. 22. Separations RP-Mode CHIRALPAK IA furoin MeOH/H 2 O 55:45 min 0 2 4 6 8
    23. 23. Separations RP-Mode CHIRALPAK IC 2,3-dibenzoyl- DL-tartaric acid HCOOH aq. (pH2.0) ACN: 55% min 0 2 4 6 8
    24. 24. Faster screening and enhanced resolution: 3-µm CSPs
    25. 25. Fast Analysis: CHIRALPAK IA-3 (4.6 x 50 mm) Hexane-EtOH 80:20 (+0.1% AE) Flow rate: 5.0 ml/min DCM-MeOH 98:2 (+0.1% AE) Flow rate: 5.0 ml/min MeOH 100% Flow rate: 5.0 ml/min 40 seconds 30 seconds 20 seconds
    26. 26. Looking for new separations: different chiral selectors
    27. 27. Different Substituents; New Enantiorecognition CHIRALCEL OD CHIRALPAK AD CHIRALCEL OZ CHIRALPAK AZ CHIRALPAK IC CHIRALPAK AY
    28. 28. Are New Selectors Needed? Metolachlor Hexane / EtOH 95:5 1ml/min, 25°C (4.6 x 250 mm) CHIRALPAK IA CHIRALPAK IB CHIRALPAK IC CHIRALPAK IA CHIRALPAK IB CHIRALPAK IC
    29. 29. Are New Selectors Needed? Metolachlor Hexane / EtOH 95:5 1ml/min, 25°C (4.6 x 250 mm) CHIRALPAK AS-H CHIRALPAK AD-H CHIRALCEL OD-H CHIRALCEL OJ-H
    30. 30. Are New Selectors Needed? Metolachlor Hexane / EtOH 95:5 1ml/min, 25°C (4.6 x 250 mm) CHIRALPAK AY-H
    31. 31. Chiral Technologies Worldwide <ul><li>Quality </li></ul><ul><li>Expertise </li></ul><ul><li>Solutions </li></ul>www.chiraltechworldwide.com Americas: 610-594-2100 Europe: 00 33 (0)3 88 79 52 00

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