1. practical chiral hplc andMethod developMent1

2. Wide variety of chiral columns available.Commercialized chiral columns:>200 TypesMost enantiomers can be readily separableMethod can be readily validated.Method can be readily scaled up tocommercial production.Chiral chromatography?2 3. Early stages of development:Fastest and more economical way to: Analyze chiral compoundsIsolate individual enantiomersObtain the two enantiomers forclinical toxicological studiesScale up from bench- to pilot plant scaleFull development / productionHighly competitive way to:Isolate single enantiomers(finaldrugs or intermediates)atproduction scale5kg to 100tonsUntil industrial scale productionmg to 10 kgChiral chromatography3 4. Enantiomeric separation:4 5. Resolution formula= (t2-t1)/0.5(tw2-tw1)Enantiomeric resolution:5 6. Enantiomeric purity= Major/(Major + Minor) 100Enantiomeric purity andenantiomeric excess(ee):6 7. Types of CSP..!7 8. Types of CSPs and their loading capacities:8 9. Which type of CSP?1999 2001 2003 20059 10. Small chiral molecules bonded to silica.More specific applications; strong 3-point interactions through 3-classes:-donor phases-acceptor phasesMixed donor-acceptor phasesBinding sites are -basic or -acidic aromatic rings(- interactions),acidic and basic sites(H-bonding), and steric interaction.Mostly used with normal phase HPLC , SFC(super critical fluid chrom).Limitations: Only works with aromatic compounds.Available columns: WhelkO1, Whelk O2,ULMO (mixed phases) etc.A= acidic groupB= acidic siteC= basic siteBrush type csp (3 point interaction model):10 11. Polymer based carbohydrates: Chiral polysaccharide derivatives, i.e.; amylose and cellulose coated on asilica support.Enantiomers form a hydrogen bonds with carbamate links b/w side chainand polysaccharide backboneSteric restrictions at polysaccharide backbone may prevent access of one ofEnantiomers to H-bonding site.Can be used with normal phase HPLC, SFC,RP-HPLCLimitations: Not compatible with a wide range of solvents other than alcohols.11 12. Available columns:12 13. Cyclodextrin Based CSPs: Alpha, Beta and Gamma-cyclodextrin bond to silicaand form chiral cavitiesThree points interaction by:1. Opening of cyclodextrin cavity contains hydroxylsfor hydrogen bonding with polar groups of analyte.2. Hydrophobic part of analyte fits into non polarcavity (inclusion complexes)One enantiomer will be able to better fit in the cavity than the other.Used in RP-HPLC and polar organic mode.Limitation: Analyte must have hydrophobic or aromatic group to fit into cavity.Available columns:Cyclobond (Alpha, Beta and Gamma cyclodextrins) from Astec. Inc.ORpak CDA(Alpha), ORpak CDB(Beta), ORpak(Gamma) from JM Sciences.13 14. Chirobiotic phases:Macrocyclic glycopeptides linked to silicaContain a large number of chiral centers togetherwith cavities for analytes to enter and interact.Potentials interactions:- complexes, H-bonding, ionic interactions.Inclusion complexation, steric interactions.Capable of running RP-HPLC, normal phase,polar organic and polar ionic modes14 15. Chirobiotic v and V2Chirobiotic T and T2Available columns:15 16. Mobile phases for coated CSPs:Normal Phase conditions: Polar mode:Alkane/IPAAlkane/Ethanol Acetonitrile Ethanol Methanol Other alcoholsReversed Phase conditions (RH-Versions):Water/Alcohol or AcetonitrilePhosphate buffer(pH 2-8)/Alcohol or AcetonitrileKPF6 pH 2/ Acetonitrile with chiralcel OD-RHBorate buffer (pH 9) Alcohol or Acetonitrile with chiral pak AD-RH16 17. Mobile phases for coated CSPs:Really! coated CSPs are not stable with all solventsChloroformEthyl acetateDCMAcetoneDMFTHFDMSONever use(even as a sample solvent)These will irreversiblydestroy the coated CSP17 18. Before connecting the column to the system:18 19. 19 20. 20 21. 21 22. 22 23. BackgroundCoated polysaccharide-derived CSPs:Highly selectiveBroad application domainHigh loading capacity23 24. 24 25. Solvent compatibility25 26. Method development.!26 27. Screening approach27 28. Method development on ChiralpakIA28 29. Applications of coated and immobilized CSPs:Non-standard mobile phase29 30. 30 31. Three immobilised CSPsElution order31 32. ExamplesAlkane/Alcohols32 33. ExamplesAlkane/Alcohols33 34. Method development on CHIRALPAK IAEffects of solvent modifiers34 35. Method development on CHIRALPAK IAEffects of solvent modifiersImportant selectivityChange can beObserved with differentSolvent modifiers35 36. Mobile phase additivesAmines, acids, salts36 37. Mobile phase additivesEffects-1Hexane/2-IPA/ Diethylamine(90:10:0.1)Hexane/2-IPA(90:10)37 38. Mobile phase additivesEffects-2n-heptane/ EtOH/AcOH(60:40:0.5)n- heptane/ EtOH/TFA(60:40:0.5)38 39. Mobile phase additivesEffects-3ACN 0.1%DEAACN 0.1% Butylamine39 40. UV cut-off of common HPLC solventsFeasibility of UV detection40 41. Regeneration in practiceProcedures:Purpose:Recover the specific supra molecular structureEnsure the consistency of the column performance41 42. Class of compound Types of CSPAcids Protein, celulose/Amylose,Pirkle, chirobioticAmino Acids Cyclodextrins,Protein,ChirobioticAmines Protein, celulose/Amylose,Pirkle, chirobiotic,CyclodextrAlcohols Protein, celulose/Amylose,Pirkle, chirobiotic,CyclodextrAmides Protein, celulose/Amylose,Pirkle,Cyclodextrin,ChirobiotEsters celulose,Pirkle, chirobiotic.Sulfoxides celulose,Pirkle, chirobiotic,Protein.Carbamates PirkleUreas PirkleCrown ethers Cyclodextrins.Metallocenes Cyclodextrins.Thiols PirkleAmino alcohols Pirkle,Chirobiotic42 43. Class of compound Types of CSPSuccinamides Pirkle.Hydantoins Pirkle,ChirobioticBi-napthol Pirkle-Lactams Parkle,CelulosePolycyclic aromatic hydrocarbons CyclodextrinsCyclic drugs ProteinAromatic drugs Protein, PirkleLactones Celulose, PirkleCyclic ketones Pirkle,CeluloseAlkaloids Celulose,PirkleDihydro pyridines Celulose, PirkleNSAIDS Pirkle,celulose,ProteinOxazolindones PirklePeptides Chirobiotic43 44. Questions???44 45. 45Thank you