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sigma-Basics of Chiral HPLCDefinitionsPrinciplesAvailable CSPsMobile phase typesT408109sigma-The Field of StereochemistryISOMERSSTRUCTURALOPTICALLY INACTIVEISOMERSSTEREOISOMERSENANTIOMERSOPTICALLY ACTIVE(ASYMMETRIC)DIASTEREOISOMERS(SYMMETRIC)OPTICALLY ACTIVEOPTICALLY INACTIVE(GEOMETRIC)EPIMERSANOMERSCIS/TRANSSYN/ANTI1.All isomers have same chemical formula but differ in the arrangement of certain chemical groups in space.2.Many types of isomers have different physical and chemical properties and can be separated by conventional phases like C18.3.Enantiomers have same physical and chemical properties and can be recognized or separated only in chiral environment.sigma-The Inclusion ComplexThe basis for many chiral separations,especially in the reversed phase mode is a phenomena called inclusion complexing.First described for the polyglucose structures,cyclodextrins,it has been identified as a mechanism for the macrocyclic glycopeptides as well as the cellulose and amylose CSPs.An understanding of this phenomena is then imperative to an understanding of how these phases separate.1.53nm0.781.37nm0.571.69nm0.950.78-cyclodextrin-cyclodextrin-cyclodextrinSchematic of one glucose unit in a cyclodextrin moleculeStructure and dimensions of the most common cyclodextrin molecules.sigma-Inclusion Complex SchematicInclusion complexing is accomplished in reversed phase systems.Its effectiveness is dependent upon the position and strength of substituentson benzene or fused ring analytes.The elution order is typically metaorthoBrClFAmine:321Carbonyl:-COOH,-CHO,-C=O,-COORSulfo-,phospho-and hydroxyl groupsOne of these functional groups must be on or alpha to the stereogenic centerPolar Organic:Structural/Functional Group Requirementssigma-Mobile phases types and mechanisms Normal PhaseReversed PhasePolar OrganicPolar Ionicsigma-Normal phase and types of interactionComposition:Hydrocarbon solvent:hexane or heptane+polar alcohol:IPA or EtOHDominant interactions:interaction,hydrogen bondingType of CSPs:CYCLOBOND I 2000 DMP,SN,RN,CHIROBIOTIC V,T,R and TAGsigma-Normal Phase Solvents MephenytoinRef:Anal.Chem.,Vol.66(9),p 1473-184,May 1994.Armstrong,Tang,S.Chen,Zhou,Bagwill and J.R.Chen.%Hexane100%Isopropanol00120102030405060708090100Hexane vs.IPAksigma-Gradient Separation in the Normal Phase5100%EtOH in Hexanet(min)6.5 16.5 26.5Derivative:3,4-DinitrobenzoylCYCLOBOND I 2000 SNHexane/EtOH Gradient2-Aminoheptane-MethylbenzylamineTryptophan methyl estersigma-Reversed PhaseComposition:Organic solvent:ACN,MeOH or THF+aqueous buffer:TEAA,NH4OAcDominant interactions:inclusion,hydrogen bondingType of CSPs:CYCLOBOND I 2000,RSP,AC,DMP,SN,RN,II,III,CHIROBIOTIC V,T,R and TAGsigma-Reversed Phase Separation5-Methyl-5-phenyl hydantoinRef:Anal.Chem.,Vol.66(9),p.1473-1484,May 1994.01020304050607080901000102030405060708090100%Buffer vs.%Organic Modifier%Buffer100%Acetonitrile0ksigma-Reversed Phase SolventsFactors Influencing a Separation?pH?Organic modifier?Buffer type and concentration?Flow rate?Temperaturesigma-Polar Organic PhaseComposition:ACN+MeOH+HOAc+TEADominant interactions:Hydrogen bonding,dipole-dipoleType of CSPs:Cyclodextrins only CYCLOBOND I 2000,RSP,AC,DMP,SN,RNsigma-Polar Organic SolventsThere are four components to the original polar organic mode(v/v/v/v):Acetonitrile 50-100 partsMethanol0-50 partsAnhydrous,Glacial Acetic Acid0.1 to 1.0 partsAnhydrous Triethylamine0.1 to 1.0 partsThis composition is primarily used for cyclodextrins and cyclodextrin derivatives.CYCLOBOND Phases OnlyFactors Influencing Retention and Selectivity?To increase selectivity alter ratio of acid to base?To decrease retention without affecting selectivity:?Increase acid/base concentration at same ratio?Increase methanol concentration?To increase retention:?Reduce or eliminate methanol?Decrease acid/base concentration at same ratiosigma-New Polar Ionic ModeComposition:MeOH+HOAc+TEADominant interactions:Ionic interaction,hydrogen bondingType of CSPs:Macrocyclic glycopeptides only-CHIROBIOTIC V,T,R and TAGsigma-Polar Ionic Mode:CHIROBIOTIC Phases Only?Faster,more efficient separations,low pressure,long column life?Faster method development,simple optimization,broad selectivity?Best use ammonia and formic acid or acetic acid modifiers(0.01-1.0 parts)in 100 parts methanol for LC/MS/MS compatibility.?Analyte salts easily disassociated?Complimentary to normal phase separations on polysaccharide CSPs?Very useful in preparative purifications to replace hexane/ethanol.?Lower boiling point than heptane or hexane,higher evaporation rate?Less toxic?Higher evaporation rateBENEFITS:sigma-POLAR IONIC MODE?Methanol/Acid/Base Components:?Methanol,anhydrous?Acid:anhydrous TFA,acetic acid,formic acid?Base:TEA,DEA,NH3?Alternatives:ammonium formate or acetate?Ratio of Acid to Base?Controls only selectivity?Rate:4:1 to 1:4,most typical 2:1?Concentration of Acid+Base?Controls retention and selectivity?Concentration range:0.001 to 1.0 part per 100,most typical 0.10.Mobile Phase Componentssigma-Conversion from Old Polar Organic Mode to New Polar IONIC ModeAlbuterol NHOHOOHHC(CH3)3Peak 1-17.04 min.Peak 2-18.37 min.Peak 1-11.28 min.Peak 2-12.45 min.70/30/0.5/0.2:CH3CN/CH3OH/HOAc/TEA100/0.01/0.01:CH3OH/TFA/NH4OHFor CHIROBIOTIC Phases Onlysigma-Acid-Base EffectsNTerbutalineOHHC(CH3)3HOOHWith the new polar organic mode different acids and bases could be used,unlike the situation with the cyclodextrin,which demonstrates selectivity only employing acetic acid and triethylamine.CH3OH/HOAc/TEA100/0.3/0.2CH3OH/20mMNH4AcCH3OH/TFA/NH4OH100/0.05/0.05=1.38=1.30=1.30sigma-Comparison Using HOAc/TEAvs ATFA in the Polar IONIC ModeCNCCH3N-Benzyl-a-methylbenzylamineCHIROBIOTIC V,250 x4.6mmPeak 1 17.09 min.Peak 2 18.82 min.Peak 1 15.21 min.Peak 2 16.66 min.HOAc/TEA in Mobile Phase100/0.02/0.01:MeOH/HOAc/TEAATFA*in Mobile Phase100/0.02 v/w:MeOH/NH4OOCCF3*Works best with CHIROBIOTIC V.sigma-Polar IONIC mode composition:Methanol+Acid+Base(100+0.1+0.1,v/v/v)or Methanol+Volatile Ammonium Salt(100+0.1%v/w)Normal Phase mode composition:Polar+Nonpolar(EtOH+Heptane)Polar organic mode composition:Polar/Nonpolar(MeOH or EtOH or ACN or combinations,eg MeOH/ACN)Reversed Phase composition:Organic+Aqueous Buffer(ACN+TEAA;ACN&NH4OAc)Comparison of Four Basic Mobile Phase Types for CHIROBIOTIC CSPsIonizableNeutralNeutral/PolarCompound is:All typessigma-Shorter analysis timeHigher efficiency17.29920.85215.40317.5323.86717.8223.15311.378ArginineCHIROBIOTIC T,25 C30/70 MeOH/10 mMNH4OAc(pH4.0)1.0 mL/min,ELSDArginineCHIROBIOTIC T,45 C30/70 MeOH/10 mMNH4OAc(pH4.0)1.0 mL/min,ELSDDiacetyl-cysteine(N15)CHIROBIOTIC T,25 C100/0.1%(v/w)MeOH/NH4OAc1.0 mL/min,ELSDDiacetyl-cysteine(N15)CHIROBIOTIC T,55 C100/0.1%(v/w)MeOH/NH4OAc1.0 mL/min,ELSDTemperature Effectssigma-Semi-Prep Application of Temperature Effect in Loading Study of Diacetyl-Cysteine(N15)Flow Rate:2.0 mL/minHigher Temp3.8837.33926.2563.8837.33926.25623 C3.8744.7928.53.8744.7928.535 CLoading:15 mgFlow Rate:1.0 mL/minLoading:15 mgFlow Rate:1.0 mL/minLess Time!Less mobile phase additive!2.68715.47623 CCHIROBIOTIC T,250 x4.6mmMobiile Phase:MeOH/NH4OAc(0.1%v/w)Detection:UV230nmsigma-Practical Guides for CSP Screeningsigma-Historical Chiral Screening Approaches?Emphasized molecular structure and analysis of analyte-CSP surface interaction as a predictive tool?Utilized databases and literature searches for similar molecules but minor structural differences often resulted in a loss of selectivity for some CSPs?Screen all CSPs!sigma-Aim Method Development Process?To quickly identify a suitable column for selectivity,in the minimum number of experimentssigma-Method Development Approaches?Single column with multi-mobile phases(mainly CHIROBIOTIC phases)?Multi-column switching with minimal number of simple mobile phasessigma-Screening StrategyChoose a small set of CSPs that:?are broad-based to increase chances of success for a wide range of molecular types?offer selectivity in a wide range of mobile phases for increased selectivity possibilities and sample solubility?are complementary to each other,minimum overlap in selectivitysigma-ComplementaryDefined as:Offering separation potential in areas not possible with a givenCSP,i.e.,CHIROBIOTIC phases handle more polar molecules better than amylose or cellulose.Offer increased selectivity in same mobile phases conditions,i.e.,substituting one CHIROBIOTIC phase for another.sigma-Complementary SeparationsCHIROBIOTIC R versus CHIROBIOTIC VMobile Phase:50/50:Hex/EtOH4-Benzyl-2-oxazolidinonePeak 1(S)6.01Peak 2(R)6.91Peak 1(R)6.54Peak 2(S)7.15CHIROBIOTIC RCHIROBIOTIC Vsigma-Complementary SeparationsCHIROBIOTIC T versus CHIROBIOTIC RNaproxenTRMobile Phase:30/70:MeOH/0.1%TEAA,pH 4.18.44 min.8.83 min.21.43min.25.85 min.sigma-Screening StrategyCurrent industry trend is for generic screening methods with:?a simple set of columns combining CHIROBIOTIC and Chiralcel/pak*CSPs?a minimum number of solvents covering all five mobile phase types*Chiracel and Chiralpak are the trademarks of Daicel.sigma-Distinct Mobile Phase Choices?Normal phase?Polar mobile phases?polar ionic mode:CHIROBIOTIC phases only?polar organic mode:CYCLOBOND phases only?Polar mode:Chiralcel/pak and CHIROBIOTIC phases?Reversed phasesigma-MetoprololCHIRACEL ODPeak 1 11.9 min.Peak 2 18.2 min.20/80/0.1:IPA/Hex/DEANormal PhaseCHIROBIOTIC TPeak 1 15.36 minPeak 2 17.11 minMeOH/0.1%ATFAPolar Ionic ModeNormal Phase vs Polar Ionic ModeONH3COOHHCH3CH3sigma-AlprenololCHIROBIOTIC VPeak 1 7.69 min.Peak 2 8.33 min.100/0.01/0.01:MeOH/HOAc/TEAONOHCH2HCH3CH3Polar Ionic ModeNormal Phase vs Polar Ionic ModeCHIRACEL ODPeak 1-12.4 min.Peak 2-16.4 min.20/80/0.1:IPA/Hex/TFANormal Phasesigma-CHIROBIOTIC V2Peak 1 8.80 minPeak 2 10.69 minPolar Ionic Mode100/0.1%:MeOH/ATFACellulosic vs CHIROBIOTIC CSPs:RP vs PIMONOHCH2HCH3CH3TolperisoneCHIRALPAK AD-RH Peak 1 5.23 minPeak 2 6.06 min60/40:ACN/20mM BorateReversed PhaseCHIRALPAK is the registered trademark of Daicel Chemical Industries,Ltdsigma-8.509.47Broad Selectivity Based on the Same StereogenicCenter with CHIROBIOTIC T Example:Amino AlcoholsMobile Phase:100/0.1/0.1:MeOH/HOAc/TEA 2.0 mL/minuteAlbuterolONHH3CCH3OHNH2OAtenolol12.6413.84OOHHNCH3CH3H3COMetoprolol6.817.48OOHHNCH3CH3OCH2OxprenololPropranololOHHONCH3CH3H3CSNHOHHNCH3CH3OOSotalol8.71 NHOHOOHHC(CH3)31.7.082.7.831.12.642.13.841.6.812.7.481.11.822.12.551.8.502.9.471.8.712.9.66sigma-Broad Selectivity Based on the Same StereogenicCenter with CHIROBIOTIC V:Example:ProfensMobile Phase:10/90:THF/10mM Na Citrate,pH 6.3 1.0 mL/min.Fenoprofen Methyl EsterFenoprofenKetoprofenIbuprofenFlurbiprofenNaproxen1.8.312.9.721.8.102.9.561.8.162.9.911.5.772.6.471.8.532.11.191.8.132.9.98 OCHCOOCH3CH3OCH3COOHOCH3COOHH3CCH3CH3COOHFCOOHCH3CH3COOHH3COsigma-Purpose of Assay?Quick analytical method(possibly suitable for later optimization and validation)?Suitable for possible small scale prep?Trace analysis of unwanted isomer?Impurity profiling?LC-MS methodGeneric screening develops options for a variety of applicationssigma-Cellulose&Amylose(CHIRALCEL/CHIRALPAK)Macrocyclic Glycopeptides(CHIROBIOTIC)Broad applicability over wide range of compound typesBroad applicability over wide range of compound typesTraditionally NP,but now also RP,POM(different columns)Designed to provide selectivity in PIM,RP,NP(same column)Chiral interaction sites via different chemistries&helical structureLarge number of chiral interaction sitesMost useful are AD,OD,AS,OJMost useful are V2,T,R,TAGCoated phasesChemically bondedComplementary Method Developmentsigma-Complementary Method DevelopmentCHIRALCEL/PAK:?Compound must be in neutral form-interaction always non-ionic?Separate samples into acids,bases and neutrals(neutrals can be screened with either acids or bases)CHIROBIOTIC:?Compound can be ionised,or can be a salt ionic interactions are a key mechanism?Same mobile phase screens are used for all samples,but can choose selective screening for acid,bases or neutralsNote:Functional group on or near stereogenic center dictates whether analyte is acid or basesigma-sigma-Typical Screening ResultsOptimized CHIROBIOTIC VMeOH/NH4TFA;100/0.02 w%Chiralyzer Optical RotationRTVCHIROBIOITC ColumnsPolar ionic mode:screen 5sigma-Best Positive Screening ResultsColumn:CHIROBIOTIC V2,250 x4.6mmMobile Phase:40/60,ACN/10 mM NH4OAc,pH 3.8Flow Rate:0.8mL/minUV:380 nmColumn:CHIROBIOTIC V2,150 x4.6mmMobile Phase:50/50,MeOH/5 mM NH4OAc,pH 3.5Flow Rate:0.8mL/minUV:380 nmOptimized Method for LC/MS applicationmin024681012mAU 02468VWD1 A,Wavelength=380 nm(SAMPLE04-04-13.D)P1:6.04P2:7.67min02468101214mAU 020406080100120140160VWD1 A,Wavelength=380 nm(SAMPLE04-04-7.D)P1:10.11P2:11.36sigma-TerbutalineOptimization Step:Polar Ionic ModeEnhanced Selectivity T T2CHIROBIOTIC T,100/0.2/0.1;MeOH/AcOH/TEACHIROBIOTIC T2,100/0.1w%;MeOH/NH4TFAPeak 1:9.72Peak 2:10.921212Peak 1:9.70Peak 2:16.06sigma-Method Development Screen?Monitor column performance regularly?Store columns in correct solvents(free of additives):?CHIROBIOTIC:100%MeOH?CYCLOBOND:100%2-PrOH?CHIRALCEL/PAK:Hexane/2-PrOH,90/10sigma- 0123456mAU 0 100 200 300 400 0 1 2 3 4 5mAU 0 25 50 75 100 125 150 175 0 1 2 3 4 56m AU 0 50 100 150 200 250 01 2345678mAU 020406080100120140160 Performance Tests for CHIROBIOTIC PhasesTo ensure the selectivity and performance of all CHIROBIOTIC LC columns,periodically test your columns.This can now be accomplished with a single compound for all the CHIROBIOTIC phases in a very simple mobile phase of 100%MeOHCHIROBIOTIC VCHIROBIOTIC TCHIROBIOTIC RCHIROBIOTIC TAGPeak 1:3.70 min.Peak 2:4.21 min.Peak 1:3.83 min.Peak 2:5.05 min.Peak 1:4.15 min.Peak 2:4.63 min.Peak 1:3.98 min.Peak 2:7.22 min.OOHCH3NHNConditions for all columns:Sample:5-Methyl-5-Phenylhydantoin(Aldrich 18,082-3)Column size:250 x4.6mmMobile phase:100%MeOHFlow rate:1 ml/min.UV:220nmsigma-Mobile Phase:Flow Rate:Injection Vol.:Sample Conc:Detection:100/0.3/0.2:ACN/HOAc/TEA1.0 mL/min.5 L5 mg/mL254 nmMobile Phase:Flow Rate:Injection Vol.:Sample Conc:Detection:10/90:ACN/0.1%TEAA,pH 4.11.5 mL/min.3 L5 mg/mL230 nm10/90:MeOH/0.1%NaAc,pH 5.51.0 mL/min.3 L5 mg/mL230 nm50/50:ACN/0.1%TEAA,pH 4.11.0 mL/min.5 L5 mg/mL254 nmMobile Phase:Flow Rate:Injection Vol.:Sample Conc:Detection:Mobile Phase:Flow Rate:Injection Vol.:Sample Conc:Detection:Performance Tests for CYCLOBONDCYCLOBOND I 2000Warfarin(Sigma#A2250)Peak 1:6.89 min.Peak 2:7.90 min.OOHOCH3OCYCLOBOND I2000RSPChlorthalidone(Sigma#C2775)Peak 1:12.04 min.Peak 2:13.73 min.ClONHSHONH2OOHOCHNH2CH2HOCYCLOBOND I 2000ACNorphenylephrine(Aldrich#11,372-7)Peak 1:7.50 min.Peak 2:8.77 min.SNNH3NO2SF3COOHCH2HCYCLOBOND I 2000 SNBendroflumethiazide(Sigma#B5775)Peak 1:6.80 min.Peak 2:7.40 min.sigma-Isocratic or Gradient?Gradients suitable for NP,unsuitable for RP?Recent papers suggest that gradient method development not much faster and has no greater success rate?In PIM,ammonium formate gradient possible from 0.01%to 0.05%in,methanol to replace HOAc which has a very broad windowsigma-Results of Complementary Generic Screening(1)Two
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