Schreier Bernreuther Huffer 60 40 5R,8R ~ S5,8R20 e0- + - - - ~ : : : : : : : : : : : ..=..== ...=..== ...=...==: ..~ .", ........ 20 -40 60 " / . : 5R,85 .. 55,85 .80-+----.----.----.---r--r----i JIJIJ 200250300350200250300350 runrun AA tt lm de Gruyter Peter Schreier Alexander Bernreuther Manfred Huffer Analysis of Chiral Organic Molecules Methodology and Applications Walter de Gruyter.Berlin'New York1995 ProfessorDr.Peter Schreier Dr.AlexanderBernreuther Dr.ManfredHuffer Institut furPharmazieundLebensmittelchemie UniversiHitWurzburg AmHubland 97074Wurzburg Thebook contains72figuresandformulasand 42tables. @Printedonacid-freepaper whichfallswithintheguidelinesof theANSI to ensurepermanenceanddurability. Libraryof CongressCataloging-in-PublicationData Schreier,Peter,1942Analysisof chiralorganicmolecules:methodologyand applicationsIPeterSchreier,AlexanderBernreuther,ManfredHuffer. p.cm. Includesbibliographicalreferences(p.)and index. ISBN3-11-013659-7 1.Opticalisomers- Analysis.2.Chirality.I. Bernreuther, Alexander,1961- II.Huffer,Manfred,1960III.Title. QD471.S381996 547'.3-dc2095-32104 CIP DieDeutscheBibliothek- Cataloging-in-PublicationData Schreier,Peter: Analysisof chiralorganicmolecules: methodologyandapplications I Peter Schreier; Alexander Bernreuther ; Manfred Huffer.- Berlin;NewYork:deGruyter,1995 ISBN3-11-013659-7 NE:Bernreuther,Alexander:;Huffer,Manfred: Copyright1995byWalterdeGruyter&Co.,0-10785Berlin Allrightsreserved,includingthoseof translationintoforeignlanguages.Nopartof thisbook maybereproducedor transmittedinanyformorbyanymeans,electronicormechanical,includingphotocopy, recordingor anyinformationstorage and retrievalsystem,without permissionin writingfromthepublisher. PrintedinGermany. Printing:GerikeGmbH,Berlin.- Binding:Liideritz&Bauer GmbH,Berlin. CoverDesign:HansberndLindemann,Berlin. PREFACE In thecourseof manyyears'workin theappliedresearchfieldoftheanalysisof volatile aroma compounds and their non-volatile precursors, we were (and still are) continuously confronted with the problem of selecting the most appropriate method forthe analysis of chiral molecules. In all areas of 'dural analysis', i.e.from classical opticalrotationtothemodernchromatographicandelectrophoreticmethods, excellentcomprehensivereviewsandmonographscanbefoundandfor applications in liquid chromatography and gas chromatography even databases are available.However,aconciseintroductiontoandguidethroughthisrapidly developingfieldcoveringallfacetsofmethodologiesintheanalysisofchiral organicmoleculesislackingtodate.Thisbookisanattempttofillthisgap,its primary objective being to introduce the practical considerations involved in 'chiral analysis',includingchiropticalmethods(polarimetry,opticalrotationdispersion, circulardichroism),nuclearmagneticresonance,chromatographic(liquid chromatography,gaschromatography,supercriticalfluidchromatography,planar chromatography, counter-current chromatography)and electrophoretictechniques. Someknowledgeoftheoryisessentialtoattainthisgoal,butneithera comprehensive nor a rigorous treatment of the theories is presented here. In order to extendtheutilityofthisbooktothelargestpossiblenumberofusers,wehave stressedsimplicity,particularlyin explanations.Wesincerelyhopethatthisbook succeedsinfacilitatingtheapproachto'chiralanalysis'andenablinganalyststo pinpoint the most appropriate analytical methods quickly and easily. We are grateful to Dr. M.Herderich, Dr. H. U.Humpf, and Dr. W.Schwab fortheir helpfuldiscussionsundcontributions.OurparticularthanksareduetoM. Kleinschnitzforhisintensiveworkinpreparingthe'camera-ready'manuscript. Finally, the kind support provided by the publisher is gratefully acknowledged. WfuzburgP. Schreier April 1995A. Bernreuther M.Huffer CONTENTS Ust of Abbreviations ......................................................................................XI 1Introduction.. ........ ................ ............ ......................... ....... ...... ............. ........ ....1 References........................................................................................................7 2The development of stereochemical concepts ..............................................9 2.1Chirality and molecular structure.................................................................10 2.1.1Molecules with asymmetric atoms................................................................11 2.1.2Other types of chiral molecule structure ......... ................... ................. .........12 2.2Definitions and nomenclature......... ....... ............. ............... ..... ............. .........13 References........................................................................................................16 3Techniques used in the analysis of optically active compounds................17 3.1Chiroptical methods .......................................................................................17 3.1.1Theoretical background of optical activity ...................................................17 3.1.2Polarimetry......................................................................................................21 3.1.3Optical rotation dispersion (ORO) ................................................................26 3.1.4Circular dichroism (CD).................................................................................27 3.1.5Magnetic circular dichroism (MCD) and magnetic optical rotatory dispersion (MORO) ........................................................................................31 3.1.6Vibrational optical activity (VOA) ................................................................32 3.1.7Detectors used in liquid chromatography....................................................33 3.1.8Enantiomeric differentiation..........................................................................37 3.1.9Analytical applications...................................................................................38 References..... .............................. ................ .................. ...... ....... .................. ....39 3.2Nuclear magnetic resonance ..........................................................................42 3.2.1Chiral derivatizing agents (CDA) .................................................................42 3.2.1.1IH and 19p NMR analysis ..............................................................................42 VIIIContents 3.2.1.2Nuclear magnetic resonance with other nuclei............................................47 3.2.2Chirallanthanide shift reagents (ClSR) .......................................................50 3.2.3Chiral solvating agents (CSA) .......................................................................53 3.2.4Practical examples ..........................................................................................55 References ........................................................................................................59 3.3General aspects of chromatography..............................................................62 3.3.1Definitions and formulas used in chromatography....................................62 3.3.2Sources of error in the determination of enantiomeric compositions (ee values) by chromatographic methods.....................................................65 References ........................................................................................................66 3.4liquid chromatography .................................................................................68 3.4.1Covalent derivatization with crural reagents to form diastereomers ........68 3.4.1.1Practical considerations of diastereomer formation....................................69 3.4.1.2The background of the chromatographic separation of diastereomers.....71 3.4.1.3The detection properties of diastereomers ...................................................74 3.4.2Addition of chiral reagents to the mobile phase..........................................75 3.4.2.1Chiral additives at metal complexation........................................................75 3.4.2.2Uncharged chiral mobile phase additives....................................................77 3.4.2.3Charged additives used in ion-pairing techniques .....................................79 3.4.3Chiral stationary phases (CSP) ......................................................................81 3.4.3.1Chiral phases of the brush type' (type 1) ......................................................93 3.4.3.2Chiralligand exchange chromatography (CLEC, type 2)............................99 3.4.3.3Chiral polymer phases with a helical structure (type 3) ..............................100 3.4.3.4Chiral phases with inclusion effects (type 4) ................................................106 3.4.3.5Protein phases (type 5) ............................ :.......................................................117 3.4.3.6Chlral ion-exchange phase (type 6)................................................................124 3.4.4Practical examples ..........................................................................................125 3.4.4.1Description of the preparation of derivatives of crural components using 'brush type' CSP ..............................................................................................125 3.4.4.2Enantiodifferentiation of crural hydroperoxides and their corresponding alcohols using a Chiracel OD column...........................................................126 References........................................................................................................127 3.5Gas chromatography ......................................................................................132 References........................................................................................................134 IXContents 3.5.1Enantiomeric separation via diastereomeric derivatives ............................135 References........................................................................................................143 3.5.2Separation of enantiomers on chiral stationary phases...............................147 3.5.2.1Amide phases ..................................................................................................151 References........................................................................................................169 3.5.2.2Metal complex phases ....................................................................................175 References..... ......... .... ...... ............. ............ .......... ........ ....... ...... ..... ...... ...... .......180 3.5.2.3Cyclodextrin phases .......................................................................................182 References ........................................................................................................222 3.5.2.4Other chiral phases........................................................................................231 References........................................................................................................232 3.5.2.5Further developments ....................................................................................232 References........................................................................................................233 3.6Supercritical fluid chromatography (SFC) ...................................................234 3.6.1Properties of the mobile phase used in SFC .................................................234 3.6.2Influence of various separation parameters .................................................236 3.6.2.1Temperature ....................................................................................................236 3.6.2.2Dimension of the columns .............................................................................237 3.6.2.3Analysis time ...................................................................................................237 3.6.3Stationary phases used in SFC.......................................................................238 3.6.3.1Packed columns ..............................................................................................238 3.6.3.2Open tubular columns....................................................................................239 References ........................................................................................................241 3.7Electrophoresis...............................................................................................243 3.7.1Introduction.....................................................................................................244 3.7.2Classical electrophoretic methods .................................................................247 3.7.2.1Paper electrophoresis (PE) .............................................................................247 3.7.2.2Isoelectric focusing (lEF) ................................................................................248 3.7.2.3Gel zone electrophoresis (GZE) .....................................................................248 3.7.3Capillary electrophoretic methods ................................................................248 3.7.3.1Capillary gel electrophoresis (CGE)..............................................................248 3.7.3.2Capillary zone electrophoresis (CZE) ...........................................................251 3.7.3.3Micellar electrokinetic capillary chromatography (MECC) ........................263 3.7.3.4Capillary isotachophoresis (CITP) ................................................................269 3.7.3.5Capillaryelectrochromatography (CEC) ......................................................270 xContents 3.7.3.6Conclusions .....................................................................................................272 References ........................................................................................................272 3.8Planar chromatography ..................................................................................279 3.8.1Fundamentals..................................................................................................279 3.8.2Paper chromatography...................................................................................281 3.8.3Thin layer chromatography ...........................................................................283 3.8.3.1Chiral derivatization agents (CDA) ..............................................................284 3.8.3.2Chiral stationary phases (CSP) ......................................................................287 3.8.3.3Chiral mobile phase additives (CMA) ..........................................................301 References........................................................................................................306 3.9Other methods.................................................................................................312 3.9.1Counter-current chromatography .......................................... ;......................312 References ............................................................................... ;........................314 3.9.2'Pseudo-racemates' ..........................................................................................315 References........................................................................................................315 3.9.3Immunological methods ................................................................................316 References ........................................................................................................317 3.9.4Electrodes, membranes and sensors .............................................................317 References........................................................................................................318 Annex Ust of chiral substances analyzed by the treated techniques .....................319 Index ................................................................................................................325 List of Abbreviations a A A ACC ACE AcOH AEC AGE AGP AHNS ala all ANA AP ara arg asn asp ASTM BE BGE BOC BSA CAE CAGE CAZE CCC CCGLC CCI CD CDA CE CEC CES CGE CGS Relative retention Absorbance Symbol for Angstrom unit N-Acetylcysteine Affinity capillary electrophoresis Acetic acid Affinity electrokinetic chromatography Affinity gel chromatography aI-Acid glycoprotein 4-Amino-5-hydroxy-2,7-naphtalene disulphonate Alanine Allose 5-Amino-2-naphtalene sulphonate 2-Aminopyridine Arabinose Arginine Asparagine Aspartic acid American society for testing and materials Butyl ester Background electrolyte tert-Butyloxycarbonyl Bovine serum albumine Capillary affinity electrophoresis Capillary affinity gel electrophoresis Capillary affinity zone electrophoresis Countercurrent chromatography Continuous countercurrent gas liquid chromatography Chiral counter-ion Circular dichroism Cydodextrin Chiral derivatizing agent Capillary electrophoresis Capillaryelectrochromatography Capillaryelectroseparation Capillary gel electrophoresis Centimeter, gram, second-system XII chromat. CIEF CITP CLC CLEC CLSR CM CMA CME CMEC cov. CSA CSP CSR CT CTA cys CZE d Da Dansyl DBT OC OCC ocqq DE def OlOP DIPTA diss DMA DMAP DNBC DNA DNB DNP DNS DOPA EC ECC ee EEOQ EKC ELISA chromatographic; chromatography Capillary isoelectric focusing Capillary isotachophoresis Centrifugal layer chromatography Chiralliquid exchange chromatography Chirallanthanide shift reagent Carboxy methyl Chiral mobile phase additive Carboxymethylethyl Capillary micellar electrokinetik chromatography covalent Chiral solvating agent Chiral stationary phase Chiral shift reagent Charge transfer Cellulose triacetate Cysteine Capillary zone electrophoresis day Dalton 5-Dimethylamino-l-naphthalenesulphonyl Dibutyl tartrate Direct current Dicyclohexyl carbodlimide Droplet counter-current chromatography Displacement electrophoresis derivative 2,s-Isopropylidene-2,3-dihydroxy-l,4-bis(diphenylphosphino)butane Dlisopropyl tartraric diamide dissolved in Dimethylamino 4-(Dimethylamino)pyridine 3,5-Dinitrobenzoyl chloride 3,5-Dinitroaniline 3,5-Dinitrobenzoyl Dinitrophenyl Dansylated, d. Dansyl 3,4-Dihydroxyphenylalanine Electrochromatography Electrokinetik capillary chromatography Enantiomeric excess % ee = (R - 5) / (R + S). 100; for R> S N-Ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline Electrokineticchromatography Enzyme linked immunosorbent assay List of Abbreviations XIIIList of Abbreviations Eu(dcm}J Eu(fodh Eu(hfc}J Eu(pvc}J Eu(tfc}J f FFPLC FFTLC FlD FMOC frc FS FSCE FTIR fuc FZCE FZE gal GC GC-CIMS GC-MS GE GITC GLC gIc gIn glu gly GPC GSC GZE h H HEC HETP HFB his HPCE HPCGE HPCZE Tris-(d,d-dicampholylmethanato)-europium(ill) [Tris-( 6,6 ,7,78,8,8-heptafl uoro-2,2-dimethyl-3,5-octanedionato)europium] Tris-[3-(heptafIuoropropyl-hydroxymethylene)-d-camphorato]europium(ill) Tris-(3-tertbutyl-hydroxymethylene-1R-camphorato)-europium(ill) Tris-[3-(trifluoromethyl-hydroxymethylene)-d-camphorato]europium(ill) Femto (= 10-15) Forced-flow planar liquid chromatography Forced-flow thin-layer chromatography Flame ionization detector 9-Fluorenylmethoxycarbonyl Fructose Fused silica Free solution capillary electrophoresis Fourier transform infrared spectroscopy Fucose Free zone capillary electrophoresis Free zone electrophoresis Galactose Gas chromatography Gas chromatography-chemical ionization mass spectrometry Gas chromatography-mass spectrometry Gel electrophoresis 2,3,4,6-Tetra-O-acetyl-f5-glucopyranosyl isothiocyanate Gas liquid chromatography Glucose Glutamine Glutamic acid Glycine Gel permeation chromatography Gas solid chromatography Gel zone electrophoresis Hour Length of a column equivalent to one theoretical plate: h =Lin (d. HETP) Hydroxyethylcellulose Height equivalent to a theoretical plate Heptafluorobutanoyl Histidine High performance capillary electrophoresis High performance capillary gel electrophoresis High performance capillary zone electrophoresis XIV HPE HPLC HPPE HPPLC HPTLC HPZE HRGC HRP HTAB HVE HVPE Hz ICDNA Ld. IEF He iPA iPC iPE IPG iPU ITP IUPAC LC LCP LE LEE leu LIF LSR lys lyx man MCD MCE MDGC MEC MECC MEEKC ME(K)C MES List of Abbreviations High performance electrophoresis High performance (pressure) liquid chromatography High performance paper electrophoresis High performance planar liquid chromatography High performance thin-layer chromatography High performance zone electrophoresis High resolution (capillary) gas chromatography Horseradish peroxidase Hexadecyltrimethylammonium bromide High voltage electrophoresis High voltage paper electrophoresis Hertz N-Imidazole-N'-carbonic acid-3,5-dinitroanilide Inner diameter lsoelectric focusing Isoleucine lsopropylamide lsopropylcarbamate lsopropylester Immobilized pH gradient lsopropylureido lsotachophoresis International union of pure and applied chemistry Partition ratio Michaelis constant Liquid chromatography Left circularly polarized light Leading electrolyte Ligand exchange chromatography Leucine Laser induced fluorescence Lanthanide shift reagent Lysine Lyxose Mannose Magnetic circular dichroism Microemulsion capillary electrophoresis Multidimensional gas chromatography Micellar electrokinetik chromatography Micellar electrokinetik capillary chromatography Microemulsion electrokinetik chromatography Micellar electrokinetik chromatography Morpholine ethansulphonic acid monohydrate List of Abbreviationsxv metMethionine Methyldopa3,4-0ihydroxy-a-methylphenylalanine MHECMethylhydroxyethylcellulose minMinute MMAMonomethylamino MOROMagnetic optical rotatory dispersion MrMolecular mass M-RPCMicrochamber rotation planar chromatography MSMass spectrometry MTHMethylthiohydantoin MTPAa-Methoxy-a-(trifluoromethyl)phenylacetic acid nTheoretical plate number (n =tR/o)2 NEffective theoretical plate number (N =t'R/s)2 NC-11-Naphthoyl chloride NEA1,1' -Naphtylethylamino NIC-11-Naphthylisothiocyanate NMA-11-Naphthalenemethylamine NMRNuclear magnetic resonance o.d.Outer diameter 00Optical density OPAo-Phthaldialdehyde OPLCOver-pressured layer chromatography OPPCOver-pressured planar chromatography OPTLCOver-pressured thin-layer chromatography OROOptical rotation dispersion ornOrnithine OVOvomucoid POptical purity PAAPolyacrylamide PAGEPolyacrylamide gel electrophoresis PEPaper electrophoresis PEAPhenylethylamine PEGPol yethylenegl ycol PFPPentafluoropropanoyl phePhenylalanine pIIsoelectric point PLCPlanar liquid chromatography PPAAPoly( ethylenephenylalanineamide) PPLPorcine pancreatic lipase Pr(hfchTris-[3-(heptafluoropropyl-hydroxymethylene)-d-camphorato]praseodym(III) Pr(tfc)3Tris-[3-(trifluoromethyl-hydroxymethylene)-d-camphorato]praseodym(III) XVIList of Abbreviations pro PSD PIFE PIH PYA PVC PVPP RS rac. RCP rha RI Ri RIA rib RLCC(C) ROA RPC (J s SA SBE 50s ser SFC SIM sor SubFC 1M tR. t'R TAC tal tBA tBC tBE TBS-HS04 TE TEAA TFA TFAE TFM thr Proline Phase sensitive detector Poly(tetrafluoroethylene) Phenylthiohydantoin Polyvinylalcohol Polyvinylchloride Polyvinylpolypyrrolidone Peak resolution racemic Right circularly polarized light Rhamnose Refraction index Retention index Radioimmunoassay Ribose Rotation locular countercurrent chromatography Raman optical activity Rotation planar chromatography Standard deviation in a Gaussian peak Second Serum albumine 5ulphobutyl ether Sodium dodecyl sulphate Serine 5upercritical fluid chromatography Single ion monitoring Sorbose Subcritical fluid chromatography Gas holdup time Retention time Adjusted retention time TriacetylceUulose Talose tert-Butylamide tert-Butylcarbamate tert-Butylester Tetra-n-butyl ammonium hydrogen sulphate Terminating electrolyte Triethylammonium acetate Trifluoroacetyl 1-(9-Anthryl)-2,2,2-trifluoroethanol Trifluoromethyl Threonine XVIIList of Abbreviations TLC TLE 1MA lMS trp tyr U-RPC UV val VCD VOA xyl Yb(hfc}J Yb(tfc}J ZE Thin-layer chromatography Thin-layer electrophoresis Tetramethylammonium TrimethylsUyl Tryptophan 1;'yrosine Ultra-microchamber rotation planar chromatography Ultraviolet Valine Vibrational circular dichroism Vibrational optical density Peak width at base Peak width at half height Xylose Tris-[3-(heptafluoropropyl-hydroxymethylene)-d-camphorato1ytterbium(ID) Tris-[3-(trifluoromethyl-hydroxymethylene)-d-camphorato]ytterbium(ID) Zone electrophoresis 1Introduction An understandingofthecurrent methodsofanalysisofchiralorganicmolecules requires afundamental knowledge of themost important advances that have been madeinanalyticalstereochemistryandseparationtechniques.Thenextchapter, therefore,willprovideabriefsummaryofstereochemicalconcepts.Thecomprehensive third chapter is devoted to the various techniques used in the analysis of optically active organic compounds. In two main parts the methods not using separations and those employing separations are treated. There are many reasons for the increasing interest in the analysis of chiral organic moleculesinrecentyears.Thephenomenaassociatedwiththeopticalrotation featuresofasymmetricmoleculeshavelongbeenstudiedbymolecularspectroscopiSts. The role of chiral compounds has been decisive in the elucidation of reaction mechanismsandtheirdynamicbehaviourinorganicchemistry.Thereaction mechanisms in organic chemistry would not be understandable without studies of optically active molecules. Theknowledge issuing fromthese investigations, which havemostlybeen basedon classicalpolarimetry, hastremendouslystimulated .organic chemistry. In addition, the increasing interest in the analysis of chiral organic moleculesisalsorelatedtotheoftenhigh biologicalactivitydevotedto diastereoselective and enantioselective reactions. Both in diastereoselective synthesis (d. [1-6]) and in the different areas of enantioselective synthesis, i.e.(i)kineticresolution of racemates[n (ii)biotransformation ofprochiralsubstrates[8),(iii)diastereoselectivereactionswithopticallypure reagents[9),and (iv)enantioselectivereactions(d. examples in Figures1.1-1.3and monographs [10-13]), it is essential to select the most appropriate analyticalmethod forstereocontroLHigh-sophisticatedsynthesesinnaturalproductchemistry,as recentlyperformed,e.g.,forcalicheamidn[14)andtaxol[15,16],implythe knowledge of all the facets of the analysis of chiral organic molecules. RHOH .. R2R3L-(+)-diethyl tartrate RlH, alkyl >90%ee R2 =H, alkyl, phenyl; R3 =H, alkyl Figure 1.1Sharpless epoxidation of allylic alcohols [17]. In addition tosynthetic organic chemicalapproachesto enantioselectivesyntheses, biotransformations have become key technologies. Representativeexamples are the chemistry/biotransformationapproachtobothD- andL-aminoacids,theuseof 2 RblR-BINAP .. RhIR-BINAP= -----...1Introduction OLi nBuLi THF OLi(1) H2C=CHCH2MgCl0 "chiralprolon source"_...1..S""Ph__L_D_A._THF ___.. =(-)-(N}lsopropyl-epbedrine 4 (2)TosOH, toluene 4S-a-damascone Figure 1.2Enantioselective synthesis of a-C>D, are viewed in such a way that D (of lowest priority)points away from the viewer. (iii)Theremainingligandsarethencounted,startingfromtheoneofhighest priority (Le.first A,second B,third C).If this operation is clockwise forthe viewer, the designation will be R (rectus), otherwise it will be 5 (sinister). Thus, the example shown is an R-configuration: 142The development of stereochemical concepts Theselection foraxialchiralityimpliesthattheatomsclosesttotheaxisareconsidered in a priority sequence, e.g., the ortho-carbon atoms in a biaryl compound. With regard to a molecule exhibiting planar chirality, a plane of chirality has first to be selected. The second step involvesthe determination of apilot atom Pwhich shouldbebounddirectlytoanatomoftheplaneandlocatedatthepreferred ("nearer")side.Pisselectedaccordingtothesequencerules.In thenextstepone passesfromPtothein-planeatomtowhich it isdirectlybound(a).Thisatomis then the atom of highest priority of the in-plane sequence. The second atom of this sequence is the in-plane atom (b), bound directly to (a), which is most preferred by thestandardsubrules.Aftercompletionofthesequencethechiralityrulecanbe applied (c). The paracyclophane 9 illustrates the principle. 9 The helicenes can be treated asaxially chlralmolecules, but they arepreferentially regarded as secondary structures. Thus, for hexahelicene 10 the (+form represented below formsalefthandedhelix[M(=minus)helicity],which isdesignatedM-(-). The opposite enantiomer is called P (plus). This M,P-nomenclature is also often employed forchiral biaryls. Firstly, an axis is drawnthroughthesinglebondaroundwhichconformationisdefinedandthe smallest torsion angIe formed between the carbon atoms bearing the groups of highest priority is used to define the helix. Thefollowingdefinitionsareusedthroughoutthisbook:Hstructuralisomers with thesameconstitutiondifferin thespatialarrangementoftheirsubstituents, they are called stereoisomers. To classify stereoisomers according to their symmetry onecandifferentiatebetweenenantiomersanddiastereomers(Figure2.2.1).Each stereoisomer can be regarded as a chlral object (from the Greek word "cheir"= hand), which means that the object is not superimposable on its mirror image. 2.2Definitions and nomenclature15 AJ::::>==< ~* ~ ~HO ANOH*..0~ *0* ClHHH OH0* 1314151617 Formoleculeswithmoreindependentchiralcentres,2n stereoisomersexist.The number of stereoisomers of molecules with dependent asymmetric atoms can be determined only empirically; e.g., theoretically there exist for lineatin (IS)24 = 16 stereoisomers, but in reality only 2; "Riesling acetal" (16)23 =8 stereoisomers, but in reality only 4; B-pinene (17)22 = 4stereoisomers, but in reality only 2. Asto open-chain molecules exhibiting a minimum of two asymmetric centres, but a symmetric constitution, 2(n-1)+2(n-2)/2stereoisomers exist, if nisan even number, and 2(n-1), if n is an odd number. A classic example of a molecule with an even number of chiral centres is tartaric acid (IS). It is easy to recognize that both asymmetric carbonatomscarrythesamesubstituents.AccordingtotheCahn-Ingold-Prelog system,theright-rotatingformshowstheR,Rconfigurationandtheleft-rotating form exhibits the S,Sconfiguration. The second expected enantiomeric pair does not exist, since both forms,R,S and S,R,arecongruent to each other and are, therefore, identicalandachlral.Suchanopticallyinactivestereoisomeriscalledthemeso form; it has a diastereomeric relation to the twoother stereoisomers. References [1]Malus, E.L. Mem.Soc.Arcueil (1809), 2, 143 [2]Herschel, J.PW. Trans Cambridge Phil.Soc.(1821), 1,43 [3]Arago, D.P. Mem.Classe Sd Math.Phys.Int.Imp.France (1811), 12, 115 [4]Biot,J.B.Bull.Soc.Philomath.Paris(1815),190;(1816),125; Mem.Acad.Roy.Sci.Inst.France (1817),2,41 [5]Pasteur, L.;Lectures from20.1.and3.2.1860 attheSoc.Chim.Paris(d. Richardson, G.M.The Foundations of Stereochemistry, Amer. Book Comp.: New York, 1921) [6]Kekule, A. Liebigs Ann. Chem.(1858), 196, 154 [7]Van't Hoff, J.H.Bull.Soc.Chim.France (1875), 23, 295(cf. Richardson, G.M.TheFoundations of Stereochemistry, Amer. Book Comp.: New York, 1921) [8]Le Bel, J.A.Bull.Soc.Chim.France (1874), 22,337 [9]Bijvoet, J.M.Peerdernan, A.F.; Van Bommel,A.J.Nature (1951),168,271 [10]Buding, H.; Deppisch, B.; Musso, H.; Snatzke, G.Angew.Chem.(1985), 97, 503 [11]Cahn, RS.; Ingold, c.K.; Prelog, V.Experientia(1956), 12,81 [12]Cahn, RS.; Ingold, C.K.; Prelog, V. Angew. Chem.Intern.Ed.(1966),5,511 [131Mislow, K. Introduction toStereochemistry, Benjamin: Menlo Park, 1965 3Techniques used in the analysis of optically active compounds 3.1Chiropticalmethods Chiropticalmethods comprisepolarimetry,opticalrotatorydispersion(ORO),and circular dichroism (CD).Detection is based on the interaction between achiral centerintheanalyteandtheincidentpolarizedelectromagneticradiation.Previous applicationsfocusedprimarilyontheelucidationofmolecularstructures,particularly of natural products forwhich atechnique capable of confirming or determining the absolute stereochemistry was critical. In recent yearstheapplication of these techniques has become more and more significant to analytical chemistry. Amongthevariousrequirementsof analyticalmethodologiesthepropertiesof analytical selectivity and breadth of application are of prime importance. Analytical selectivity depends on thestructural properties of the analyteand the ability of the selected detector to differentiate between the analyte and a potentially high number of interfering compounds.The optimum number of molecularproperties necessary to achieve an acceptable level of selectivity appears to be two. If only one property is necessary,separation isessentialunlessamoresophisticatedprocedure,whichis either time- or phase-sensitive, is used. If three or more properties are necessary, the number of potential analytesisgreatly diminished. Themost widelyused chiroptical method is CD, which measures both rotation and absorbance simultaneously. Severalcomprehensivearticleson thephysicalphenomenaofchiralityandthe marufestation of its interaction with polarized light are available[1-5].For chemical analysis,an elementaryunderstandingofthenatureof theinteractionsandtheir relationshipstoeachother,aswellasthedependenceoftheexperimentally measuredparametersontheconcentrationoftheopticallyactivespecies,issufficient[6]. 3.1.1Theoreticalbackground ofoptical activity A molecule will absorb light strongly only if the transition from ground state to excitedstateinvolvesatranslationofcharge.Thisisthebasisoflineardichroism. Thus,thelowest energysingletvalenceelectronictransitioninbicyclohexylidene can be shown to be polarized along the doublebond, with light linearlypolarized alongthedouble bond being preferentially absorbed tolight with aperpendicular linearpolarization(Figure3.1.1).Foraknowntransitionpolarization,lineardichroismmeasurementscan supply information about theorientation of theabsorbing group with respect to the axes of the linearly polarized light.Linear dichroism 183Techniques used in the analysis of optically active compounds isconcernedwiththerelationshipbetweenelectronicmovementsinamolecule (chromophore) and the oscillating electric vector of electromagnetic radiation. In II ~ In not absorbedI[ ' , '" ~ 200300400nm linear dichroism Figure 3.1.1The origin of linear dichroism for the example of bicydohexylidene [7]. Opticalactivityresults fromdifferencesin theability of achromophore in achiral molecule to absorb the two hands of circularly polarized light.In case of AL>AR a positive CD will be obtained (where AL,Rare the absorbances for left and right circularlypolarizedlight).Themoleculehasinteractedpreferentiallywithleftcircularly polarized light (the converse would be true for the enantiomer with AR>AL)' All theories of optical activity are concerned with matching electronic movements in a chromophore (or infrared vibration) with the oscillating electric field of one of the handsofcircularlypolarizedlight.Thiselectronicchiralityoriginatesfrominteractionsbetween thechromophoreanditsassociatedmolecularstructure(absolute configuration, conformation).To exhibit opticalactivity atransition must have collinear electric and magnetic transition dipolemoments.Figure 3.1.2showsthetwo possibleelectricchiralities.Parallelelectric(Il)andmagnetic(m)dipolemoments givearight-handed electronicchirality(positiveCD);antiparallelmomentsgivea left-handed electronic chirality (negative CD). 3.1Chiroptical methods19 ab Figure 3.1.2Electronicchiralitiesofaspectroscopictransition.(a)Collinearparalleland(b) antiparallel electric (11) and magnetic (m) dipole moments [7]. In electronicspectroscopy,themechanismsforgeneratingthesechiralitiescanbe grouped into four classes (Figure 3.1.3). Inherently dissymetrlcCoupled oscillatorSymmetric singleVlbronic effects chromopbore(Exciton Ibeory)cbromopbore In dissymmetric environment Hexahelicene a) Amide units ofa) Cbiral ketones Dlmelbylallene a-helix b) Biaryls )W o b) Amino acids R-.!CH-COOH I NH2 Qass 1Class 2Class 3Qass4 Figure 3.1.3Four classes of chromophores capable of generating electronic chirality [7]. Class 1 includes molecules where the chromophore itself is chiral and the associated electronictransitionsinherentlypossesstransitionelectricandmagneticdipole moments.Class3comprisesmoleculeswithisolatedchromophoreswhosetran203Techniques used in the analysis of optically active compounds sitionshave only onemoment or neither of therequired moments.Forexample,a ketone has an n-1t*transition at approximately 300 nm which is magnetic dipoleallowed(rotationofcharge)withalowordinary extinctioncoefficient;themissing collinear electric dipole moment can begenerated by electrostatic interactions with the polarizability of the various bonds in the surrounding molecular structure. This has been taken as the theoretical basis for different rules [5].In particular, the 'octant rule'hasto be mentioned, which often allows, especially forsteroid ketones, exact prediction of the sign of thecotton effect. Bythethree nodal planes of thenand 1t* orbitals the space around the C=O group is divided up into the octants of Cartesian coordinate system(Figure3.1.4a).Lookingin thedirectionoftheO=Caxisof the carbonyl group, the fourrear, more important octants arerepresented in projection (Figure 3.1.4b).Atoms arranged in anodal plane do not contribute to CD; the signs of the contributions of atoms within the various octants are outlined in Figure 3.1.4b. It may be assumed that the disymmetric disturbance of the basically symmetric C=O chromophor iscaused by Coulomb interactions of the other nuclei of themolecule whichareinsufficientlyshieldedbytheirelectronshells.Theoretically,twosubstituentsin neighboringoctantscontributeoppositesignstothecottoneffect.The octantdistributionofthesignswasdeterminedsemi-empiricallybymeansof various calculations and a large number of collected data. The 'octant rule' cannot be appliedto2,3-unsaturatedketones.Inthiscase,theexperimentallyconfirmed heHcity rule is valid. ab e ~ - - - o - -z e I Figure 3.1.4The'octant rule'of saturatedketones.(a)Alleight octants,(b)thefourrear octants with the sign of the CD effect. Nonetheless, the number and relative importance of chromophore-bond interactions makes the assessment of electronic chirality uncertain. Therefore, an absolute determination of configurationisnotgenerallyattemptedforthisclass.It isnormally 3.1Chiroptical methods21 undertakenrelativelybycomparison oftheunknownwith a'library'ofreference standards. The most important class in electronic optical activity (class 2)includes molecules in whichtherequiredcollinearmomentsarederivedfromtheinteractionoftwo electricdipoleallowedtransitions(,ExcitonCoupling').Forexample,theabsolute configuration of thealcohol 1belongs to class 3iit hasan optical activitythat happenstobeexceedinglysmall.Itsbenzoylatedderivative2containstwochromophores with electric dipole allowedtransitionsaround 239nm, one on the phenanthrene and the other on the benzoate which is a charge transfer transition polarized alongthelongaxis(determinedfromlineardichroismmeasurements).Thetwo transitionmomentscanbe'in-phase'or'out-of-phase',givingrisetothecharacteristic 'Exciton Coupling' with a CD sign pattern deriving from the electronic chiralityofthetwotransitionelectronicconfigurations,whichthereforedeterminesthe absolutestereochemistryofthealcohol1.Thisaspectof opticalactivityhasbeen discussed extensively in Nakanishi's books [8]. 1 Polarimetry and ORD both determine the extent to which a beam of linearly polarizedlightisrotatedontransmissionthroughthemediumcontainingthechiral sample.The two techniques are entirely equivalent fornonabsorbing chiralspecies anddifferonlyinthatORDyieldsaspectralresponse,whereaspolarimetric measurementsusuallyarerestrictedtoalimitednumberofpreselectedwavelengths. 3.1.2Polarimetry Thecomparison of the chiroptical properties of an optically activecompound of given enantiomeric composition with that of the pure enantiomer (of either sign of opticalrotation)representsadirectquantitativemeasureforopticalpurity.Since polarimetric equipment is availableat most research facilitiesand themeasurement of optical rotations goes back almost two centuries, the determination of the optical purityPbypolarimetryisthemostpopularmethodforevaluatingenantiomeric compositions. Optical rotation is the angle by which the polarization plane is rotated asplane-polarizedlightpassesthroughasampleofopticallyactivemolecules. Figure 3.1.5 explains the expression 'plane-polarized light'. 223Techniques used in the analysis of optically active compounds Ordinary visible light is radiant energy, of a certain range of frequencies or wavelengths, which is transmitted asa result of vibrations of an electromagnetic character.Accordingtophysicaltheorythesevibrationsoccurinalldirectionsatright angles to the direction of propagation of the light. By passing a beam of light from a mono- or polychromatic sourcethrough certain optical devices,e.g.,aNicolprism (the so-called polarizer), allthevibrationsexcept thosein oneparticularplaneare absorbed. This emergent beam is then said to be plane-polarized. crossed circle scale in degrees a angle of rotation polarizer Nicol prism sample in polarimeter tube detector analyzer Figure 3.1.5Basic elements of a polarimeter. Thislightwillpassthrough asecondNicolprism(theso-calledanalyzer),if it is held at exactly the same orientation to the polarizer because they both transmit light in the sameplane.H this secondprism isrotatedthrough 90aboutan axisin the direction of the beam of light, it willnow absorbthevibrationstransmitted by the firstprism.Htheanalyzer of apolarimeter containingwater orsome other achiral solventisrotateduntilnolightpassesthrough,thisisthezeropointforthe instrument. H an optically active compound is now placed in the flowcell, a certain rotation of the light will take place. To measure this rotation the analyzer prism can be rotated again until zero is found. This gives the optical rotation a of the solution. For a solution of the optically active sample the well-known expression formulated by Biot is used: [a]TA= specific rotation at a given temperature and wavelength a=opticalrotation in degrees(theobservedangle by which thepolarizationplaneisrotatedasplane-polarizedlightpassesthrougha sample of optically active molecules) T=temperature in degrees Centigrade A;:wavelength (for historical reasonsthe sodiumD-line, 589 nm) I= cell path length in decimeters (;: 10 cm) c=concentration in grams per 100 ml solution at the temperature T. 3.1Chiroptical methods23 The magnitude and sign of [a]are functionsof these variables. Under defined conditions the specific rotation of one enantiomer has the same magnitude, but opposite sign asthat of itsantipode.When thespecificrotationof apureliquidiscited in literature itsdensity dmust also becited. An error isintroduced into [a]when the densitymeasurementisinaccurate.An alternativewaytoreporttherotationofa pureliquidwouldbebytheobservedrotationa.Sincetheobservedrotationis dependent on cell length, the path length must also be given [10]. The specific rotation can be converted from percent composition tothe molecular rotation [fb] by relating it to the molecular mass, M.This expression is used frequently in ORO, but not in polarimetry. Theterms'opticallyactive'and'chiral'areoftenusedsynonymously,althougha chiralmoleculeshowsoptical activityonly when exposedtoplane-polarizedlight and occasionally a chiralmolecule may possess no measurable optical activity.The optical purity P is defined as follows:, P =[a]/[a]max [a]= specific rotation in degrees without CGS dimensions [a]max=specificrotationindegreesofthepureenantiomer(absolute rotation). It has to be stressed that literature data must be carefully evaluated before the degreeof enantiomeric purityisaccepted, sincethepotential forerror in obtainingan optical rotation is significant [11]. The sources of errors in the determination of enantiomericcompositionswithregardtotheopticalpuritywillnowbediscussedin detail. (i)Concentration dependence:Errors may arise from the concentration-dependence ofthespecificrotation.Forexample,thespecificrotationofmalicacidinwater changes its sign with increasing concentration [12].Dependence of thespecific rotationondilutionhasbeenobservedfor2-phenylpropanal(hydratropaldehyde)in benzene [13]. Non-linear rotations may be observed in highly dilute solutions [14]. In someextremecases,achangein sign of therotationmayoccurondilution; these changes become more pronounced at wavelengths close to the optically active absorption bands[11].It has, therefore, been recommended tomeasure at different wavelength when the specific rotation of a new compound is reported [11].For the purposeofdeterminingtheopticalpurityP,itisessentialtousethesameconcentration of the given solvent when comparing solutions on an absolute basis. (ii)Nature of the solvent: The nature of the solvent decisively influences the magnitudeandsignof[a],owingtotheinteractionsbetweentherotatorypowerand molecularactionsbetweenthesoluteandsolvent,formationofsolvates,conformational changes and variations in ionic species. For example, the sign of the optical 243Techniques used in the analysis of optically active compounds rotation of tartaric acidis positive when it is dissolved in water and negative when dissolved in benzene/ethanol. In practice,thesolventwilloften beselectedaccordingtoliteraturedata;such properties as the pH, the solubility of the solute, the magnitude of optical rotation, theabsenceof molecularassociationandchemicalreactionshould be considered. Another parameter is the purity of the solvent: (iii)Purityof thesolvent:Ahighpuritygradeofthesoluteisalsoimportant in polarimetry. Small impurities in a chiral compound exhibiting avery high rotatory powermay strongly influencetheaccuracyof polarimetric measurements.Achiral impurities as wellcan change, sometimes even increase,thevalue of specificrotations through chemical interaction with the solute. For instance, traces of water may alterthespecificrotationofsolutespronetohydrogen-bondingsuchasamines, alcohols and carboxylic acids. Thus, impurities both in the solute and in the solvent mayimpairtheaccuracyofspecificrotations.Carefulpurificationofanisolated sample of unknown enantiomeric purity istherefore of prime importance. Care has to be taken, in particular, not to involve any crystallization steps for solids, since accidental optical fractionation can alter (in most cases increase) the enantiomeric composition ascompared to the original ratio.Distillation or chromatographic methods in an achiral environment are recommended purification steps. (iv)Temperature:Thespecificrotationistemperature-dependent.Theeffectof temperature on [a]arises fromat least threemainsources[11,15]:thedensityand concentration, the equilibrium constants formolecular association and dissociation, andtherelativepopulationof thechiralconformationschangewithtemperature. Thus,foraccuratemeasurementsofcertaincompounds,e.g.,tartaricacidderivatives, precise control of thetemperature is very important [11].The specific rotations[a]and[a]maxmust bemeasuredatthesametemperaturewhen theoptical purity P is determined. (v)Molecularself-association:Theopticalpurity(which describestheratioof the specificrotation of amixtureof enantiomerstothat of thepure enantiomer)islinearly relatedtothe enantiomeric purity(which describestheactualcomposition) only when the enantiomers do not interact with each other. Ithas been shownthat the optical purity markedly deviates from thetrue enantiomeric composition if the enantiomersundergomolecularself-association[16].Theoligomersformedin solution display their own individual rotatory power and, depending on their concentration, contribute to the overall specific rotation. Theprerequisitefordeterminingenantiomericcompositionsviaopticalpurity measurementsisamediumtohighrotatorypower of thesample,permittingthe correct determination of small differencesin enantiomeric excess, 'ee'[defintion,% ee =(R-S)j(R+S)'100; forlOS]. Specific rotations may range fromvery high values (e.g.,helicenes)to very lowvalues(e.g.,moleculesowing their chirality to isotopic substitution).Somechiralhydrocarbonsareevenopticallyinactiveunderconventional conditions, for example 5-ethyl-5-propyl-undecane [17]. The determination of the optical purity of a chiral sample requiresthat the specificrotationof thepure enantiomer,[a]max(absoluterotation),be known.Theab3.1Chiroptical methods25 soluterotation may be established bycalculation ordetermineddirectly.Whereas thesemi-empiricalcalculationofopticalrotationspresentsdifficulties,Horeau [18,19]and Schoofs and Guette [20]have described methods forcalculating absolute rotations based on the principle of kinetic resolution. The maximum specific rotation of an enantiomer can be calculated by themethod of using asymmetric destruction ofthecorrespondingracemicmixtureorbythemethodofusingtworeciprocal kinetic resolutions. Adirectestimationoftheabsoluterotationcanbeachievedbyenzymatic destructionofoneenantiomer.Thekineticresolutionmethodsusingenzymes require that one enantiomer reactsquantitatively in the presence of the other enantiomer which is completely inert. Natural products originating from enzymatic reactions) are usually believed to be enantiomerically pure and may,therefore, serve as referencestandardsforthedeterminationoftheabsoluterotation.Thereisincreasing evidence, however, that natural products (e.g., pheromones [21,22])are not always enantiomerically pure. Theabsoluterotationofasamplemay beascertainedbycrystallizationtoconstantrotation.Inrarecases,however,theconstantrotationofasamplemayalso conform to a composition below 100% ee.The classical resolution by crystallization has been reviewed in detail [23,24]. Since many direct non-chiroptical methods are available fordetermining enantiomeric purities, absolute rotationscan be extrapolated fromthe specific rotation of a sample of known ee value. This procedure requires that the optical purity - based on the rotation - and the enantiomeric purity - based on independent physical methods - be identical.The uncritical use of literature data of absoluterotationsmay lead to errors in predicting the optical yield of enantioselective syntheses. For accurate determination of the optical purity Pthe specificrotation aand the absoluterotation a max haveto be determined underthesame experimentalconditions[25,26],such asthe pH of thesolvent, the purity grade of sample and solvent, instrumentation and cellparameters. Theanalyst should not relyon literaturedata for[a]max but determine the standard on his own and check the enantiomeric purity by anindependent method.It hasbeen recommendedthattheopticalrotation be preferably measured at several wavelengths and in at least two solvents [11]. Even when these precautions are observed, the determination of the optic;al purityPwillbeaffectedbysystematicerror.Errorsinmeasurementsoftheoptical rotation resulting from temperature and concentration effects have been reported to beatleast+/- 4%.Themainsystematicerrorarisesfrominstrumentreading,in particular, when low specific rotations are recorded, which may be due to low rotatorypowerorlowenantiomericpurityofasample.Thus,polarimetricmeasurementsarenot recommendedforcompoundsoflowopticalrotatorypoweror for near-racemicmixtures.But opticalyields of greaterthan 97%may bequestionable as well, unless experimental conditions are clearly stated. Polarimetryrepresentsaconvenientandpopularroutinemethodforobtaining opticalpuritydata, but itsuseforthecorrectdetermination of enantiomericcomposition is limited by the conditions summarized in the following [28]: 263Techniques used in the analysis of optically active compounds (i)Theaccurateknowledgeofthemaximumopticalrotation[a]maxofthepure enantiomer (absolute optical rotation) is essential. (ii)Relatively large sample sizes are required. (iii) The substance must exhibit medium to high optical rotatory power, permitting the correct determination of small differences in ee. (iv)Isolation and purification of the chiral substance have to be performed without accidental enantiomer enrichment. (v)The accuracy of optical rotation depends on temperature, solvents and traces of optically active (or inactive) impurities. (vi)'Optical purity' may not, a priori, conform to enantiomeric composition. 3.1.3Optical rotation dispersion (ORD) The degree of rotation depends on the rotatory strength of the chiral center, the concentrationofthechirophore,andthepathlength.Previously,unusualtermsand concentration unitshave been formulated[2-4]when dealing with solution media, such as. [ell]=10-2 M [a]and [a] = 100 a f(c d) where a,[a]and[ell]are therotation,specificrotation, and molecularrotation,respectively; M is the molecular mass; disthe sample path length; and c isthe solute concentration, expressed either as a percent or as gfdL (!UPACrecommends retaining this concentration unit because of the high number of references employing it in theliterature).Combiningtheseequationsyieldsan equationthatisanalogousto the Beer-Lambert law, namely a= [ell]d c. Experimental values foraare usually on the order of millidegrees (mO)unless laser sourcesareused,in whichcasemicrodegreescan bemeasured.In theabsenceof absorption,theplain ORDspectrumchangesmonotonicallywiththewavelength. This change can be eitherpositive or negative(seeFigure 3.1.6a).Forchiralmedia that absorb the polarized light beam, anomalous rotations in the ORD spectrum are produced if thechiral center and the chromophore are structurally adjacent to each other in an arrangement called a chirophore.Thisanomalous behaviour isreferred to as the Cotton effect [29]and is limited to the wavelength range of the absorption band,whereit isseen superimposedonthemonotonicallychangingplaincurve. Theanomalytakestheformof aSigmoidalcurvewith peakand through extrema whosewavelength values are bisected at an intermediatecrossoverpoint at which the rotation iszero (Figure 3.1.6b). In the simplest case, where a single Cotton effect exists, the height between the extremacan be used forquantitativemeasurements. 3.1Chiroptical methods27 ORDhasnotbeenextensivelyusedasan effectivemethodinanalyticalorganic chemistry becauseof alackof specificityin differentiation and becauseof theuncertainty in defining the baseline, which is theundeveloped part of theplain curve under the Cotton band. + Na-O ~ e (589 run) , ! Figure 3.1.6Typical OROcurves.(a)Plain ORO curve, (b) OROcurve with asinglecotton effect (for ellipticity, d. Section 3.1.4). 3.1.4Circular dichroism (CD) Circular dichroism (CD) is the most sophisticated of the three chiroptical methods in that therotation and absorbance measurementsaremade simultaneously.Linearly polarizedlightconsistsoftwo beamsof circularlypolarizedlightpropagatingin phasebutinoppositerotationalsenses.Incruralmediathebeamsarephase differentiated because they'see'two different refractiveindexes and, consequently, travel at different speeds, a phenomenon that produces the rotation effect. ab c Figure 3.1.7Phaserelationsassociatedwiththepassageofcircularlypolarizedlightthrough different media [30]; a-c, d. explanations in the text. 283Techniques used in the analysis of optically active compounds CD represents the differential absorption of left circularly polarized (LCP)and right circularly polarized (RCP) light. The effect of the differential absorption is that when the electric vector projections associated with the LCP and RCP light are recombined after leaving the 240 run, where molar absorbances are small compared with the strong bands observed in the far UV; CD bands are still sufficientlylarge.Analyteconcentrationsaretypically10-4molorlessforwavelengths> 240 run. CD signals in the far UV can be very large, but the signal-ta-noise quality is poor whenever strong absorbers are present. Because 6. is much smaller than the average e value, the CD signal is a very small millivoltquantityridingontopofarelativelylargevalue.Despitethelarge difference in signal size, detection limits are 0.1Ilglml foranalytes with eMvalues of approximately 200mO Imol em at the band maxima. This value can be improved by introducing fluorescencedetection [31].The use of fluorescence,however, introduces the need fora third structural requirement in theanalyte molecule, which in effectdecreasestheapplicabilityofCD.Instrumentoperatingconditionsand solution concentration variables are chosen to givetheoptimum ratio of CDto the total absorption, although one has to consider that a mixture may contain several absorbingspecies.Whenfluorescenceisthedetectorofchoice,moreseriousinterference from the emissions of CD-passive fluorophores can be expected. To predict whether an analyte will be optically activeandcan be determined by CD, the presence of chirality and absorbance must beconfirmed. Onemust remember,however,thatalthoughthemolecularstructuremaysuggestthatchiralityis present,thesubstancemayonly beavailableasaracemicmixtureand,therefore, undetectable. AlthoughtheserequirementsmayseemtomakeCDtooselectiveforpractical analyticaluse,theapplicabilityofCDcanbeincreasedbyaddingthemissing molecularproperty byin situderivatization.Onecanmakeanachiralabsorbing analyteCD-activeby reactingit with acruralpartner,preferablyonethatisnonabsorbing, and a chromophore can be introduced in a way that either does or does not affect the overall chirality of the molecule. TheseCDinductionreactionsshouldnot beconsideredexclusivelyaspossible precolumn or postcolumn derivatization reactionsin chromatographicapplications using CDdetection.Theyare,instead,regardedtobeso specificthattheycan be used for the analysis of unseparated mixtures. Most instrumentation suitable formeasuring CDisbased on thedesign of Grosjean andLegrand[32].Ablockdiagramoftheirbasicdesignisshownin Figure 3.1.9.Linearlypolarizedlightispassedthroughadynamicquarterwaveplate, which modulatesit alternativelyintoleftandrightcircularlypolarized(LCPand RCP)light.Thequarterwaveplateisapieceofisotropicmaterialrenderedanisotropic through the external application of stress.Thedevicecan be a Pockelscell (inwhichstressiscreatedinacrystalofammoniumdideuteriumphosphate throughtheapplicationofalternatingcurrentathighvoltage),oraphotoelastic modulator(inwhichthestressisinducedbythepiezoelectriceffect).Thelight leavingthecellisdetectedbyaphotomultipliertubewhosecurrentoutputis converted to voltage and then split. One signal consists of an alternating signalpraportional to the CD;it isdue tothedifferentialabsorption of onecircularlypolari3.1Chlroptical methCKis31 zed component over the other. This signal isamplified by means of phase-sensitive detection.The other signal isaveraged and isrelated to themean light absorption. The ratio of thesesignalsvaries linearlyasa function of the CD amplitude,and is the recorded signal of interest. The small signal intensity requiresthat theincident power be verylargeia500WXelampisusuallyemployed.Thissourcemust be water-cooled and oxygen must be removed fromthe instrument toreduce the production of ozone, which isdetrimental to theoptics.The volume of atypical1..an path length cellisabout 3.5 Wi smaller path length cells,which may require focusing of the incident beam, are available for analyses requiring smaller volumes. 0SourceMono- Circular chromatorpolarizer SampleDetector system MCKiulator power supply DisplayAnalog deviceratio device Figure 3.1.9Block diagram of a CD spectrometer. 3.1.5Magneticcirculardichroism(MeD)andmagneticopticalrotatorydispersion (MORD). Magnetic circular dichroism(MCD)is induced in allmatter by auniform magnetic fieldapplied paralleltothedirection of propagation of themeasuring light beam. AlthoughphenomenologicallysimilartonaturalCD,themolecularoriginofthe effect (called'Faraday effect') isdifferent.Both MCDand CDcan bepresent in an optically active molecule in a magnetic field. The two effects are additive. Within the last years an increasing number of experimental MCDdata has been collected and thetheoreticalanalysisiswellfounded.MCDspectroscopyhasfoundinterest for applications in chemistry, physics and biochemistry [34]. The origin of the 'Faraday effect'depends on two facts:(i)Degenerate electronic states aresplit in thepresence of a magnetic field(the first-orderZeeman effect)to yield a set of sub-levels called Zeeman components. All states are mixed together by 323Techniques used in the analysis of opticaUy active compounds anappliedmagneticfield(thesecond-orderZeemaneffect).(ii)ElectronictransitionsfromtheZeeman sub-levels of theground statetothoseof an excitedstate arecircularypolarizedifthemagneticfieldisparallel(oranti-parallel)tothe direction of the light beam. The origin of MCD can be illustrated by the followingexample. Let usconsider an electronic transition froma25ground state, with spin 5=1/2 andzero orbital moment, to a 2P1/2 excited state as shown in Figure 3.1.10. When a magnetic field is appliedthedegeneraciesareliftedbytheZeemaneffect.Theselectionrulesfor electric-dipoleallowedtransitionsbetweentheZeemansub-levelsoftheground and excited statesareL1mL=+1fortheabsorption of LCPlightand L1mL=-1for RCP:L1ms=O.Thus theleft- and right circularly polarized photons impart angular moments of opposite sign tothe system.At temperatures of 300K thetwocomponents of the 25 ground state are almost equally populated. When the temperature is lowered, the population is frozen into the lower component of the ground state and the LCPtransition gains in intensity at the expense of theRCPintensity.Therefore,theMCDintensityofaparamagnetistemperature(and field-) dependent, increasing in intensity as the temperature is lowered . ....-.---- mJ=+ 1/2 Electronic ~ m L = +1~ m L-1absorption LCPRCPtransition ...--+--,---'-- Ins =+ 1/2 EPR transition ' ~ - - ' - - ' - - - Ins =-1/2 B=O BolO Figure 3.1.10The origin of the MCDin the atomic transition 2Sto 2p.The figureshows the allowed opticaltransitionsbetweentheZeemansub-levelsofthegroundandexcitedstates [33]. 3.1.6Vibrational optiCilI activity(VOA) [33J Vibrationalopticalactivity(VOA)comprisesbothvibrationalcirculardichroism (VCD)andRamanopticalactivity(ROA).VCDmeasuresthedifferencein absor3.1Chiroptica\ methods33 bance of LCP and RCP infrared light in the region of vibrational absorption bands of opticallyactivemolecules.ROAmeasuresthedifferenceinscatteredintensityof LCPandRCPincidentlaserradiation.Vibrationalopticalactivityisbecominga powerful tool for determining the stereochemistry of chilal molecules - both the conformation and the absolute configuration. Unlike electronic CO spectroscopy, which provides information only about chromophores and their immediate environments, in VCOeverypart of amoleculecancontributetothespectrum.Theoretically,it should be possible to determine both absolute configuration and conformation from theVCOorROAalone.TheprimaryexperimentaldifficultyisthatYOAisvery weak, with signals being fouror fiveorders of magnitudesmallerthan theparent effects,i.e.infraredabsorptionandRamanscattering.However,withinthelast years great progress has been made in both areas [35-39]. The highest and lowest frequenciesat which VCOhasbeen reportedusingdispersiveinstrumentsareapprox.6000em-Iand900em-I,respectively.Overthis range the sensitivity limit in terms of absorbanceA= Cd(wherec isthe concentration and dthe path-length) can be IJ.A= 10-5 - 10-6at a bandwith of 5 - 10em-I, sufficienttoresolvemostroomtemperatureliquid-phaseVCOspectra.Thelowfrequencylimit ofVCOmeasurements,usingFouriertransformation instrumentation,isnowapproximately600cm-l,StephensandLowe[38]havedescribeda general theory of VCO,the so-callednon-adiabatictheory, permitting prediction of vibrational rotational strengths and spectra. There are also a variety of heuristic models, including coupled oscillators and fixed partial charges. ForROAnosuch frequencylimitsexist,although thelargest effectscommonly occuratfrequenciesbelow1000cm-l.Furthermore,thedifficultyofobtaining measurable signals in ROAhaslimitedexperimentstoveryconcentratedsamples, eitherpureliquidsorsaturatedsolutions,whereintermoleculareffectsmaybe dominant. VCO, on the other hand, has been measured on sample concentrations of 0.1- 0.01mol. Since the demonstration that YCOspectra can be measured with high reliability, theoretical analysis has advanced rapidly. The field is entering a phase of collecting spectra and making comparisons with theoretical models. 3.1.7Detectors used in liquid chromntography The chiroptical detectorsused in liquid chromatography (LC)areprimarily singlewavelength detectors.Because of theconstraints of both signalsizeand small elution volumes,lasersarethemostsuitablelightsourcesforthesedetectors.Yeung andco-workershavedescribedboth polarimetric andCOdetectorsforLC[31,40], Stopped-flow CO spectral detection for LC has been described both by development engineers from TASCO, Inc.[41]and by Westwood et a1.[42]. Chiropticaldetectorsareparticularlyusefulinstudyingsubstancesofnatural origin, and their usecan complement the more common chromatographic detectors ininvestigatingcomplexmixtures.Themajorityoftheapplicationsdevelopedto 343Techniques used in the analysis of optically active compounds date have involved laboratory preparations; the number of real samples investigated is very small. H total separation of amixture ispossible, polarimetry is the chiroptical detector of choice. It has been used, for example, to identify and quantitate structurally relatedcarbohydratesinamixture[43].Polarimetricinstrumentationisinexpensive both to purchaseand to operate,and the detectorresponds equally welltoabsorbingandnonabsorbinganalytes.Furthermore,theeffectivenessofapolarimetric detector has been demonstrated in both the direct mode, where the rotation caused by the analyte ismeasured[40],and in the indirect mode, where the change in the measuredbackgroundrotationforanopticallyactivemobilephaseisusedto quantitate the analyte [44]. A detector for high performance liquid chromatography (HPLC) based on optical activitywouldseemtopossessseveraladvantagesin manyattractiveareasof organicanalysis.Sincemost chromatographic eluentsarenot opticallyactive,oneis not limited in the choice of eluents or gradients. Such adetector is extremely selective, so that complex samples can be analyzed. The availability of a sensitive micropolarimeterwill,therefore,benefitorganicanalysiswhencoupledtoHPLC,and will broaden the applicability of spectropolarimetry in general. For over acentury, mechanical polarimetershave been constructed with sensitivities on the order of 0.01.An example of this class of polarimeter is the model 241 LCfromPerkinElmer.Monochromaticlightispassedthroughthepolarizer,the flow cell (40 or 80 J.Ll)with the sample and through the analyzer to a photomultiplier as detector (d. Figure 3.1.5).The polarizer, which also means the polarization plane of the light, is modulated at 50 Hz at an inclination of 0.7around the optical axis of the system. In theunbalanced state of the system a 50Hz signal is produced in the photomultiplier which is intensified and transmitted with the correct sign to a servomotor. This motor turns the mechanically connected analyzer until the 50 Hz signal is reduced to zero. Thus, the system becomes balanced (optical zero balance) and the polarizationplanesofpolarizerandanalyzerformanangleofexactly90.An optically active sample placed into the light beam rotates the polarization plane; the analyzer isagain balanced by the servomotor (at aspeed of approximately1.3 Is). Therotationoftheanalyzeristransformedintoelectricimpulsesbyanoptical encoderandtheimpulsesareevaluated.Whenmechanicalpolarimetersareused thepossibilitiesof chromatographiclossofresolutionpowercausedbythefinite balancevelocityhasto betaken into consideration.In addition,theadjustment of the flow cell is very time-consuming. Currently availablecommercial polarimeters usethetechniqueof Faraday compensation, resulting in sensitivities on the order of 0.001.Examples of thisclassof instrumentaretheChiraMonitor(ACS- AppliedChromatographySystems,UK) andthe Chiralyser(IBZ,HannoverandKnauer,Berlin;both Germany).Howthey functionisshown below,usingtheChiraMonitorasexample(Figure3.1.11).The instrument consists of a solid state near-infrared laser (820nm) chosen so that there isverylittleinterferencefromtheabsorbingcharacteristicsofcompounds.The radiation fromthe laser passes, after being focusedto lessthan 1mm in diameter, 3.1Chiroptical methods35 through apolarizing prism to acalibrator/modulator with theFaraday rod(made fromaspecialglassmaterial)whichgivesrisetoa1kHz(f1)polarisation modulation of the laser beam varying about 10 around the polarizer/analyzer cross point (Figure 3.1.12; angle a). Modulator Polarizer(with FaradayCellCalibratorAnalyzerDetector Power amplifier Figure 3.1.11Schematic diagram of the polarimetric LC detector ChiraMonitor (ACS, UK). This 1 kHz signal is also the referencesignal forthephase sensitive detector (PSD). The calibrator is controlled by a DC power supply and it feeds a signal to the detectorto checkthecalibration of the detector.Afterpassing through the flowcellthe lightenterstheanalyzer.Withpolarizerandanalyzercrossedatexactly90the exciting light is apure 2 kHz (2(1)amplitude-modulated carrier signal of constant polarization. Any optical rotation due to the sample (Figure 3.1.12; angle 0)deflects the system away fromthecross point and thereis aresultant 1 kHz amplitudemodulation of the carrier. This signal is then recovered using a phase-sensitive detector. The phase anomalygeneratesacompensationcurrentintheFaradaycoilviathepower amplifierwhichcreatesanelectricalcompensationfieldandcompensatesthe influence of the optically active substance until the phase anomaly disappears. The flow cell is the most critical component for optimization of the signal-to-noise ratio.The dimensions of the cellalways represent acompromise between having a longlightpathandasmallvolumeandmustallowalaminarflowdistribution. Wheneverthereisasudden, largechange in therefractiveindex,such aswhena high concentration of material passesthrough,thelaser beam isdistorted and will not pass through theanalyzer and the apertures properly.A similar problem exists when absorption processes cause thermal lensing. This can, in principle, be avoided by choosingan appropriate laserwavelength.Bubblestrapped inside thecellmay also be a problem, particularly when the cell is used initially. Phase sensitivedEr Recorder 363Techniques used in the analysis of optically active compounds plane of analyzer plane of polarizer vector diagram Figure 3.1.12Function principle of the polarimetric LC detector ChiraMonitor (ACS, UK). Scatteringandreflectionsofthecellwallscanalsoincreasethenoiselevelsubstantially.A flowdirection againstthelight sourcecan act asa' hydrostatic lensto improve the signal/noise ratio of the instrument. Using the Drude relationship, it is possible to compare as followsthe rotations measured at 820nm (wavelength of the laser in the ChiraMonitor) with tabled data at 589 nm. aD = rotation at Na-D-line (589 nm) aM = rotation at 820 nm A.M= wavelength of the laser used (820 nm) AD=wavelength of the Na-D-line (589 nm) AA= absorption wavelength of the chromophore. This relationship only holds for molecules with one optically active chromophore, or wherethemajorcontributiontoasubstance'sopticalactivityisfromonechromophore. More complex molecules can not be so easily described. Example:A typical saturated lactone shows a AAaround 200 nm. Using the Drude equation the quotient aD/aM =2.06 results. This means the rotation measured at 820 nm is only half the rotation at 589 nm' The differences between theChiraMonitorandtheChiralyseraremainlyin the construction of the flowcelland the light source. Thecellof the ChiraMonitorconsists of a stainless steel block through which a1 romdiameter sample cell(opening out to 1.5 rom) is machined. The volume of the cell is approximately 20~ .The flow cellof theChiralyserconsistsof aglass-coatedstainlesssteeltubewith an optical length of 200 rom and a volume of approximately 40 Ill. The reflecting surface of the glass coating of theflowcellallowsoptimal light transmission and alaminarflow distribution within the celL 3.1ChiropticaJ methods37 In contrast to the ChiraMonitor, which uses alaser diode, the light source of the Chiralyser consists of a halogen lamp allowing only polychromatic determination of theopticalrotations.Theadvantageofthistechniqueistheincreasedsensitivity compared with the older 'Chiraldetector' distributed by the same companies. Limiting CD detection to a single-wavelength measurement reduces it to no more than a very expensivepolarimeter with a much smaller range of application, since thenonabsorbingchiralcompoundsarenowtransparenttothedetector.Butif separation isnot complete, thenthedifferentiation capabilityofthefull-rangeCD detector becomes necessary. As the need arises, convenient instrumentation for fastscan measurements may become available.Earlyattemptstodevelopsuchdevices have been described in the literature [45,46]. 3.1.8Enantiomeri.c differentiaton Enantiomeric differentiation is a two.-Ievelproblem. If the identity of the substance isknown and only one isomer ispresent, then thesign of the rotation easily establishes its stereochemical identity, in which case polarimetric detection is sufficient. If both enantiomersarepresent,which isnormallythecase,theanalysistakeson a different dimension; one has to detet:mine the ee value or optical purity. Polarimetry is the best choice to determine the enantiomeric enrichment at the exploratory level, where eluted volumes are small. When chromatographic procedures aredevelopedtothepointwherelarge-scaleseparationsarepossible,CDisthe betterdetector,becausedifferencesin thefullspectrumoftheanalytecompared with that of the standard signify the presence of a coeluted chiral interference. Because nonderivatized racemic mixtures coelute from conventional LC columns, neitheraconventionaldetectornorachiropticaldetectoraloneisadequateto determinetheee.If thedetectorsareplacedinseries,however,aquantitative distinction can be made.Data from either an absorbance or RI detector provides the sum oftheconcentrationsofthetwoisomersandthesignalfromthechiroptical detector(which is eithertherotationaldifference,[Cl(+)- Cl(-),forthepolarimetric detector, or the difference in ellipticity, ['lI(+)- 'lI(-)], for CD) provides informationto calculatetheconcentrationdifference[47,48].Theconcentrationof eachisomeris then readily obtained from the simultaneous solution of these equations. In manysituationswhere CDisthedetector of choice, itsselectivityissogreat that it can be used as a stand-alone detector, providing the concentration difference information without separation.Thisisespeciallyimportantwhenevertheeluted volumes are small, because of the small CD signal. An aliquot is injected onto a conventional column and the total concentration of both enantiomers is measured using absorption detection. The concentration difference is calculated simultaneously from theCDspectrumofanotheraliquotoftheunseparatedmixture.Theeeisthen calculated as described earlier. Whichever method is used to determine the ee, the quality of the results depends decisivelyontheopticalpurityofthestandardmaterials.Thesecanneverbe 383Techniques used in the analysis of optically active compounds consideredto beopticallypure,sincetheinstrumentalorseparationmethodsare limited by their resolution capabilities. Theoretically, forspectroscopic analysis it is necessary to have only one of the isomers forinstrument calibration, provided that diastereoisomerization is not aprerequisite forthe determination, asit isin NMR Tohaveboth isomersof equivalent opticalpurity asan internalcheckof thecalibration in chiroptical methods isan unrealisticexpectation.Reportsof enantiomer ratio determinations should emphasize the fact that the ratio is relative to the purity of the best available standard reference materiaL Thedetection limitsobtainedwithchiropticaldetectorsareequivalenttothose obtainedwith absorbancedetectorsforbulkmeasurements.In conventionalchromatographic systems, nanogram detection limits are usual; in state-of-the-art detectordevelopment, picogram or even femtogramlimitshave been reported[48].The abilitytodetectsuchsmallquantitiesisofcriticalimportanceonlywhenthe physicalsizeofthesampleislimited,asisthecaseinmicroboreLC,wherethe elutedvolumesareverysmall.If thesamplesizeisnotlimited,thesimplealternative is to scale up the experiment. Chiroptical methods are not yet competitive within the lowest of these achievable detectionranges,showingatypicalcutoffinthenanogram-permilliliterrange, unlessfluorescenceisused forsignalenhancement orlasersourcesareused[40]. When sample sizes are not a problem and a typical working sample volume is a few milliliters,detectionlimitsontheorderofmicrogramspernlilliliterarereadily achieved using CO. 3.1.9Analytical applications Polarimetry andOROhavenopotentialasselectivedetectors;theyarefunctional only when all interference has been removed. CD is in the same category as long as its use is limited to single-wavelength chromatographic detection. In many cases the mostusefulwavelengthrangeisfrom240to400nm,whichcomprisesthe transitionsfromthearomaticringandunsaturedketonechromophores.Fewsubstances are CD-active in the visible range;at wavelengths < 240nm signal-to-noise ratios are significantly decreased because of the extremely intense absorption bands. In addition, CD bands are observed to be broad and featureless and usually of only onesign,resultinginspectrathatarethesameasthecorrespondingabsorption spectra. Theneed fortwo detectorsin thedetermination of eeor optical purity wasdiscussed above. Some early examples combined UV or RIwith polarimetric detection; for instance, cocaine and codeine [49],epinephrine [50],and 0- and L-penicillamine [51]were investigated in this manner. UV and CD were successfully used in series for prepared mixtures of R- and S-nicotinein whichsolutionsofthenaturalisomerwerespikedwithaliquotsofthe other [1]. Subsequently, leaf extracts were spiked withthe unnatural isomer and the eewasdeterminedusingconventionalLC.Thetotalnicotineconcentrationwas 3.1Chiroptical methods39 measuredby LCusingan absorbancedetector,and theCDspectrum of eitheran aliquot of the unseparated mixture or eluate from a conventional LCcolumn yielded the data from which the concentration difference was calculated. Theconcentration of each was obtained by the simple solution of the simultaneous equations. Theassayofglycosidesisarelativelyunexploredareawithseveralattractive possibilitiesfortheapplicationofchiropticaldetectors.Structurally,thesecompoundsfulfilltherequirementsforCDactivitybyhavingthechiralcenterin the sugarmoietyandanaromaticchromophoreindose juxtaposition;theconnection between the two parts is through either carbon (cyanogenics), nitrogen (nudeosides and nudeotides), oxygen (saponins and flavonoids), or sulphur (glucosinolates). The magnitude of the CDsignalwilldepend on howadjacentthenearest chiralcenter on the sugar is to the chromophore. Nakanishiandcoworkershavedeveloped- basedon CDexcitonchirality- a microscalemethod forcharacterizingthestructuresofmonosaccharidesandtheir linkagesinoligosaccharides.SugarcomponentswereidentifiedbyUVandCD spectroscopyof chromophoric degradationproducts.CDspectraldata ofapproximately 150 different reference glycopyranosides have been published [52]. Theexciton chirality method isalsouseful forthestereochemicalanalysisofthe aglyconepartinglycosides.Recently,thedeterminationoftheabsolutestereochemistryofnatural3,4-dihydroxy-15-iononeglycosideshasestablishedtheconfiguration as being 315,4lS(3S,4R)[53]. Furthermore,alotofORDandCDspectroscopyhasbeendoneinthefieldof carotenoid chemistry. hl a fundamental paper by Klyne'sand Weedon's groups [54] ORO spectra of carotenoids have been studied systematically. Extensive CD studies ofcarotenoidshavebeenperformedbyNoak,Liaaen-Jensenandothers[55-57]. Theoreticalpredictions and experimentaldatawereshown by Sturzenberger et a1. [58]to conform to the 'C2-rule' [59]. References [1]Purdie, N.; Swallows, K.A. Anal.Chem.(1989),61,77A [2]Crabbe, P. ORD and CD in Chemistry andBiDchemistry: An Introduction;Academic Press: New York,1972 [3]Charney, E. The Molecular Basis ofOptical Activity; Wiley: New York, 1979 [4]Mason, S. F. Q.Rev.Chem.Soc.(1%1), 15,287 [5]Snatzke, G. Chemie in unserer Zeit (1981), 3, 78; (1982),5,160 [6]Purdie, N. Prog.Anal.At.Spectrosc.(1987),10,345 [7]Drake, A. F.Eur.Spectrosc.News (1986), 69, 10 [8]Harada,N.;Nakanishi,K.CircularDichroismSpectroscopy-ExcitonCouplinginOrganic Chemistry;University Science Books:MillValley,1983;Nakanishi,K.;Berova,N.;Woody, R.W.Circular Dichroism-Principles and Applications, VCH Verlagsgesellschaft: New York, 1994 [9]Schurig, V.Kontakte (Darmstadt) (1985), 1,54 [10]Eliel, E. 1. Stereochemistry of CarbonCompounds; McGraw-Hili: New York; 1962 403Techniques used in the analysis of optically active compounds [11]Lyle, G.G.;Lyle, RE.Asymmetric Synthesis(J.D.Morrison, ed.) Vol.1,13Academic Press: New York, 1983 [12]Beilsteins Handbuch tier Organischen Chemie, 4. Auflage, Band 3; Springer: Berlin, 1921 [13]Consiglio, G.;Pino, P.;Flowers, L.I.; Pittman jr.; C.U. J.Chem.Soc.,Chem.Commun.(1983), 612 [14]Heller,W.;Curme,H.G.PhysicalMethodsof Chemistry(Weissgerber,A.;Rossiter,B.W., eds.), Wiley: New York, part III C, 51 [15]Raban, M.; Mislow, K.Top.Stereochem.(1967),1,1 [16]Horeau, A. TetrahedronLett.(1969),3121 [17]Hoeve, W. T.; Wynberg, H. J.Org.Chem.(1980), 45,2754 [18]Horeau, A. J.Am. Chem.Soc.(1964),86,3171 [19]Horeau, A.Bull.Soc.Chim.Fr.(1964),2673 [20]Schoofs, A. R, Guette, J.-P. Asymmetric Synthesis(J.D.Morrison, ed.); Academic Press: New York, 1983, Vol. I, 29 [21]Weber, R; Schurig, V.Naturwissenschaften, (1984) [22]Mori, K.Technique of PheromoneResearch(Hummel, H.E.;Miller, T.A.,eds), Springer:New York, 1984,323 [23]Jaques, J.; Collet, A.; Wilen, S. H. Enantiomers,Racemates and Resolutions, Wiley: New York, 1981 [24]Leitich, J.TetrahedronLett.(1978),3589 [25]Hom, D.H. S.; Pretorius, Y.Y. J.Chem.Soc.(1954), 1460 [26]Klyne, W.; Buckingham, J.Atlas of Stereochemistry, Vol.I, Chapman Hall: London, 1974 [27]Plattner, P.A.; Heusser, H. Helv.Chim.Acta (1944), 27, 748 [28]Schurig, V.Asymmetric Synthesis(J.D.Morrison, ed.), Vol 1, 59,Academic Press: New York, 1983 [29]Cotton, A.Compt.Rend.(1895),120,989 [30]Brittain, H. G. Spectrosc.Int.(1991),3,12 [31]Synovec, RE.; Yeung, E. S. J.Chromatogr.(1986),368,85 [32]Velluz, L.;Legrand, M.; Grosjean, M.OpticalCircularDichroism:Principles,Measurement,and Application; Verlag Chemie: Weinheim, 1965 [33]Thomson,A.J.PerspectivesinModernChemicalSpectroscopy(Andrews,D.L.,ed.),255, Springer: Berlin, New York, 1990 [34]Piepho, S.; Schatz, P.N. Group Theory in Spectroscopy, Wiley: New York, 1983 [35]Osborne, G. 0.; Cheng, J. c.; Stephens, P. J.Rev.Sci.Inst.(1973),44,10 [36]Nafie, L. A.; Keiderling, T.A.; Stephens, P. J. J.Am.Chem.Soc.(1976),98,2715 [37]Annamalai, A.; Keiderling, T.A. J.Am. Chem.Soc.(1984), 106, 6254 [38]Stephens, P. J.;Lowe, M.A. Ann.Rev.Phys.Chem.(1985),36,213 [39]Nafie, L. A.; Diem, M.Acc.Chem.Res.(1979), 12,296 [40]Synovec, RE.; Yeung, E. S. Anal.Chem.(1986),58,1237A [41]Takakuwa, T.;Kurosu, Y.;Sakayanagi, N.;Kaneuchi,F.;Takeuchi, N.;Wada, A.;Senda, M. J.Liquid Chromatogr.(1987), 10,2759 [42]Westwood, S.A.; Games, D. E.; Sheen, L. J.Chromatogr.(1981),204,103 [43]DiCesare, J.L.; Ettre, L.S.Chromatogr.Rev.(1982),220,1 [44]Yeung, E.S. J.Pharm.Biomed.Ana/.(1984),2,255 3.1Chiroptical methoos41 [45]Anson, M.; Bayley, P.M. J.Phys.E (1974), 7, 481 [46]Hatano,M.;Nozawa,T.;Murakami,T.;Yamamoto,T.;Shigehisa,M.;Kimura,S.; Kakakuwa, T.; Sakayanagi, N.; Yano, T.; Watanabe, A. Rev.Sci.Instrum.(1981),52,1311 [47]Boehme, W. Chromatogr. Newsl.(1980),8,38 [48)Meinard, c.; Bruneau, P.; Perronnett, J.}. Chrmnatogr.(1985),349,109 (49)Palma, R. J.;Young, J.M.; Espenscheid, M. W. Anal.Letters (1985),18,641 [50]Scott, B.S.; Dunn, D. L. J.Chromatogr.(1985),319,419 [51]DiCesare, J.L.; Ettre, L. S.}. Chrmnatogr.(1962),251,1 [52]Wiesler, W.T.; Berova, N.; Ojika, M.; Meyers, H.V.; Chang, M.;Zhou, P.; Lo, L.c.; Niwa, M.; Takeda, R.;Nakanishi, K. Helv.Chim.Acta (1990), 27, 748 [53]Humpf, H.U.; Zhao, N.; Berova, N.; Nakanishi, K.; Schreier, P.}. Nat.Prod.(1994),57,1761 [541Bartlett,L.;Klyne,W.;Mose,W.P.;Scopes,P.M.;Galasko,G.;Mallams,A.K.;Weedon, B.C.L.; Szabolcs, J.; Toth, G.J.Chem.Soc.C (1969), 2527 [55]Noak,K.InCarotenoidChemistryandBiochemistry,Britton,G.;Goodwin,T.W.,Eds., Pergamon: Oxford, 1982, p. 135 [56]Liaaen-Jensen, S. In Proc.Intern.Conference on Circular Dichroism, Bonn, 1991, p. 47 [57]Buchecker, R.;Marti, U.;Eugster, C.H. Helv.Chim.Acta (1980), 65,8% [581Sturzenberger, V.; Buchecker, R.; Wagniere, G. Helv.Chim.Acta (1980), 63, 1074 [59]Wagniere, G.;Hug, W.TetrahedronLetters(1970),4765;Hug, W.;Wagniere, G.Helv.Chim. Acta (1971),54,633; Hug, W.; Wagniere, G. Tetrahedron(1972), 28,1241 423Techniques used in the analysis of optically active compounds 3.2Nuclear magnetic resonance Nuclear magnetic resonance(NMR)doesnot allowtodifferentiate between enantiomers, since the resonances of enantiotopic nuclei are isochronous. The determinationofenantiomericcompositionsbyNMRspectroscopy,therefore,requiresthe conversion of the enantiomers into diastereomers by means of a chira! auxiliary. The chemical shift nonequivalence of diastereotopic nuclei in diastereoisomers in which the stereogenic centers are covalently linked in a single molecule was first noted by Cram[2].Underappropriateexperimentalconditionsthechemicalshiftnonequivalence provides adirect measure of diastereomeric composition which can be related directly to the enantiomeric composition of the original mixture. Threetypesofchiralauxiliaryareused.(i)Chirallanthanideshiftreagents (CLSR)[3,4]and(ii)chiralsolvatingagents(CSA)[5,6]formdiastereomericcomplexes in situ with substrate enantiomers and may be employed directly.(iii) Chiral derivatizing agents(CDA)[7]requiretheseparateformationof discretediastereoisomers prior to NMR analysis.With CDA it hastobe ensured that neither kinetic resolution nor racemization of the derivatizing agent occurs during derivatization. 3.2.1Chiral derivatizing agents (CDA) Derivatization of enantiomerswithan enantiomericallypurecompound(CDA)is the most widely used NMR technique fortheassay of enantiomeric purity. In contrasttochirallanthanideshiftreagents(CLSR)andchiralsolvatingagents(CSA), which formdiastereomericcomplexesthat arein fastexchangeon theNMRtime scale, derivatization yields discretediastereomers forwhich the observed chemical shiftnonequivalenceAoistypicallyfivetimesgreaterthanforrelatedcomplexes with a CSA Several prerequisites exist forthe CDAmethod:The derivatizing agent must be enantiopurei the presence of a small amount of the enantiomeric compound reduces the enantiomeric purity. During the formation ofdiastereomersracemization must be excluded.For instance, racemization during ester formationhad been observed by Raban and Mislow[8]as they first reported the chemical shift nonequivalence in the1HNMRspectraofdiastereomeric2-phenylpropionicacidestersof1-(2fluorophenyl)ethanol. In addition, the possibility of kinetic resolution due to differential reaction rates of the substrate enantiomers must be excluded. This danger can be minimized by using an excess of the derivatizing agent. 3.2.1.1IH and 19F NMR analysis Alcohols and amines SelectedexamplesofusefulCDAsforIH and/or19Fanalysisarerepresentedin Table3.2.1.Themostwidelyusedisa-methoxy-a-(trifluoromethyl)phenylacetic 32Nuclear magnetic resonance43 acid(M1PA)(1),introduced by Mosherin 1969[9,10].Sincethereisno hydrogen atom at thechiralcenter, racemization during derlvatization isexcluded.M1PA is available commercially in enantiomerically pure form, either as the acid or the acid chloride. Reaction with primary and secondary alcohols or amines formsdiastereomerlc ami des or esters that may be analyzed by 1H or 19FNMR [9-11].In IH NMR analysischemicalshiftnonequivalenceistypically0.1to0.2ppm(CDCI3;298K). Problemswithkineticresolutionhavebeenreported[12,13],however,NMR analysisafterM1PA derivatization remainsthe method ofchoiceforsimplechiral aminesandalcohols[14-16].OftenthediastereomerscanbeseparatedbyGCor HPLC aswell(see Sections3.4and 3.5),permitting independent verification of enantiomeric purity. Table 32.1Selected chiral derivatizing reagents for IH and 19p NMR analysis R-O-Acetylmandelic acid R,R-2,3-Butanediol Camphanic acid R-2-Pluoro-2-phenylethylamine S-O-Methylmandelic acid S-a-Methoxy-a-(trifluoromethyl)phenylacetic acid (MTPA) S-a-Naphthylethylamine S-a-Phenylethylamine The accuracy of the measured values depends upon the instrumental conditions, the methods of data handling, and the size of the shift nonequivalence. The error can be estimatedtobe+/-1%.AlthoughseveralanaloguesofM1PAhavebeenstudied, e.g.,2a-e[17)and3a-d[18],theysufferfromracemizationundertheforcing conditions required to form ester derivatives of sterically hindered alcohols. Ph PhRPh P3c1.....hCOOH H ~ ...."COOHH-1...."COOH P3c1'''''NCO OMe RF OMe 1la-e3a-d 4 R:a)OMeR:a)SPh b)t-Bu b) Ph c)CF3 c)OPh d)OH d)CH2Ph e)CI Some success has been achieved with the isocyanate 4 [19].The isocyanate does not reactwithhinderedalcohols,butwithprimaryandsecondarychiralaminesit 443Techniques used in the analysis of optically active compounds yields diastereomeric ureas that show higher chemical shift nonequivalence than the correspondingMTP Aderivatives.Withimprovementsinthesynthesisofesters (alsowithhinderedalcohols[20])oramidesundernoruacemizingconditions, derivatizing agents other than MTPA may b