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.}.
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[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
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65,8% [581Sturzenberger, V.; Buchecker, R.; Wagniere, G.
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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