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Anintroductiontocirculardichroismspectroscopy

Circulardichroism(CD)isthedifferenceintheabsorptionoflefthandedcircularlypolarised light(LCPL)andrighthandedcircularlypolarisedlight(RCPL)andoccurswhenamolecule containsoneormorechiralchromophores(lightabsorbinggroups). Circulardichroism=A()=A()LCPLA()RCPL,whereisthewavelength Circulardichroism(CD)spectroscopyisaspectroscopictechniquewheretheCDofmoleculesis measuredoverarangeofwavelengths.CDspectroscopyisusedextensivelytostudychiral moleculesofalltypesandsizes,butitisinthestudyoflargebiologicalmoleculeswhereit findsitsmostimportantapplications.Aprimaryuseisinanalysingthesecondarystructureor conformationofmacromolecules,particularlyproteinsassecondarystructureissensitivetoits environment,temperatureorpH,circulardichroismcanbeusedtoobservehowsecondary structurechangeswithenvironmentalconditionsoroninteractionwithothermolecules. Structural,kineticandthermodynamicinformationaboutmacromoleculescanbederivedfrom circulardichroismspectroscopy. Measurementscarriedoutinthevisibleandultravioletregionoftheelectromagnetic spectrummonitorelectronictransitions,and,ifthemoleculeunderstudycontainschiral chromophoresthenoneCPLstatewillbeabsorbedtoagreaterextentthantheotherandthe CDsignaloverthecorrespondingwavelengthswillbenonzero.Acirculardichroismsignalcan bepositiveornegative,dependingonwhetherLCPLisabsorbedtoagreaterextentthanR CPL(CDsignalpositive)ortoalesserextent(CDsignalnegative).Anexamplecircular dichroismspectrumofasamplewithmultipleCDpeaksisshownbelow,demonstratinghow CDvariesasafunctionofwavelength,andthataCDspectrummayexhibitbothpositiveand negativepeaks.

Circulardichroismspectraaremeasuredusingacirculardichroismspectrometer,suchasthe Chirascan,whichisahighlyspecialisedderivativeofanordinaryabsorptionspectrometer.CD

spectrometersmeasurealternatelytheabsorptionofLandRCPL,usuallyatafrequencyof 50kHz,andthencalculatethecirculardichroismsignal.

1. Understanding Circular Dichroism


Thebasicsofpolarisation Toreallyunderstandcirculardichroism,onemustfirstunderstandthebasicsofpolarisation. Linearlypolarisedlightislightwhoseoscillationsareconfinedtoasingleplane.Allpolarised lightstatescanbedescribedasasumoftwolinearlypolarisedstatesatrightanglestoeach other,usuallyreferencedtotheviewerasverticallyandhorizontallypolarisedlight.

Vertically Polarised Light

Horizontally Polarised Light

Ifforinstancewetakehorizontallyandverticallypolarisedlightwavesofequalamplitudethat areinphasewitheachother,theresultantlightwave(blue)islinearlypolarisedat45degrees.

45 Degree Polarised Light

Ifthetwopolarisationstatesareoutofphase,theresultantwaveceasestobelinearly polarised.Forexample,ifoneofthepolarisedstatesisoutofphasewiththeotherbya quarterwave,theresultantwillbeahelixandisknownascircularlypolarisedlight(CPL).The helicescanbeeitherrighthanded(RCPL)orlefthanded(LCPL)andarenonsuperimposable mirrorimages. Theopticalelementthatconvertsbetweenlinearlypolarisedlightandcircularlypolarisedlight istermedaquarterwaveplate.Aquarterwaveplateisbirefringent,i.e.therefractiveindices seenbyhorizontallyandverticallypolarisedlightaredifferent.Asuitablyorientedplatewill convertlinearlypolarisedlightintocircularlypolarisedlightbyslowingoneofthelinear componentsofthebeamwithrespecttotheothersothattheyareonequarterwaveoutof phase.ThiswillproduceabeamofeitherleftorrightCPL.

Left Circularly Polarised (LCP) Light

Right Circularly Polarised (RCP) Light

Thedifferenceinabsorbanceoflefthandandrighthandcircularlypolarisedlightisthebasis ofcirculardichroism.AmoleculethatabsorbsLCPandRCPdifferentlyisopticallyactive,or chiral.

2. Chiral Molecules
Theoriginofopticalactivity Inthefirstsectionofthistutorial,westatedthatcirculardichroismisthedifferential absorbanceoflefthandcircularlypolarisedandrighthandcircularlypolarisedlight,andthat chiralmoleculesexhibitcirculardichroism.Butwhatarechiralmolecules? Chiralmoleculesexistaspairsofmirrorimageisomers.Thesemirrorimageisomersarenot superimposableandareknownasenantiomers.Thephysicalandchemicalpropertiesofapair ofenantiomersareidenticalwithtwoexceptions:thewaythattheyinteractwithpolarised lightandthewaythattheyinteractwithotherchiralmolecules. Circularbirefringenceandopticalrotation Chiralmoleculesexhibitcircularbirefringence,whichmeansthatasolutionofachiral substancepresentsananisotropicmediumthroughwhichleftcircularlypolarised(LCPL)and rightcircularlypolarised(RCPL)propagateatdifferentspeeds.Alinearlypolarisedwavecan bethoughtofastheresultantofthesuperpositionoftwocircularlypolarisedwaves,oneleft circularlypolarised,theotherrightcircularlypolarised.Ontraversingthecircularlybirefringent medium,thephaserelationshipbetweenthecircularlypolarisedwaveschangesandthe resultantlinearlypolarisedwaverotates.Thisistheoriginofthephenomenonknownas opticalrotation,whichismeasuredusingapolarimeter.Measuringopticalrotationasa functionofwavelengthistermedopticalrotatorydispersion(ORD)spectroscopy.


Circular birefringence - the orange cuboid represents the sample

Circulardichroism Unlikeopticalrotation,circulardichroismonlyoccursatwavelengthsoflightthatcanbe absorbedbyachiralmolecule.AtthesewavelengthsLeftandrightcircularlypolarisedlight willbeabsorbedtodifferentextents.Forinstance,achiralchromophoremayabsorb90%ofR CPLand88%ofLCPL.Thiseffectiscalledcirculardichroismandisthedifferenceinabsorption ofLCPLandRCPL.Circulardichroismmeasuredasafunctionofwavelengthistermedcircular dichroism(CD)spectroscopyandistheprimaryspectroscopicpropertymeasuredbyacircular dichroismspectrometersuchastheChirascan.

Circular dichroism - the orange cuboid represents the sample

Opticalrotationandcirculardichroismstemfromthesamequantummechanicalphenomena andonecanbederivedmathematicallyfromtheotherifallspectralinformationisprovided. Therelationshipbetweenopticalrotatorydispersion,circulardichroism,absorptionspectra

andchiralityareshownbelow,withacomparisonofthetwoenantiomersofcamphor sulphonicacid.

CD, ORD and Absorbance spectra of R and S forms of camphor sulphonic acid

AlthoughORDspectraandCDspectracantheoreticallyprovideequivalentinformation,each techniquehasbeenusedforverydistinctapplications.Opticalrotationatasinglewavelength isusedasageneralmeasurementtoolforchiralmolecules,todetermineconcentrationandas adeterminantofchiralpuritycomparedtoaknownstandard.Thesimplicityandlowcostof theexperimentandinstrumentationmakesitidealforthisapplication.Circulardichroism spectraontheotherhandarebetterspectrallyresolvedthanORDspectra,andconsequently moresuitableforadvancedspectralanalysis.

3. Chirality and Biology


Circulardichroismandthestudyofbiologicalmolecules Circulardichroismisaconsequenceoftheinteractionofpolarisedlightwithchiralmolecules. Thevastmajorityofbiologicalmoleculesarechiral.Forinstance,19ofthe20commonamino acidsthatformproteinsarethemselveschiral,asareahostofotherbiologicallyimportant molecules,togetherwiththehigherstructuresofproteins,DNAandRNA.Thehighlychiral chemistryofbiologicalmoleculeslendsitselfwelltoanalysisbycirculardichroismandthe studyofbiologicalmoleculesisthemainapplicationofthetechnique. Alargesubsetoftheuseofcirculardichroisminbiochemistryisintheunderstandingofthe higherorderstructuresofchiralmacromoleculessuchasproteinsandDNA.Thereasonforthis isthattheCDspectrumofaproteinorDNAmoleculeisnotasumoftheCDspectraofthe individualresiduesorbases,butisgreatlyinfluencedbythe3dimensionstructureofthe macromoleculeitself.Eachstructurehasaspecificcirculardichroismsignature,andthiscanbe usedtoidentifystructuralelementsandtofollowchangesinthestructureofchiral macromolecules. Themostwidelystudiedcirculardichroismsignaturesarethevarioussecondarystructural elementsofproteinssuchasthehelixandthesheet.Thisisunderstoodtothepointthat

CDspectrainthefarUV(below260nm)canbeusedtopredictthepercentagesofeach secondarystructuralelementinthestructureofaprotein.Someofthecommonprotein secondarystructuralelementsandtheCDspectraassociatedwiththemareshownbelow:

The secondary structure conformation and the CD spectra of protein structural elements. Right is an example of the backbone conformation of a peptide in an -helix and left is the conformation of a peptide in a -sheet. In the centre are the associated CD spectra for these different conformations.

Therearemanyalgorithmsdesignedforfittingthecirculardichroismspectraofproteinsto provideestimatesofsecondarystructure.TheproteinsecondarystructureCDanalysis softwaredistributedwiththeChirascanisCDNN. Secondarystructurepredictionisonlypartofthepowerofcirculardichroismspectroscopy. Changesincirculardichroismspectraareverygoodproxiesforchangesinthestructureofa molecule.Couplethiswiththefactsthat(i)spectracanberecordedinminutesand(ii)single wavelengthkineticscanberecordedfrommillisecondsonwards,CDisaparticularlypowerful tooltofollowdynamicchangesinproteinstructure.Forinstancechangesinducedbychanging temperature,pH,ligands,ordenaturantsareallcommonlyused. Apowerfulapplicationofcirculardichroismistocomparetwomacromolecules,orthesame moleculeunderdifferentconditions,anddetermineiftheyhaveasimilarstructure.Thiscan beusedsimplytoascertainifanewlypurifiedproteiniscorrectlyfolded,determineifa mutantproteinhasfoldedcorrectlyincomparisontothewildtype,orfortheanalysisof biopharmaceuticalproductstoconfirmthattheyarestillinacorrectlyfoldedactive conformation.

4. CD Spectrometer Operating Principles


Allthedetailsforafullunderstanding Circulardichroism(CD)isthedifferenceinlightabsorbancebetweenleft(LCPL)andright circularlypolarisedlight(RCPL)andcirculardichroismspectrometers(spectrophotometers) arehighlyspecialisedvariationsoftheabsorbancespectrophotometer. Acirculardichroismspectrophotometerisalsocommonlytermedacirculardichroism spectropolarimeteroracirculardichrograph.Mostmoderncirculardichroisminstruments operateonthesameprinciples,whichisdemonstratedintheslideshowatthebottomofthe page.Thereisasourceofmonochromaticlinearlypolarisedlightwhichcanbeturnedinto

eitherleftorrightcircularlypolarisedlightbypassingitthroughaquarterwaveplatewhose uniqueaxisisat45degreestothelinearpolarisationplaneasdescribedinthesectionabout polarisedlight. Insteadofastaticquarterwaveplate,acirculardichroismspectrophotometerhasa specialisedopticalelementcalledaphotoelasticmodulator(PEM).Thisisapiezoelectric elementcementedtoablockoffusedsilica.Atrest,whenthepiezoelectricelementisnot oscillating,thesilicablockisnotbirefringent;whendriven,thepiezoelectricelementoscillates atitsresonancefrequency(typicallyaround50kHz),andinducesstressinthesilicainsucha waythatitbecomesbirefringent.Thealternatingstressturnsthefusedsilicaelementintoa dynamicquarterwaveplate,retardingfirstverticalwithrespecttohorizontalcomponentsof theincidentlinearlypolarisedlightbyaquarterwaveandthenviceversa,producingleftand thenrightcircularlypolarisedlightatthedrivefrequency.Theamplitudeoftheoscillationis tunedsothattheretardationisappropriateforthewavelengthoflightpassingthroughthe silicablock. Ontheothersideofthesamplepositionthereisalightdetector.Whenthereisnocircularly dichroicsampleinthelightpath,thelighthittingthedetectorisconstant.Ifthereisacircularly dichroicsampleinthelightpath,therecordedlightintensitywillbedifferentforrightand leftCPL.UsingalockinamplifiertunedtothefrequencyofthePEM,itispossibletomeasure thedifferenceinintensitybetweenthetwocircularpolarisations(vAC).Theaveragetotallight intensityacrossmanyPEMoscillations(vDC)canbeusedtoscalethesizeofthelockin amplifiersignaltotakeintoaccountvariationsintotallightlevel.Bothsignalscanberecorded andfromthemthecirculardichroismsignalcanbecalculatedeasilybydividingthevAC componentbythevDCsignal.

Gisacalibrationscalingfactortoprovideeitherellipticityordifferentialabsorbance.The sectionaboutCDUnitsandtheirinterconversionexplainshowellipticityanddifferential absorbancearerelated.

5. CD Spectrometer Performance
Thedesignanditseffectonoperation Thelimitofdetectionofacirculardichroism(CD)spectrophotometer(orany spectrophotometer)isdeterminedbyitssignaltonoise(S/N)characteristics:thebetterthe S/N,thebetteritslimitofdetection. Thesignaltonoiseratioislimitedbyphotonshotnoise,whichisthestatisticalvariationabout anaverageinthenumberofphotonsperunittimedetectedbythelightdetector.The quantisednatureofphotonsandtheirrandomarrivalatthedetectormeansthatalthoughthe averagenumberofphotonsdetectedpersecondmaybesay5,thenumberinanyparticular onesecondintervalmaybe0,2,7orsomeothernumber.Thus,ameasurementmustbe madeoverasufficientlylongperiodoftimetodeterminethetrueaverageandthetimetaken todeterminethetrueaveragewillbeinverselyproportionaltoS/N.Itisthereforeimportantto designacirculardichroismspectrophotometertomaximiseitsS/Ncharacteristics.

Ageneralrelationshipbetweenthecontributingfactorstothesignaltonoiseinanoptical spectrometercanbewrittenas:

whereQ=detectorquantumefficiency,I=lightintensity,t=timescaleofthemeasurement. Fromthisitisapparenttherearethreewaystoimprovethesignaltonoiseofacircular dichroismspectrophotometer:increasetheintensityoftheincidentlinearlypolarised monochromaticlight,increasethequantumefficiencyofthedetector,orspendmoretime collectingandaveragingdatapoints. Thefirsttwofactors,lightintensityanddetectorperformance,arethosethatcanbe influencedbythedesignofacirculardichroismspectrophotometerandworktogetherto lowerthelastfactor,thetimerequiredtocarryoutameasurement.Thehigherthelight throughputandbetterthedetectorefficiency,thelesstimeittakestocollectqualitydataor, equally,thehigherthequalityofdatathatcanbecollectedintimelimitedexperimentssuch asstoppedflowmeasurements. Increasingtheintensityoftheincidentlightisthemainavenueforincreasingtheperformance ofcirculardichroismspectrophotometersandthisfindsitsultimateexpressionintheuseof synchrotronlightsourcesforCDspectroscopy.Synchrotronfacilitiesprovidetremendouslight intensityacrossaverywidespectralrangeofwavelengthsbutaccesstothemisexpensiveand limitedandtheiruseisrestrictedtothemorecuttingedgeapplicationsofcirculardichroism. ForthevastmajorityofCDexperiments,ahighintensitybenchtopsourceistheonlypractical option:AppliedPhotophysicsChirascanhasbeendesignedfromthegrounduptomaximise thelightthroughputfromitsXearclampsourcetothesample. TheChirascanmonochromatorusestwosynthetic,singlecrystalquartzprismsinsteadofthe diffractiongratingsthatmostpeoplearefamiliarwithfromnormalabsorbance spectrophotometers.Quartzprismsaremoreefficientthandiffractiongratingsforaverywide rangeofwavelengths,particularlyintheUV.Quartzisalsobirefringentandtheprismsnotonly disperselightintothecomponentwavelengthsbutalso,becauseoftheirbirefringence, dispersethelinearlypolarisedcomponents,oneofwhichisselectedforconversionto circularlypolarisedlight.Afurtheradvantageofprismsistheydonotpasssecondorder multiplesofthedesiredwavelength,whichisamajorsourceofstraylightingratingbased monochromators. Unlikethedispersionofagrating,whichislinearandhighlycustomisable,thedispersionofa prismisnonlinearandissetbythepropertiesoftheprismmaterial.Consequentlytheoptics andmechanicsofaprismmonochromatorhavetobemorecomplexthanagrating monochromator,withtheneedtoconstantlyvarytheslitwidthasafunctionofwavelengthto maintainaconstantbandpass,andacomplexrelationshipbetweenprismmovementand wavelength.However,thelargewavelengthdispersionintheUVmeansthatwiderslitscanbe usedevenatasmallspectralbandpass,whichmeansgreaterlightcollectionefficiency throughouttheUVregion.Thelargemajorityofcirculardichroismapplicationsarecarriedout intheUVanditisatthesewavelengthsthatthecharacteristicsofaprismareamajor advantage.

Inadditiontothehighintensitytoimprovesignaltonoise,thelightfromthemonochromator musthaveaverylowstraylightcontentandaveryhighpurityoflinearpolarisationtoprovide accuratemeasurements.Allofthesethreekeyelementshavebeenoptimisedinthe Chirascancirculardichroismspectrophotometerandhavebeenachievedbykeydesign featuresoftheChirascanmonochromator. ThesecondinfluenceontheS/Nisthequantumefficiencyofthedetectorused.i.e.its efficiencyinturninganincidentphotonintoanelectronicsignal.Incirculardichroism spectrophotometers,thedetectorofchoiceinthelastfewdecadeshasbeenthe photomultipliertube.Thequantumefficiencyofthesedetectors,whicharetraditionallyused inspectroscopyforUVandvisiblelightdetection,hasremainedfairlystaticoverthatperiod. Recently,advancesinphotodiodetechnologyhaveresultedinnew,highgain,largeareasolid statedetectors,whichprovidesignificantimprovementsinquantumefficiencyintheultra violet,visibleandnearinfraredregionswhencomparedwithphotomultipliertubes(see Figure1).Oneofthesenewhighperformancesolidstatedetectorsisfeaturedinthenew ChirascanplusanditgivesfurtherS/Nimprovementsoverthephotomultiplierbased Chirascanspectrometer.Thequantumefficiencyimprovementoutlinedbelowresultsin significantsignaltonoiseimprovementsoverandabovethealreadyhighperformanceofthe standardChirascan.

Figure 1. Quantum efficiency of Chirascan Plus detector vs a standard photomultiplier tube

6. CD Signatures of Structural Elements


Circulardichroismsignaturesofsecondarystructuralelements

7. CD Units & Conversions


Allrelationshipsexplained Circulardichroism(CD)isusuallyunderstoodandactuallymeasuredasthedifferential absorbanceofleft(ALCP)andrightcircularlypolarised(ARCP)light,andsocanbeexpressed as: A=ALCPARCP Takingintoaccountcellpathlengthandcompoundconcentration,wecanarriveatamolar circulardichroism(). =LCPRCP=A/(Cxl) WhereLCPandRCParethemolarextinctioncoefficientsforLCPandRCPlightrespectively, C=molarconcentration,andl=pathlengthincentimeters. AnotherimportantunitismeanresiduemolarcirculardichroismMR.Thisisaunitspecific forproteins,andreportsthemolarcirculardichroismforindividualproteinresiduesinsteadof wholeproteinmolecules.Thisallowseasycomparisonofdifferentproteinswithvastly differentmoleculeweights.Therearetwowaystocalculatethisdependingonhowmuch informationisknownabouttheprotein. Theconcentrationofprotein(C)inmolarismultipliedbythenumberofaminoacids(N)inthe proteintoprovidethemeanresidueconcentration(CMR): CMR=CxN MR=A/(CMRxl) AnestimatecanbedeterminedforCMRifthesequenceoftheproteinisn'tknown,usingthe averageaminoacidresidueweightof113daltons,andtheconcentrationofprotein(P)ingL1 CMR=P/113 Aandarethemostintuitiveunitsformanybiochemists,astheyarederivedfromthe familiarconceptofUV/Visabsorbancespectroscopy,anditisalsohowmodernCDinstruments actuallymeasurecirculardichroism.CDcanalsobeexpressedasdegreesofellipticity(), whichisalegacyofpolarimetry,andsuchunitsarefrequentlyusedintheliterature.Inthe contextofmodernCDspectroscopy,theseunitsarearchaicandcanbeconfusing.The relationshipbetweenAandareexplainedbelow. ThedescriptionofellipticityissomewhatmorecomplexthanA.Linearlypolarisedlightwhen passedthroughacirculardichroicsamplewillbecomeellipticallypolarised.Elliptically polarisedlightislightthatisnotfullycircularpolarised,butinsteadisellipticalinshape.Thisis becausethecircularpolarisedcomponentsoftheoriginallinearpolarisedlightarenownotof equalmagnitudesduetodifferentialabsorbance(circulardichroism).Theeffectis demonstratedbelow:

Circular dichroism as ellipticity - the orange cuboid represents the sample

Thedegreeofellipticity()isdefinedasthetangentoftheratiooftheminortomajorelliptical axis,andisillustratedbelow:

Linear polarised light has 0 degrees of ellipticity (), while fully LCP or RCP will have + or - 45 degrees respectively.

Theadvantageofcirculardichroismellipticityasameasurementunitisthatitismoreeasily relatedtoopticalrotationmeasurementsandpolarimetry.Bothellipticityandopticalrotation aremeasurementsofchangesinpolarisationstateofalinearpolarisedanalyzerbeam,and bothhavethesameunitsandsimilaramplitudesforagivensample.Thissimilarityaidsin comparisonofopticalrotationandcirculardichroismmeasurements,ausefulabilitywhen circulardichroismspectroscopyfirststartedtobewidelyused,backinthe1960's. FortunatelyitisveryeasytointerconvertbetweenandA: A=/32.982 Note:Duetothesmallsizeofmanymeasurements,isoftenquotedasmillidegrees(m)or 1/1000ofadegree. MolarellipticitycanbemanipulatedinthesamewayasA.Forinstancetakingintoaccount concentrationandcellpathlengthaccordingtoBeerLambertslaw,wecanderivea measurementofmolarellipticity[].Followingpolarimetricconventions,molarellipticityis reportedindegreescm2dmol1,ordegreesM1m1whichareequivalentunitsasshown below.

Molarellipticitycanbecalculatedusingthefollowingequation: []=100x/(Cxl) Cistheconcentrationinmolar,andlthecellpathlengthincm.Thefactorof100convertsto pathlengthinmeters. MolarCirculardichroismandmolarellipticitycanbeconverteddirectlyby: =[]/3298.2 Thisfactorisahundredfoldlargerthanbetweenrawabsorbanceandellipticityduetothe conversionbetweenmolarextinctiondefiningpathlengthsincentimetersandellipticityhaving pathlengthdefinedinmeters. Anotherimportantunitismeanresidueellipticity[]MR.Thisisaunitspecificforproteins,and reportsthemolarellipticityforindividualproteinresiduesinsteadofwholeproteinmolecules. Thisallowseasycomparisonofdifferentproteinswithvastlydifferentmoleculeweights.There aretwowaystocalculatethisdependingonhowmuchinformationisknownaboutthe protein. Theconcentrationorprotein(C)inmolarismultipliedbythenumberofaminoacids(N)inthe proteintoprovidethemeanresidueconcentration(CMR): CMR=CxN []MR=100x/(CMRxl)

AnestimatecanbedeterminedforCMRifthesequenceoftheproteinisn'tknown,usingthe averageaminoacidresidueweightof113daltons,andtheconcentrationofprotein(P)ingL1 CMR=P/113 FortunatelytheProDatasoftwareforChirascancanconverteasilybetweenalltheseunits, withaminimumofuserintervention. WeatAppliedPhotophysicshopethatyouhavefoundthetutorialincirculardichroisma usefulresource.IfyouwanttofindoutmoreaboutAppliedPhotophysics'rangeofcircular dichroismspectrometers,thenpleasecontactsales@photophysics.com.

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