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CD Spectros
CD Spectros
CD Spectros
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.
Ifforinstancewetakehorizontallyandverticallypolarisedlightwavesofequalamplitudethat areinphasewitheachother,theresultantlightwave(blue)islinearlypolarisedat45degrees.
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.
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
andchiralityareshownbelow,withacomparisonofthetwoenantiomersofcamphor sulphonicacid.
CD, ORD and Absorbance spectra of R and S forms of camphor sulphonic acid
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.
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.
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.
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)