RamanSpectroscopy

AnintroductiontoRamanSpectroscopy

Simplified energy diagram

Itistheshiftinwavelengthoftheinelasticallyscatteredradiationthatprovidesthechemicaland
structuralinformation.Ramanshiftedphotonscanbeofeitherhigherorlowerenergy,dependingupon
thevibrationalstateofthemoleculeunderstudy.Asimplifiedenergydiagramthatillustratesthese
conceptsisshownontheright.

Stokesradiationoccursatlowerenergy(longerwavelength)thantheRayleighradiation,andanti
Stokesradiationhasgreaterenergy.Theenergyincreaseordecreaseisrelatedtothevibrationalenergy
levelsinthegroundelectronicstateofthemolecule,andassuch,theobservedRamanshiftofthe
StokesandantiStokesfeaturesareadirectmeasureofthevibrationalenergiesofthemolecule.A
schematicRamanspectrummayappearasshownbelow.

TheenergyofthescatteredradiationislessthantheincidentradiationfortheStokeslineandthe
energyofthescatteredradiationismorethantheincidentradiationfortheantiStokesline.Theenergy
increaseordecreasefromtheexcitationisrelatedtothevibrationalenergyspacingintheground
electronicstateofthemoleculeandthereforethewavenumberoftheStokesandantiStokeslinesarea
directmeasureofthevibrationalenergiesofthemolecule.

Intheexamplespectrum,noticethattheStokesandantiStokeslinesareequallydisplacedfromthe
Rayleighline.Thisoccursbecauseineithercaseonevibrationalquantumofenergyisgainedorlost.
Also,notethattheantiStokeslineismuchlessintensethantheStokesline.Thisoccursbecauseonly
moleculesthatarevibrationallyexcitedpriortoirradiationcangiverisetotheantiStokesline.Hence,
inRamanspectroscopy,onlythemoreintenseStokeslineisnormallymeasuredRamanscatteringisa
relativelyweakprocess.ThenumberofphotonsRamanscatteredisquitesmall.However,thereare
severalprocesswhichcanbeusedtoenhancethesensitivityofaRamanmeasurement.

Schematic Raman spectrum

Ifthewavelengthoftheexcitinglasercoincideswithanelectronicabsorptionofamolecule,the
intensityofRamanactivevibrationsassociatedwiththeabsorbingchromophoreareenhancedbya
factorof102to104.ThisresonanceenhancementorresonanceRamaneffectcanbeextremelyuseful,
notjustinsignificantlyloweringthedetectionlimits,butalsoinintroducingelectronicselectivety.
ThustheresonanceRamantechniqueisusedforprovidingbothstructuralandelectronicinsightinto
speciesofinterest.Metalloporphyrins,carotenoidsandseveralotherclassesofbiologicallyimportant
moleculeshavestronglyallowedelectronictransitionsinthevisible,makingthemidealcandidatesfor
resonanceRamanspectroscopy.Resonanceselectivityhasafurtherpracticaluse,inthatspectrumof
thechromophoricmoietyisresonanceenhancedandthatofthesurroundingenvironmentisnot.For
biologicalchromophores,thismeansthatabsorbingactivecentrescanbespecificallyprobedbyvisible
excitationwavelengths,andnotthesurroundingproteinmatrix(whichwouldrequireUVlasersto
bringintoresonance).ResonanceRamanspectroscopyisalsoanimportantprobeofthechemistryof
metalcentredcomplexes,fullerenes,polydiacetylenesandother"exotic"moleculeswhichstrongly
absorbinthevisible.Althoughmanymoremoleculesabsorbintheultraviolet,thehighcostoflasers
andopticsforthisspectralregionhavelimitedultraviolet(UV)resonanceRamanspectroscopytoa
smallnumberofspecialistgroups.

Vibrationswhichareresonantlyenhancedfallintotwoorthreegeneralmechanisticclasses.Themost
commoncaseisFranckCondonenhancement,inwhichacomponentofthenormalcoordinateofthe
vibrationoccursinadirectioninwhichthemoleculeexpandsduringanelectronicexcitation.Themore
themoleculeexpandsalongthisaxiswhenitabsorbslight,thelargertheenhancementfactor.The
easilyvisualizedringbreathing(inplaneexpansion)modesofporphyrinsfallintothisclass.Vibrations
whichcoupletwoelectronicexcitedstatesarealsoresonantlyenhanced,throughamechanismcalled
vibronicenhancement.Inbothcases,enhancementfactorsroughlyfollowtheintensitiesofthe
absorptionspectrum.Thefullertheoryofresonanceenhancementisbeyondthescopeofthissection.

Resonanceenhancementdoesnotbeginatasharplydefinedwavelength.Infact,enhancementof5xto
10xisobservediftheexcitinglaseriswithinevenafew100wavenumbersbelowtheelectronic
transitionofamolecule.This"preresonance"enhancementcanbeexperimentallyuseful.

TheRamanscatteringfromacompound(orion)adsorbedonorevenwithinafewAngstromsofa
structuredmetalsurfacecanbe103to106xgreaterthaninsolution.ThissurfaceenhancedRaman
scatteringisstrongestonsilver,butisobservableongoldandcopperaswell.Atpracticalexcitation
wavelengths,enhancementonothermetalsisunimportant.SERSarisesfromtwomechanisms:The
firstisanenhancedelectromagneticfieldproducedatthesurfaceofthemetal.Whenthewavelengthof
theincidentlightisclosetotheplasmawavelengthofthemetal,conductionelectronsinthemetal
surfaceareexcitedintoanextendedsurfaceelectronicexcitedstatecalledasurfaceplasmonresonance.
Moleculesadsorbedorincloseproximitytothesurfaceexperienceanexceptionallylarge
electromagneticfield.Vibrationalmodesnormaltothesurfacearemoststronglyenhanced.The
secondmodeofenhancementisbytheformationofachargetransfercomplexbetweenthesurfaceand
analytemolecule.Theelectronictransitionsofmanychargetransfercomplexesareinthevisible,so
thatresonanceenhancementoccurs.Moleculeswithlonepairelectronsorpicloudsshowthestrongest
SERS.Theeffectwasfirstdiscoveredwithpyridine.Otheraromaticnitrogenoroxygencontaining
compounds,suchasaromaticaminesorphenols,arestronglySERSactive.Theeffectcanalsobeen
seenwithotherelectronrichfunctionalitiessuchascarboxylicacids.Theintensityofthesurface
plasmonresonanceisdependentonmanyfactorsincludingthewavelengthoftheincidentlightandthe
morphologyofthemetalsurface.Thewavelengthshouldmatchtheplasmawavelengthofthemetal.
Thisisabout382nmfora5msilverparticle,butcanbeashighas600nmforlargerellipsoidalsilver
particles.Theplasmawavelengthistotheredof650nmforcopperandgold,theothertwometals
whichshowSERSatwavelengthsinthe3501000nmregion.Thebestmorphologyforsurface
plasmonresonanceexcitationisasmall(<100nm)particleoranatomicallyroughsurface.SERSis
commonlyemployedtostudymonolayersofmaterialsadsorbedonmetals,includingelectrodes.Other
popularsurfacesincludecolloids,metalfilmsondielectricsubstratesand,recently,arraysofmetal
particlesboundtometalordielectriccolloidsthroughshortlinkages.AlthoughSERSallowseasy
observationofRamanspectrafromsolutionconcentrationsinthemicromolar(10x6)range,non
reproducabilityofquantitativemeasurementshasinthepastmarreditsutilityforanalyticalpurposes.
However,standardizationinproductionofSERSactivemediaissteadilyimprovingitspotentialinthis
areaalso.
UVRRSisapowerfultoolinthemolecularanalysisofcomplexbiologicalsystems.Mostbiological
systemsabsorbUVradiationandhencehavetheabilitytoofferresonancewithUVRaman
excitation.ThisresultsinthehighlyselectiveresonanceRamaneffectenablingenhancementof
importantbiologicaltargetssuchasproteinorDNA.Forexample,excitationaround200nmenhances
theRamanpeaksfromvibrationsofamidegroups;excitationaround220nmenhancespeaksfrom
certainaromaticresidues.TheRamanscatterfromwaterisweak,allowingforanalysisofveryweak
aqueoussystems.

Fiber optic
UVRRS configuration

DuetotheselectivenatureofUVRRS,atunablelaseristypicallyrequiredastheexcitationsource.
Sincetrulytunablecontinuouswavelasersarenotyetavailable,aNd:YAGpumpeddyelaserwith
frequencydoubledoutputisonesuitableUVRRSsystem.Dependingonthedyesused,thislasersetup
cangivealmostanyrequiredUVwavelength.IntensifiedCCDs(ICCDs)withUVphotocathodes,back
illuminatedCCDsorCCDswithUVenhancing(BASFlumogen)coatingscanbeusedasdetectorsfor
UVRRS.Thesedetectorsareusedonaccountoftheirhighdetectionefficiencyandmultichannel
capabilities.TheprimaryobstacletothemergingoftheworldsofUVRRSandfiberopticspectroscopy
issolarization,theprocessbywhichUVradiationcausesopacityoffiberoptics(evenquitepuresilica
fibers).Thisopacityimpairstransmission,renderingstandardfiberopticsuselessforUVRRS.

VibrationalRamanspectroscopyisnowwidelyrecognisedasausefultechniqueforchemicalanalysis
ofstablespecies,sincethetechnologywhichunderpinsRamanmeasurementshasmatured.Similarly,
timeresolvedRamanspectroscopyhasalsobecomeestablishedasanexcellentmethodforthe
characterizationoftransientchemicalspecies.Manyofthetechnicaladvanceswhichhavereducedthe
costandincreasedthereliabilityofconventionalRamansystemscanalsobeexploitedinstudiesof
transientspecies.

Species of Interest

Pulsedlasersaretypicallyutilizedinthestudyofshortlivedspecies.Alaserpulsecanbesuppliedtoa
molecularsystemwithenoughenergytoredistributetheelectronsinamoleculecausingtheformation
ofanexcitedstateasillustratedontheright.TheRamanspectrumofthisexcitedstatemoleculecanbe
studiedeitherusingthesamelaserpulseoradifferentpulsefromasecondlaser(singlecolorandtwo
colorpulsedRaman).Excitedstatesofinterestcanhavelifetimes,frompicosecondstomilliseconds,
butthemajoritycanbestudiedusinggatingintheorderof5ns.Asthemajorityofexcitedstatesare
generatedusingUVandvisiblelasers,photocathodeswithhighUVandvisibleQuantumEfficiencies
(QE's)aretypicallysuitable.

Schematic of basic pulsed excited state

Raman

Thesimplestpulsedlaserexperimentsaresocalledsinglecolorexperimentswherehighirradiance
laserpulsesareusedbothtoinitiatethephotoreaction,andthentoRamanprobethetransientspecies
createdwithinthepulsewidth.Byopeningtheintensifiertubeasshownontheright,onlytheRaman
spectrumoftheexcitedstatewillberecorded.Thispulse/ICCDgatecombinationwillberepeatedand
accumulatedhundredstothousandsoftimesinordertoachieveagoodoverallsignaltonoiseratio
withhighdynamicrange.

InTimeResolvedResonanceRaman(TR3)spectroscopy,pairsoflaserpulsesofdifferentwavelength
areusedtophotolyse(optically"pump")andthentoRamanprobethetransientspeciesofinterest.The
spectralwindowofthespectrograph/detectorischosensothatitcorrespondstothefrequencyrangeof
theRamanscatteringfromtheprobelaser.

Schematic of pump-probe (two color)

Raman

InTimeResolvedResonanceRaman(TR3)spectroscopy,pairsoflaserpulsesofdifferentwavelength
areusedtophotolyse(optically"pump")andthentoRamanprobethetransientspeciesofinterest.The
spectralwindowofthespectrograph/detectorischosensothatitcorrespondstothefrequencyrangeof
theRamanscatteringfromtheprobelaser.

Thetimeevolutionofthetransientsignalismonitoredbyrecordingaseriesofspectraatdifferent
delaysafterthephotolysisevent,i.e.ataseriesoftimedelaysbetweentheexcitationandprobepulses.
TheICCDcameraoreitherofthelaserscansupplythetrigger.Adelaygeneratorisusedtocontrolsthe
delays.

Pulsed two color Raman layout with delays

under the control of a delay generator

InRamanmicroscopy,aresearchgradeopticalmicroscopeiscoupledtotheexcitationlaserandthe
spectrometer,thusproducingaplatformcapableofobtainingbothconventionalimagesandinaddition
generatingRamanSpectrafromsampleareasapproachingthediffractionlimit(~1micron).Imaging
andspectroscopycanbecombinedtogenerate"Ramancubes",3dimensionaldatasets,yielding
spectralinformationateverypixelofthe2Dimage.Amotorizedxyzmicroscopestagecanbeusedto
automaticallyrecordspectralfiles,whichwillconstitutethebasisofRamanimages,Ramanmapsora
setofRamanspectrarecordedfrompreselectedpoints.Specificsoftwareroutineswillallowthequick
andeasyreconstructionofthesemaps.Thepossibilityofgeneratingtwodimensionalandthree
dimensionalimagesofasample,usingvariousspecialfeatures,isanevidentadvantageovereither
traditionalspectroscopyormicroscopy.

Tim
e delay sequences

ThefirsteverRaman"instrument"wasconstructedin1928.Thisinstrumentusedmonochromatized
sunlightasalightsourceandahumaneyeasadetector.Ramaninstrumentationwasdeveloped(based
aroundarclampsandphotographicplates)andsoonbecameverypopularupuntilthe1950's.Since
theseearlydays,Ramaninstrumentationhasevolvedmarkedly.Moderninstrumentationtypically
consistsofalaser,Rayleighfilter,afewlenses,aspectrographandadetector(typicallyaCCDor
ICCD).
 Typical
Continuous Wave (CW) Raman layout

OneofthemajoradvantagesofdispersiveRamanisthatitoffersthepossibilitytoselecttheoptimal
laserexcitationwavelengthtopermittherecordingofthebestRamaninformation.Forexample,
wavelengthscanbeselectedtoofferthebestresonancewiththesampleunderinvestigation.

Onemightalsoneedtotunewavelengthtoavoidfluorescenceandthermalemission
backgrounds.Nowadays,itispossibletouselaserlinesfromUV,(downto200nm)uptotheinfrared,
(1.06mNd:YAGlaserline),frommicroWattsuptoseveralWatts..