拉曼光譜資料

拉曼光譜OVERVIEW1.Ramanspectragiveinformationonmolecularvibrationsandareobtainedfromchangesinthefrequencyoflightobservedinascatteringexperiment(inelasticscattering).2.Thephysicalpicturearisesfromconsideringchangesinpolarizability(induceddipolemoment)thatariseifavibrationoccursduringthetimetheelectronsareoscillatinginresponsetotheappliedradiation.3.Thegrossselectionruleisthatthevibrationalmotionmustproduceachangeinthepolarizabilityofthemolecule.4.Theanisotropyofthepolarizationofthescatteringcanbemeasured.Comparisonofthespectrapolarizedperpendicularandparalleltotheincidentradiationgivesinformationonthesymmetryofthevibrationalmotions.5.Ramanspectracanbeobtainedinwater.Thisisamajoradvantageoverinfraredspectra.6.ResonanceRamanspectraresultwhenthewavelengthoftheexcitinglightfallswithinanelectronicabsorptionbandofachromophoreinthemolecule.Somevibrationsassociatedwithsuchachromophoremaybeenhancedbyfactorsof1000ormore.7.Theexperimentalparametersofabandinaspectrumareitsposition()(whichisindependentofthefrequencyoftheexcitinglight),itsintensity(whichisdirectlyproportionaltoconcentration),anditspolarization.8.ThemainbiologicalapplicationsofconventionalRamanareverysimilartothoseforinfrared.ResonanceRamanaffordsameansofprobingselectivesitesinmolecules.Forexample,inmetalloproteins,Ramancangiveinformationonthenatureoftheliganddirectlyattachedtothemetal.6.1引言拉曼光譜和紅外光譜都反映了分子振動(dòng)的信息，但其原理卻有很大差別：紅外光譜是吸收光譜，而拉曼光譜是散射光譜。紅外光譜的信息是從分子對(duì)入射電磁波的吸收得到的，而拉曼光譜的信息是從入射光與散射光頻率的差別得到的。拉曼光譜的突出優(yōu)點(diǎn)是可以很容易地測(cè)量含水的樣品，而且拉曼散射光可以在紫外和可見(jiàn)光波段量測(cè)。由于紫外光和可見(jiàn)光能量很強(qiáng)，因此其量測(cè)比紅外波段要容易和優(yōu)越得多。拉曼光譜得名于印度物理學(xué)家拉曼(Raman)。1928年，拉曼首先從實(shí)驗(yàn)觀察到單色的入射光投射到物質(zhì)中后產(chǎn)生的散射，通過(guò)對(duì)散射光進(jìn)行譜分析，首先發(fā)現(xiàn)散射光除了含有與入射光相同頻率的光外，還包含有與入射光頻率不同的光。以后人們將這種散射光與入射光頻率不同的現(xiàn)象稱(chēng)為拉曼散射。拉曼因此獲得諾貝爾獎(jiǎng)。當(dāng)一束入射光通過(guò)樣品時(shí)，在各個(gè)方向上都發(fā)生散射。拉曼光譜儀收集和檢測(cè)與入射光成直角的散射光。由于收集和檢測(cè)的散射光強(qiáng)度非常低，因此拉曼光譜的應(yīng)用和發(fā)展受到很大限制。六十年代激光開(kāi)始廣泛應(yīng)用，拉曼光譜儀以激光作光源，光的單色性和強(qiáng)度都大大提高，拉曼散射儀的信號(hào)強(qiáng)度因而大大提高，拉曼光譜技術(shù)得以迅速發(fā)展，應(yīng)用領(lǐng)域遍及物理，材料，化學(xué)，生物等學(xué)科，并已成為光譜學(xué)的一個(gè)分支拉曼光譜學(xué)。6.2拉曼光譜原理6.2.1光的散射：入射光通過(guò)樣品后，除了被吸收的光之外，大部分沿入射方向穿過(guò)樣品，一小部分光則改變方向，發(fā)生散射。一部分散射光的波長(zhǎng)與入射光波長(zhǎng)相同，這種散射稱(chēng)為瑞利散射(Rayleighscattering)。1899年，瑞利從實(shí)驗(yàn)中得出結(jié)論：晴天時(shí)天空呈蘭色的原因是大氣分子對(duì)陽(yáng)光的散射。瑞利還證實(shí)：散射光的強(qiáng)度與波長(zhǎng)的四次方成反比。這就是瑞利散射定律。由于組成白光的各種顏色的光中，蘭光的波長(zhǎng)最短，因而散射光強(qiáng)度最大。天空因而呈現(xiàn)蘭色。瑞利當(dāng)時(shí)并沒(méi)有考慮到散射光的頻率變化。他認(rèn)為散射光與入射光的頻率是相同的。所以后來(lái)把與入射光波長(zhǎng)相同的散射稱(chēng)為瑞利散射，而把波長(zhǎng)與入射光不同的散射稱(chēng)為拉曼散射。6.2.2拉曼散射的產(chǎn)生機(jī)械力學(xué)的解釋光由光子組成，這是光的微粒性。光子與樣品分子間的相互作用，可以用光子與樣品分子之間的碰撞來(lái)解釋。光照射樣品時(shí)，光子和樣品分子之間發(fā)生碰撞。如果碰撞時(shí)只是運(yùn)動(dòng)方向改變而未發(fā)生能量交換即發(fā)生了彈性碰撞，則光子的能量不變。由E=h，能量不變頻率也就不變。這就是瑞利散射產(chǎn)生的原因。如果光子和樣品分子間發(fā)生非彈性碰撞，即光子除改變運(yùn)動(dòng)方向外還有能量的改變，一部分能量碰撞時(shí)在光子和樣品之間發(fā)生交換，光子的能量有所增減，則光的頻率發(fā)生改變。從能級(jí)之間的躍遷來(lái)分析光子和樣品分子之間的作用也可以從能級(jí)之間的躍遷來(lái)分析。Figure9.1Processesleadingtonormal,preresonance,andresonanceRamanscattering.(Forcomparison,theprocessesinvolvedinIRandfluorescenceareshown.)Thehorizontallinesrepresentdifferentvibrationalenergylevelsinthetwoelectronicstates.TheRamanscatteringspectrumisalsoindicated.NotethattheintensityoftheStokeslinesisgreaterthanthatoftheanti-StokesP240Fig9.1樣品分子處于電子能級(jí)和振動(dòng)能級(jí)的基態(tài)，入射光子的能量遠(yuǎn)大于振動(dòng)能級(jí)躍遷所需要的能量，但又不足以將分子激發(fā)到電子能級(jí)激發(fā)態(tài)。這樣，樣品分子吸收光子后到達(dá)一種準(zhǔn)激發(fā)狀態(tài)，又稱(chēng)為虛能態(tài)。樣品分子在準(zhǔn)激發(fā)態(tài)時(shí)是不穩(wěn)定的，它將回到電子能級(jí)的基態(tài)。若分子回到電子能級(jí)基態(tài)中的振動(dòng)能級(jí)基態(tài)，則光子的能量未發(fā)生改變，發(fā)生瑞利散射。如果樣品分子回到電子能級(jí)基態(tài)中的較高振動(dòng)能級(jí)即某些振動(dòng)激發(fā)態(tài)，則散射的光子能量小于入射光子的能量，其波長(zhǎng)大于入射光。這時(shí)散射光譜的瑞利散射譜線(xiàn)較低頻率側(cè)將出現(xiàn)一根拉曼散射光的譜線(xiàn)，稱(chēng)為Stokes線(xiàn)。如果樣品分子在與入射光子作用前的瞬間不是處于電子能級(jí)基態(tài)的最低振動(dòng)能級(jí)，而是處于電子能級(jí)基態(tài)中的某個(gè)振動(dòng)能級(jí)激發(fā)態(tài)，則入射光光子作用使之躍遷到準(zhǔn)激發(fā)態(tài)后，該分子退激回到電子能級(jí)基態(tài)的振動(dòng)能級(jí)基態(tài)，這樣散射光能量大于入射光子能量，其譜線(xiàn)位于瑞利譜線(xiàn)的高頻側(cè)，稱(chēng)為anti-Stokes線(xiàn)。Stokes線(xiàn)和anti-Stokes線(xiàn)位于瑞利譜線(xiàn)兩側(cè)，間距相等，如圖9.1所示。Stokes線(xiàn)和anti-Stokes線(xiàn)統(tǒng)稱(chēng)為拉曼譜線(xiàn)。由于振動(dòng)能級(jí)間距還是比較大的，因此，根據(jù)波爾茲曼定律，在室溫下，分子絕大多數(shù)處于振動(dòng)能級(jí)基態(tài)，所以Stokes線(xiàn)的強(qiáng)度遠(yuǎn)遠(yuǎn)強(qiáng)于anti-Stokes線(xiàn)。拉曼光譜儀一般記錄的都只是Stokes線(xiàn)。P241WorkedExample9.1OnlyStokesLinesAreUsuallyMeasuredExperimentallyUseFigure9.1tohelpexplainthestatementinthetitleoftheproblem.SolutionAnti-Stokeslinesinvolvetransitionsfromexcitedvibrationalstatesofthesamples.Thepopulationsofthesestatesareverymuchlessthanthepopulationofthegroundstate(seeChapter2).Fewermoleculesarethereforeavailabletogiveupaquantumofvibrationalenergy;thus，itiscustomarytomeasuretheStokeslinesinRamanexperiments.從光的波動(dòng)性來(lái)分析由于光同時(shí)具有波動(dòng)性，因此也可以從光的波動(dòng)性分析拉曼散射的產(chǎn)生：電磁波的交變電場(chǎng)可以用E=E0cos(2't)表示，其中E是任意時(shí)刻t的電場(chǎng)強(qiáng)度，E0為交變電場(chǎng)的振幅，'為頻率。樣品分子的電荷分布在交變電場(chǎng)的作用下會(huì)發(fā)生變形，其正電荷和負(fù)電荷的中心會(huì)發(fā)生位置上的相對(duì)移動(dòng)或分離，產(chǎn)生誘導(dǎo)偶極矩μ，μ=E，其中E為入射光的交變電場(chǎng)強(qiáng)度，是分子的極化率(polarizability)。分子極化率是衡量分子在電場(chǎng)作用下電荷分布發(fā)生改變的難易程度或誘導(dǎo)偶極矩(induceddipolemoment)的大小，也就是單位電場(chǎng)強(qiáng)度誘導(dǎo)產(chǎn)生的偶極矩的大小。如果分子的振動(dòng)引起分子極化率的改變，則分子具有拉曼活性。以雙原子分子為例，設(shè)分子極化率隨分子振動(dòng)而變化，則可按臺(tái)勞級(jí)數(shù)展開(kāi)。忽略高次項(xiàng)，可得到：=0+(d/dq)0q式中0是分子在平衡位置時(shí)的極化率，q=rre，是雙原子分子核間距r與平衡位置時(shí)核間距r0的差。(d/dq)0表示平衡位置上對(duì)q的導(dǎo)數(shù)。由=E=[0+(d/dq)0q]E0cos(2't)根據(jù)前面紅外原理中所推得的方程d2q/dt2=kq/的解q=q0cos2t可以有=[0+(d/dq)0q0cos2t]E0cos2't=0E0cos2't+q0E0(d/dq)0cos2tcos2't=0E0cos2't+(1/2)q0E0(d/dq)0[cos2('+)t+cos2(')t]式中前一項(xiàng)0E0cos2't對(duì)應(yīng)于樣品分子產(chǎn)生的波長(zhǎng)未變化的散射即瑞利散射，第二項(xiàng)反映分子極化率隨分子振動(dòng)而改變(即(d/dq)0不為零)時(shí)分子產(chǎn)生的與入射光頻率不同的散射光。散射光與入射光頻率的差值即分子的振動(dòng)頻率，這就是拉曼散射。紅外吸收的光頻率是分子的振動(dòng)頻率，拉曼散射光與入射光的頻率差也反映了分子振動(dòng)能級(jí)之間的差。6.2.3拉曼散射的選擇定則（參考書(shū)P242）外加交變電磁場(chǎng)作用于分子內(nèi)的原子核和核外電子，可以使分子電荷分布的形狀發(fā)生畸變，產(chǎn)生誘導(dǎo)偶極矩。極化率是分子在外加交變電磁場(chǎng)作用下產(chǎn)生誘導(dǎo)偶極矩大小的一種度量。極化率高，表明分子電荷分布容易發(fā)生變化。=E如果分子的振動(dòng)過(guò)程中分子極化率也發(fā)生變化，則分子能對(duì)電磁波產(chǎn)生拉曼散射，稱(chēng)分子有拉曼活性。有紅外活性的分子振動(dòng)過(guò)程中有偶極矩的變化，而有拉曼活性的分子振動(dòng)時(shí)伴隨著分子極化率的改變。因此，具有固有偶極矩的極化基團(tuán)，一般有明顯的紅外活性，而非極化基團(tuán)沒(méi)有明顯的紅外活性。拉曼光譜恰恰與紅外光譜具有互補(bǔ)性。凡是具有對(duì)稱(chēng)中心的分子或基團(tuán)，如果有紅外活性，則沒(méi)有拉曼活性;反之，如果沒(méi)有紅外活性，則拉曼活性比較明顯。(Thisisanotherimportantprincipleofvibrationalspectroscopy,theruleofmutualexclusion.Thisrulestatesthatforanymoleculecontainingatrueinversioncenterofsymmetry,theinfraredactivevibrationsareRamaninactiveandviceversa.)一般分子或基團(tuán)多數(shù)是沒(méi)有對(duì)稱(chēng)中心的，因而很多基團(tuán)常常同時(shí)具有紅外和拉曼活性。當(dāng)然，具體到某個(gè)基團(tuán)的某個(gè)振動(dòng)，紅外活性和拉曼活性強(qiáng)弱可能有所不同。有的基團(tuán)如乙烯分子的扭曲振動(dòng)，則既無(wú)紅外活性又無(wú)拉曼活性。下圖顯示了甲苯的紅外譜和拉曼譜?？梢钥吹剑涸谀承╊l率處兩者是吻合的，而在另一些頻率上，只有一種譜上有峰。

FIGURE1.12.ComparisonofIR(top)andRaman(bottom)spectraoftoluene.SomelinesappearatthesamefrequencyinboththeIRandtheRamanspectrum.However,somelinesshowintheIRbutnotintheRamanspectrum.TheintensitiesoftheIRlinesarcdifferentfromthoseoftheRamanlines,althoughmanyofthemappearatthesamefrequency.Thisreflectsthedifferenceinselectivityoftwofundamentallydifferentprocesses.例.包含兩個(gè)相同原子的雙原子分子的紅外活性和拉曼活性如何?(參考書(shū)P242例9.2)答：具有紅外活性的分子振動(dòng)必須引起分子固有偶極矩的變化。對(duì)于含兩個(gè)相同原子的分子來(lái)說(shuō)，由于它沒(méi)有固有偶極矩，因此這個(gè)振動(dòng)不可能發(fā)生固有偶極矩的變化，這個(gè)振動(dòng)沒(méi)有紅外活性。但是在分子振動(dòng)過(guò)程中，分子會(huì)變形，這就會(huì)引起電子相對(duì)于核的分布的變化。分子的極化率會(huì)反映這種變形，因而在分子振動(dòng)中極化率會(huì)發(fā)生變化，這種振動(dòng)因此有拉曼活性。例.CO2的拉曼光譜(參考書(shū)P242例9.3)。Figure9.2CO2有四個(gè)基本的振動(dòng)模式，但其中只有一個(gè)有拉曼活性，為什麼?答：二氧化碳的對(duì)稱(chēng)伸縮振動(dòng)改變了核和電子的相對(duì)位置，因而分子的極化率也發(fā)生改變，分子具有拉曼活性。在不對(duì)稱(chēng)伸縮振動(dòng)中，一個(gè)鍵的伸展效果被另一個(gè)鍵的收縮抵消了，因此極化率總體上沒(méi)有變化。另外兩種振動(dòng)模式也是如此。這里要注意的是：具有拉曼活性的振動(dòng)是無(wú)紅外活性的。事實(shí)上，如果一個(gè)分子具有對(duì)稱(chēng)中心，則具有紅外活性的振動(dòng)沒(méi)有拉曼活性。反之，具有拉曼活性的振動(dòng)沒(méi)有紅外活性。6.2拉曼譜的量測(cè)及參數(shù)RAMANINSTRUMENTATION6.2.1拉曼譜儀：TypesofInstrumentationandTrendsinthe1990sRamaninstrumentationhasaninterestingheritage,andinsomewaysthetechniquehasseenmorechanges,andmorediversitythananyotheranalyticaltechnique.Thismaybeduetotwoaspectsofitsevolutiononetechnicalandtheotherperceptionversusacceptance.TheRamaneffectisextremelyweak,andRamanspectroscopyhasobviouslygainedovertheyearsbytechnologythatimprovessignalanddetectability.Notallthesetechnological"breakthroughs"followthesamedevelopmentaltrail.Secondly,thetechniqueisinsomewaystoocloselyrelatedtoinfraredspectroscopy,andistreatedasapoorrelative.Ramanspectroscopy,inpracticaltermsandforspecificapplications,canbedemonstratedtohaveconsiderableadvantagesoverinfraredspectroscopy.However,asageneralanalyticaltechnique,itisdifficulttodemonstratethatitoffersanynetadvantages.And,ithasbeenseentohavesomecleardisadvantages,inparticularthecommoninterferencefrombroad-bandsamplefluorescence,whichcantotallymaskthespectrum.Consequently,whenpeoplejustifythepurchaseofnewlaboratoryinstrumentation,thesafedecisiontendstobeinfraredspectroscopy,whichiswellestablished,andhasevolvedconsistentlyoverthepast40+years.WithRaman,thereislesspracticalhistory,andtheinstrumentplatformsareconstantlychanging.Today,thereareacoupleoftechnologychoiceswhicharenotnecessarilymutuallyexclusivethereareprosandconstotheselection.Whenitcomestoaspecificapplication,however,itcanbeeasytodemonstratewhetherRamanorinfraredisthebetterchoice.TherearecurrentlytwomaintechnologicalapproachestothedesignofRamaninstrumentationamonochromatororspectrograph-basedCCDsystemandFT-Raman.Asmentionedabove,becauseofpracticalandtechnicalconstraints,thesetwoapproachesdonotnecessarilyproduceexactlythesameendresult.Qualitatively,bothgeneratethesamefundamentalspectrumforagivenmaterial,however,theoverallappearanceofthespectrumortheimpactofthesample,maybedifferent.Thissectionwillprovideanoverviewofinstrumentation asitexistsinthe1990s,anditwillprovideageneraldiscussionofthetrendsandapplications.ThegreatesttechnologicaldifficultyforRamanspectroscopyhasbeentheweaknessoftheRamanspectralsignal,comparedtothemagnitudeofthemainexcitationwavelength.TheintensityofaparticularRamanlinecanbeintherange10-6to10-10ofthemainexcitationline(possiblyevenaslowas10-12).Theissuesare:howtomeasuresuchalowsignalinthepresenceofadominantsignal,howtoremoveeffectivelytheinterferencefromthedominantsignal,and,ifpossiblehowtoenhancetheweakersignal.Oneapproachtoincreasingtheabsoluteintensityofthesignalistoincreasethepowerofthesourcethelaserpower.Whilethismaybepossible,itdoesnotremovethefundamentaldynamicrangeproblem,ortheinterferenceproblem,whichonlybecomesworsewithincreasedlaserpower.Foryears,thetraditionalRamaninstrumentsfeaturedscanningmonochromators.Theimportantcriteriaforthemonochromatordesignweretominimizestraylight,toenabletheveryweaksignalstobemeasured,andtodesignformaximumRayleighlinerejection.Theoriginalsolutionwastousemorethanonemonochromator,withcommercialsystemsbeingbasedondoubleandtriplemonochromators.Whilethesehadthedesiredopticalproperties,theuseofuptothreemonochromatorsmadetheseinstrumentsmechanicallycomplex,andseverelylimitedtheopticalthroughputoftheinstrument(downto5%orlessoverallefficiency).典型的拉曼譜儀裝置圖如下圖所示。(A.T.Tu,FIGURE2.5.)EssentialpartsofaRamanspectrometer.Normallythescatteredlightat90isexamined.(instfig.pcx)由于散射效率大約為10-6~10-7，所以通常使用激光器作為光源，通過(guò)濾片和聚焦鏡投射到樣品上。這時(shí)光向各個(gè)方向散射。散射光包括瑞利散射(彈性散射)和拉曼散射(非彈性散射)。彈性光散射強(qiáng)度比拉曼散射高出103以上，所以色散系統(tǒng)必須精心設(shè)計(jì)，消除種種雜散光。為了達(dá)到高分辨率，一般采用雙聯(lián)或三聯(lián)單色儀作為分光系統(tǒng)。一般在與入射光成90度的方向上接收散射光，采用光電倍增管作為接收器，然后經(jīng)過(guò)信號(hào)處理電子系統(tǒng)和計(jì)算機(jī)，從顯示屏或記錄儀輸出。輸出參數(shù)為各個(gè)波數(shù)上的散射光絕對(duì)值，散射光波數(shù)的絕對(duì)值和拉曼波數(shù)(即入射光與散射光的波數(shù)差)，可以直接顯示在屏幕上。Withtheintroductionoflaserlinerejectionfilters,andinparticulartheholographicnotchfilters,theneedforthesecondandthirdmonochromatorswaseffectivelyeliminated.Asidebenefitoftheuseoftheserejectionfiltersandtheeliminationoftheadditionalmonochromators,wastheassociatedreductionininstrumentsize. Thenextsignificanttechnologyboostwasprovidedbythemovetowardsdetectorarraysinitiallywithintensifiedphotodiodearrays,andmorerecentlywiththecooledCCD(charge-coupleddevice)arrays.Withsuchdevices,theneedtoscanthemonochromatormechanicallyisremoved.Theresultisahighefficiencyspectrographicsystemwithnomovingparts.Today,thelimitationsofthetechnologyarethecostofhigh-performancespectroscopicCCDarraycameras,andtheoverheadassociatedwithcooling.However,withmajoradvancesinimagingtechnologiesinthe1990stherehasbeenacorrespondingexpansioninCCDtechnology.Asaresult,itisanticipatedthatCCDdeviceswithimprovedperformanceandlowercostswillcontinuetobecomeavailable.ThetradesthathavetobemadewithaCCDdevicearespectralrangeversusspectralbandwidth,andtheimpactofthesignalcut-offbetween1000and1100nmforsilicon.Theseissueswillbediscussedingreaterdetail,later.WiththegainsinperformanceexperiencedwithFTIRinstrumentationcomparedtodispersiveinfraredinstruments,therehasbeenanaturaldesiretodeterminewhetherornotthesamelevelofperformancecanbeachievedforRamanspectroscopy.Originally,thisexperimentwasconsideredtobeimpracticalbecauseofnoiseconsiderationsandtheextraordinarilylargedynamicrangeinvolvedbetweentheexcitation(Rayleigh)lineandtheRamansignals.However,withtheadventofthelaserlinerejectionfiltersmentionedabove,HirschfeldandChasedemonstratedthefeasibilityofFT-Ramanspectroscopy.Originalexperimentswereperformedwiththe647.1nmlineofaKryptongaslaser,andwithasilicondetector.However,therealjustificationforthemovetoFT-Ramanspectroscopywastheabilitytousenear-infraredlasers.MovingfromvisibletoNIRexcitationhelpedtoremoveoneofthemajorinterferencesencounteredwithRamanspectroscopytheoccurrenceofbroadbandfluorescence.This,plustheexpectedgaininperformancefromthemultiplexadvantage,offeredbyFourierspectroscopy,madethetechniqueofFT-Ramananattractivealternativetotheconventionaldispersive-basedmethodsofmeasurement.Asecond,practicaladvantageforauseristhatRamancanbeperformedonanexistingFTIRinstrument,withoutsignificantredesign.Infact,mostofthemajorFTIRvendorsofferRamanasanaccessoryfortheirhigh-endinstruments.Inmostcases,the1064nmlineoftheNd:YAGlaser,operatingwithpowersofupto4W,areusedforexcitation.FollowingthesuccessoftheFT-Ramanaccessories,somededicatedFT-Ramansinstrumentswereproduced,withnotablegainsinperformancelinkedtotheoptimizationoftheopticalsystem.Inparticular,gainswereexperiencedbytheuseofhighreflectivityoptics,thereductioninthenumberofopticalelements,andtheuseofhighsensitivitydetectors,matchedtothelaserimage.Figure22SchematicdiagramofaFT-RamanSpectrometerOneofthemajorapplicationsofRamanspectroscopyhasbeenmicroscopy,withthebenefitsofthespatialresolutionofthelaserlightsource.Ramanmicroscopygainedpopularityinthemid1970sfromthepioneeringworkofDelhaye,etal.andbytheintroductionoftheMOLEbyInstrumentsSA.Later,followingthegaininpopularityofinfraredmicroscopy,withmicroscopeaccessoriesoptimizedforcommercialFTIRinstruments,aparallelimplementationwasmadeforFT-Raman.Likewise,commercialRamanmicroscopesareofferedeitherasaccessoriesorasdedicatedsystemsforusewithCCD-basedtechnology.AnimportantrecentdevelopmentisintheareaofRamanspectroscopicimagingmicroscopy,atwo-dimensionalexperiment,wheretheRamanspectrumisscannedwithanAOTFdevice,andthemainimageisgeneratedbyaCCDarray.Thistechnologyisexpectedtohavesignificantimpactonstudiesinmaterialsscience,inpolymerchemistry,andintheareaofbiologicalandmedicalresearch.OneofthemajorbenefitsofRamanspectroscopyisthefactthattheprimarymeasurementinvolvesvisibleorNIRradiation.Thispermitstheuseofconventionalglassorquartzopticsforimaging.Furthermore,itopensuptheopportunitytouseopticalfibersforremotesampling.Insuchanarrangement,asinglefiberisusedforthetransmissionoflaserradiationtothesample,andsecondfiber,oraseriesoffibers(Figure19),transmitstheRamanscatteredradiationbacktothespectrometer.Figure19SchematicdiagramofaRamanfiber-opticsamplingprobefeaturingafiber-opticBundleTheconstructionofthesample-lightinterfaceinthiscaseisveryimportanttomaximizethecouplingbetweenthelaserandthesample,andthesubsequentcollectionofthescatteredradiation.Silicafibersarenormallyused,whichcantransmitvisibleornearinfraredradiationoverrelativelylongdistanceswithoutsignificantlightloss.Usually,theRamanspectrumfromsilicaisveryweak,however,overthedistancecoveredbyopticalfibers,thecontributioncanbesignificant.Toovercomethisproblem,samplingprobesfeaturingopticalfilteringinthemeasurementheadareutilized.AnexampleofsuchaprobeheadisshowninFigure20.Figure20SchematicdiagramofaRamanfiber-opticsamplingprobefeaturingfilterelementsinthemeasurementhead(CourtesyofKaiserOpticalSystems,Inc.)Asnotedearlier,thereareseveralimportantareasofapplicationwhereRamanexcelsoverinfrared.Often,thesearebasedonpracticalissues,suchastheabilitytouseglassintheopticalsystem,thelowerRamanscatteringofwater,whichpermitsthestudyofaqueousmedia,andtheopportunitytohavenoncontactsampling.Asecondaryissueisthatunlikemid-infraredspectroscopy,thereisnointerferencefromatmosphericwatervapororcarbondioxide.This,coupledtothescalingdownininstrumentsize,theabilitytoperformremotemeasurements,andtheavailabilityofmechanicallysimpleinstruments,hasmadeRamanapracticaltoolforprocessapplications.Applicationshaverangedfromrawmaterialscreeningtoreactionmonitoring,withamajorfocusontheanalysisofpolymericproducts.6.2.2.拉曼譜參數(shù)：(參考書(shū)P243－245)拉曼譜的參數(shù)主要是譜峰的位置和強(qiáng)度。譜峰(譜線(xiàn))位置：峰位是樣品分子電子能級(jí)基態(tài)的振動(dòng)態(tài)性質(zhì)的一種反映。它是用入射光與散射光的波數(shù)差來(lái)表示的。峰位的移動(dòng)與激發(fā)光的頻率無(wú)關(guān)強(qiáng)度：Unlikethetraditionalinfraredmeasurement,theRamaneffectisanemissionphenomenon,andisnotconstrainedbythelawsofabsorption.TheintensityofarecordedspectralfeatureisalinearfunctionofthecontributionoftheRamanscatteringcenter,andtheintensityoftheincidentlightsource.Themeasuredintensityfunctionisnotconstantacrossaspectrum,andisconstrainedbytheresponseofthedetectorattheabsolutefrequency(wavelength)ofthepointofmeasurement.Also,Ramanscatteringisnotalineareffect,andvariesasafunctionof4,whereistheabsolutewavelengthofthescatteredradiation.WhileRamanintensitymaybeusedanalyticallytomeasuretheconcentrationofananalyte,itisnecessarytostandardizetheoutputintermsoftheincidentlightintensity,thedetectorresponseandtheRamanscatteringterm,bothasafunctionoftheabsolutemeasuredwavelength.拉曼散射強(qiáng)度與產(chǎn)生譜線(xiàn)的特定物質(zhì)的濃度有關(guān)，成正比例關(guān)系。而在紅外譜中，譜的強(qiáng)度與樣品濃度成指數(shù)關(guān)系。)樣品分子量也與拉曼散射有關(guān)，樣品分子量增加，拉曼散射強(qiáng)度一般也會(huì)增加。對(duì)于一定的樣品，強(qiáng)度I與入射光強(qiáng)度I0、散射光頻率s、分子極化率有如下關(guān)系：I=CI0s42這里C是一個(gè)常數(shù)。在共振拉曼譜中，譜的加強(qiáng)是由于極化率的增加引起的。退偏比(depolarizationratio)如參考書(shū)P242中所說(shuō)，一個(gè)分子的電荷在某一個(gè)方向上可能比在另一個(gè)方向上容易變形，稱(chēng)這種分子的極化率各向異性；相反，則稱(chēng)為各向同性。把一個(gè)朝向與偏振光平行的偏振片放在偏振光的路徑上，則偏振光可以通過(guò)偏振片。若偏振片轉(zhuǎn)動(dòng)90度，則偏振光不能通過(guò)偏振片。如果一個(gè)分子位于原點(diǎn)0，對(duì)入射光進(jìn)行散射。下圖只表示了沿Y方向散射的射向觀測(cè)者的光。對(duì)于高度對(duì)稱(chēng)的分子如CH4和SF6而言，極化率是各向同性的。當(dāng)這類(lèi)分子的完全對(duì)稱(chēng)的振動(dòng)模式(例如CH4中C-H鍵的伸縮振動(dòng))與在XZ平面上偏振的入射光相互作用時(shí)，散射光將在YZ平面上偏振。這樣，當(dāng)一個(gè)偏振片平行于YZ平面放置時(shí)，只有這個(gè)平面上的散射光能夠通過(guò)。這部分光的強(qiáng)度稱(chēng)為I∥。若偏振片轉(zhuǎn)動(dòng)90度，則YZ平面上的光將無(wú)法通過(guò)。偏振片處于這個(gè)位置時(shí)測(cè)得的散射光強(qiáng)度稱(chēng)為I⊥。=I⊥/I∥定義為退偏比。對(duì)于CH4的對(duì)稱(chēng)C-H伸縮振動(dòng)來(lái)說(shuō)，退偏比=0。

(A.T.TuFIGURE1.32.)Theintensityofscatteredtightcanbemeasuredtwodifferentways.(A)Lightcomesthroughaparallel-orientedpolarizerandisparalleltotheincidentlight(I).(B)Lightcomesthroughaperpendicularlyorientedpolarizerandisperpendiculartotheincidentlight(I).TheratioofItoIiscalledthedepolarizationratio,anditisrelatedtosymmetryofvibrationalmodes.大多數(shù)分子的對(duì)稱(chēng)程度比CH4或SF6小，因此，極化率是各向異性的。一般來(lái)說(shuō)，分子散射的光在XZ和ZY平面上都有偏振。在平行于入射光偏振方向的方向上(如圖中的Z方向)，測(cè)到的散射光強(qiáng)度與垂直于入射光偏振方向的方向(如圖中所示的X方向)上測(cè)得的強(qiáng)度是不同的，但其比值一般不為零。 如果入射光是平面偏振光，則退偏比=I⊥/I∥=32a/(45i+42a)其中i是極化率的各向同性部分，a是極化率的各向異性部分，I⊥是垂直于入射光的方向上偏振的散射光強(qiáng)度，I∥是平行于入射光的方向上偏振的散射光強(qiáng)度。 對(duì)于平面偏振光來(lái)說(shuō)，退偏比與振動(dòng)的不對(duì)稱(chēng)程度有關(guān)，其值在0到3/4之間。任何分子的不完全對(duì)稱(chēng)的振動(dòng)，其退偏比為3/4(i=0)。對(duì)于完全對(duì)稱(chēng)的振動(dòng)，≤3/4。上面提到的CH4的例子中，退偏比為。但一般來(lái)說(shuō)，值在0到3/4之間，其大小取決于分子極化性質(zhì)的變化和分子鍵的對(duì)稱(chēng)性。一個(gè)完全對(duì)稱(chēng)的振動(dòng)在進(jìn)行任何對(duì)稱(chēng)操作后不變，這些對(duì)稱(chēng)操作只是交換了分子中對(duì)等原子的平衡位置。因此，退偏比的量測(cè)可以提供有關(guān)分子對(duì)稱(chēng)性的信息，而且有助于拉曼譜線(xiàn)的指認(rèn)。6.2.3增強(qiáng)拉曼光譜：TherearesomeenhancedRamanmethods,whichforsomecompoundsproducesignificantlyintensifiedRamanspectra,andwhichovercomesomeaspectsofthisdichotomy.TwosuchtechniquesareresonanceRaman（參考書(shū)P243）andsurface-enhancedRamanspectroscopy(SERS).OneotherRaman-basedtechniqueworthyofmentioniscoherentanti-StokesRamanspectroscopy(CARS)furtherdiscussionofthistechniqueisbeyondthescopeofthisbook.ResonanceRamanisparticularlyinterestingbecauseitcanturnRamanintoahighlyspecificprobeforcertainfunctionalgroupsorchemicalsites.ResonanceRamanoccurswhenthelaserexcitationfrequencycoincideswithanelectronicabsorptionband.Inthiscase,thevibrationsassociatedwiththeabsorbingchromophoreareenhancedbyasmuchas103to106timesthenormalRamanintensity.TheseintensifiedRamanlinesarelinkedtothespecificchromophoresiteandfunctionalgroupsorsites,withinthemolecule,thatinteractwiththechromophoricgroup.TheearlyresonanceRamanexperimentswerewiththevisiblelinesoftheargonionlaser,andthisobviouslyconstrainedthetechniquetoalimitedsetofcoloredcompounds.Ofthese,theworkwiththehemechromophoreofthehemeglobinmoleculewerethemostsignificant.Itispossibletoobservetheinfluenceofexternalmolecularligands,suchasoxygen,carbonmonoxideandcyanide,onthecriticalhemesite,freeofinterferencefromtheremainingoftheproteinstructures.Movingtoshorterwavelengthsfromthevisibletowards,andintotheultravioletregionsmight,atfirstsite,seemtobeimpracticalbecauseonenormallyequateshighlevelsofnativefluorescencewiththeuseofUVexcitation.However,manycompoundsabsorbintheultravioletspectralregion,andsothereisahighprobabilityfortheresonanceRamaneffecttooccur.Also,ithasbeenobservedthatbelow260nmexcitationthatthereisvirtuallynointerferencefromfluorescence.Oneofthemainissuesherehasbeentheappropriateselectionofa laseroperatingintheUVrange.OneapproachistouseadyelaserpumpedbyaNd:YAGoraXeCIexcimerlaser,coupledtofrequencydoublingandtriplingcrystalstoprovideawavelengthselectablerangeof200to750nm(Nd:YAG)and206-950nm(excimer).Boththeselasersystemsprovideapulsedlaseroutput.Apracticalalternative,wherecontinuouswavelengthtuningisnotarequirement,isanintracavityfrequencydoubledargonionlaserwhichprovidescontinuousoutputoffiveexcitationlinesinrange230nmto260nm.當(dāng)入射光引起分子中電荷的平移時(shí)，則發(fā)生散射。電荷的平移通過(guò)分子極化率反映出來(lái)。散射的強(qiáng)度與極化率的平方成正比。當(dāng)激發(fā)光的頻率接近且小于兩個(gè)電子能級(jí)之間的頻率差時(shí)，則產(chǎn)生所謂的preresonance，當(dāng)激發(fā)光的頻率等于兩個(gè)電子能級(jí)之間的頻率差時(shí)，則會(huì)發(fā)生共振，這時(shí)產(chǎn)生很大的電子電荷頻移或分子變形的概率很高，這時(shí)就會(huì)產(chǎn)生對(duì)光的吸收。這也說(shuō)明對(duì)于某些振動(dòng)，當(dāng)入射光接近或等于某個(gè)吸收躍遷頻率時(shí)，極化率會(huì)變得比較大。這種振動(dòng)稱(chēng)為共振加強(qiáng)的(resonanceenhanced)振動(dòng)。這種加強(qiáng)取決于電子躍遷的強(qiáng)度及振動(dòng)的對(duì)稱(chēng)性。如果只有一個(gè)單一的電子態(tài)，則振動(dòng)加強(qiáng)必須是對(duì)稱(chēng)的。即這種振動(dòng)不能改變分子的對(duì)稱(chēng)性。如果某個(gè)被激發(fā)的生色團(tuán)有不止一個(gè)電子躍遷，則振動(dòng)的對(duì)稱(chēng)性就不那么重要了。共振拉曼譜典型的增強(qiáng)倍數(shù)是102~103，因此共振拉曼譜在10-4moldm-3或更低的樣品濃度下即可測(cè)得。這樣，共振拉曼譜就提供了一種以接近紫外光譜的靈敏度選擇性地探測(cè)生色團(tuán)振動(dòng)頻率的手段。共振拉曼在研究生物大分子的結(jié)構(gòu)和功能時(shí)很有用，多生物分子都含有能給出共振拉曼譜的基團(tuán)，如類(lèi)胡蘿卜素、黃素、視紫紅質(zhì)、各種含銅與鐵的化合物、葉綠素等。共振拉曼譜儀的使用范圍主要受到激光光源頻率有限這一現(xiàn)實(shí)情況的限制。 Asnoted,anothertechniquethatprovidesanenhancedRamanspectral outputisSERSsurface-enhancedRamanspectroscopy.UnlikeresonanceRaman,thelaserwavelengthisnotimportant,unlessaqueous-basedmeasurementsaremadewithnear-infraredexcitation.Asthenameimplies,themeasurementisspecificinnature,andsomewhatlimitedinapplicationtosurfacesorinterfaces.MoststudiesofSERShavebeenperformedonelectrodesurfaces.Ansignal enhancement,intheorderof106isobservedforadsorbedspeciesoncertainmetallicelectrodesurfaces,notablymetalssuchasgold,silver,platinumandto someextentcopper.Theoriginalexperimentsinvolvedcalomel(Hg2C12)ona mercurysurface,andlaterworkinvolvedorganics,suchaspyridineadsorbedonroughenedsilverelectrodesurfaces.Theenhancementphenomenonisnotrestrictedtoelectrodes,andsimilareffectshavebeenobservedforothersubstratesinvolvingmetals,suchascolloidalsuspensionsofmetalsandmetalsdepositedorembeddedinoxides.Acriticalfactorinalltheexperimentsisthesurfaceroughness,whichisnominallyattheatomicscale.Theoriginoftheenhancementisbelievedtobeassociatedwithanincreasedelectricfieldintheregionofthemoleculeunderstudy.Althoughexperimentsinvolvingchargedmetalsurfaces mightappeartobelimited,thephenomenondoesopenupinterestingapplications intheareaofmetalliccorrosion,thestudyofbatteriesmaterials,andnew electrolyticstudiesinthecontestedareaofcoldfusion.6.3拉曼光譜的特點(diǎn)和應(yīng)用6.3.1.優(yōu)缺點(diǎn)：AdvantagesandDisadvantagesofVibrationalSpectroscopyRamanandinfraredspectroscopyforproteinandnucleicacidstructureanalysishavethefollowingnotableadvantages:1.Ramanandinfraredspectroscopyarenondestructivetechniques.Ordinarilythesamplemayberecoveredandassayedforbiologicalactivityafterspectroscopicexamination.2.Ramanandinfraredmethodsareapplicabletosamplesofvirtuallyanymorphologicalform.Forproteinsandnucleicacids,thisincludessolutions(aqueousandnonaqueous),suspensions,precipitates,gels,films,fibers,singlecrystals,andpolycrystallineandamorphoussolids.Dataobtainedfromagivensampleinonemorphologicalstatearegenerallytransferabletoanothermorphologicalstateofthesamesample.Thishasimportantpracticalbenefitsforexample,incomparingthemolecularstructureofaproteininthecrystalwiththatprevailinginsolution.3.Asmallsamplevolumeisrequiredforthesemethods.Approximately1lissufficientforconventionalRamanspectroscopyandapproximately10lforFouriertransforminfraredspectroscopy.Thisrepresentsanadvantageovermanyotherstructuralmethods,includingX-raycrystallographyandmagneticresonancespectroscopy.4.Ramanscatteringandinfraredabsorptionprocessesoccuronatimescalethatisveryshort(1015sec)incomparisontothetimescalesoffluorescence(>109sec)andnuclearmagneticresonancephenomena((106sec).Thus,vibrationalspectroscopyissuitablefortime-resolvedstudiesofbiologicalprocessesthatareinaccessiblebyfluorescenceandmagneticresonancemethods.5.ThereexistsalargedatabaseofinfraredandRamanspectraofproteins,nucleicacids,andtheirconstituents,forwhichreliablebandassignments,normalmodeanalyses,andspectra-structurecorrelationshavebeenmade.Thisfacilitatesinterpretationoftheoftencomplexvibrationalspectraobtainedfromproteins,nucleicacids,andtheircomplexes.ThefollowingadvantagesarespecifictoRamanspectroscopy:1.BothH2OandD2OgenerateveryweakRamanscattering,thusproducingrelativelylittleinterferencewiththeRamanspectrumofthedissolvedsolute.Thisconstitutesasignificantad