uvvisiblespectroscopyppt：uvvisiblespectroscopyppt.ppt

uvvisiblespectroscopy dr parasshahm pharm q a whatisspectroscopy spectrum scopies whenabeamoflightisallowedtopassthroughaprismorgrating itwilldispersedintosevencolorsfromredtovioletandthesetofcolorsorbandproducediscalledspectrum examination spectroscopyisthebranchofthesciencedealtwiththestudyofinteractionofelectromagneticradiation emr withmatter sothespectroscopymeansexaminationofspectrum fromthetypeofradiation whichisabsorbed wecangetideaaboutthenature type ofthecompoundandfromtheamountoftheradiation whichisabsorbed wecangetideaabouttheconcentration amount ofthesubstance sothespectroscopyisusedforqualitativeandquantitativeanalysis whenabeamoflightispassedthroughatransparentcellcontainingasolutionofanabsorbingsubstance reductionoftheintensityofthelightmayoccur thisisdueto1 reflectionattheinnerandoutersurfacesofthecell2 scatteredbytheparticlespresentinthesolution3 absorptionofthelightbythemoleculesinthesolution interactionofemrwithmatter classificationofspectroscopy 1 absorptionspectroscopy thetypeandamountoftheradiation whichisabsorbeddependuponthestructureofthemoleculesandthenumbersofmoleculesinteractingwiththeradiation thestudyofthesedependenciesiscalledabsorptionspectroscopy uv ir nmr x ray esr 2 emissionspectroscopy ifsufficientenergygetsimpingeduponasample theouterelectronsinthespecieswillberaisedfromtheirstablegroundstatetohigherenergylevel unstableinnature theseexcitedspeciesrapidlyemitsaphotonandreturntotheirgroundstableenergylevel thetypeandamountofradiation whichisemitted isstudied thistypeofspectroscopyiscalledemissionspectroscopy aes mes fluorimetry 3 scatteringspectroscopy iftheincomingradiationstrikeswiththesolidparticlessuspendedinthesolution thelighttransmittedatanangleotherthan1800fromtheincidentlight thisspectroscopyiscalledscatteringspectroscopy turbidimetry nephelometry whatisemr emrisaformofenergythatistransmittedthroughspaceatanenormousvelocity itcantravelinspacewiththesamespeedatthatoflight asthenameimpliesanemrisanalternatingelectricalandassociatedmagneticforcefieldinspace itcontainselectricalandmagneticcomponents thetwocomponentsoscillateinplanesperpendiculartoeachotherandperpendiculartothedirectionofpropagationoftheradiation emrconsistofastreamofdiscretepackets particles ofpureenergy whichiscalledphotonsorquanta theenergyofphotonisproportionaltothefrequencye h wheree energyofphotons h plank sconstant 6 624x10 27erg sec and frequencyofradiationincycles second wavelength itisthedistancebetweentwosuccessivemaximaonanelectromagneticwave m cm mm m nm anda0 frequency isthenumbersofwavespassingthroughagivenpointinunittime t 1 sec 1 cycles second hertz fresnel wavenumbers isthenumbersofwavespercentimeterinvacuum cm 1 velocity v istheproductofwavelengthandfrequency x v cm sec m sec classificationofemr emrisarbitrarilyclassifiedintodifferentregionsaccordingtowavelength energyassociatedwiththemolecules 1 themoleculeasawholemaymovethisiscalledtranslationandtheenergyassociatewiththismovementiscalledtransnationalenergy etrans 2 thepartofthemolecules thatisatomorgroupsofatoms maymovewithrespecttoeachother thismotioniscalledvibrationandtheassociatedenergyiscalledvibrationalenergy evib 3 themoleculemayrotateaboutanaxis andsuchrotationischaracterizedbytherotationalenergy erot 4 besidesthesemodesofmovements themoleculepossessesanelectronicconfigurationandtheenergyassociatedwiththisconfigurationiscalledelectronicenergy eele etotal etrans evib erot eele theoreticalprinciplesifamoleculeisallowedtointeractwiththeemrofaproperfrequency theenergyofthemoleculeisraisedfromoneleveltoahigherone wesaythatabsorptionofradiationtakesplace inorderforabsorptiontooccur theenergydifferencebetweenthetwoenergylevelmustbeequaltotheenergyofthephotonabsorbede2 e1 h wheree1isenergyoflowerlevelande2istheenergyofupperlevel thisenergyjumpfromoneleveltoanotheriscalledtransition thegraphofthelightabsorptionagainstthefrequencyiscalledabsorptionspectra visibleandultravioletlightprovidesenoughenergyforelectronictransitionthereforcalledelectronicspectra onabsorptionofenergybyamoleculeintheultravioletregion changesareproducedintheelectronicenergyofthemoleculeduetotransitionsofvalenceelectronsinthemolecule e 6 anti bonding anti bonding nnon bonding bonding 6bonding typesoftransitions 1 66 atransitionsofelectronsfromabondingsigmaorbitaltothehigherenergyantibondingorbitals eg alkane sigmabondsare ingeneral verystrong thereforetheyrequirehighenergyforthetransitionsandthistransitionsrequiresveryshortwavelength nearabout150nm 2 n6 thistransitioninvolvessaturatedcompoundswithoneheteroatomwithunsharedpairofelectrons nelectrons correspondingbandappearsat180 200nm 3 thistransitionisavailableincompoundswithun saturation eg alkene correspondingbandappearsat170 190nm 4 n thistypeoftransitionsareshownbytheunsaturatedmoleculescontainingoneormoreheteroatoms o n s 5 conjugatedsystem inconjugateddienes the orbitalsoftheseparatealkenegroupcombinetogiveneworbitalsi e thetwonewbondingorbitalswhicharedesignated 1and 2andnewtwoanti bondingorbitalsdesignatedas 3 and 4 soforthe 2 3 transitionverylowenergyisrequirescorrespondingtothehigherwavelength someimportantterms 1 chromophore itisagroupofmolecules whichisresponsiblefortheabsorptionoflightbymolecules itisconjugateddienes itisminimumstructuralrequirementsfortheabsorptionofradiationinuvrange 2 auxochrome itisasaturatedgroupcontainingunsharedelectronswhichwhenattachedtoachromophorechangesbothintensityaswellasthewavelengthoftheabsorptionmaxima e g oh nh2 cletc 3 max itisawavelengthatwhichthereisamaximumabsorptionorabsorptionintensity itisaphysicalconstantandcharacteristicofstructureandsousefulforidentificationofcompounds itisindependentofconcentration 4 bathochromicshift theshiftingofabsorptiontoalongerwavelengthduetosubstitutionorsolventiscalledasbathochromicshift itisalsocalledasredshift e g maxofascorbicacid 243nm maxofascorbicacidinalkalimedium 299nm 5 hypsochromicshift blueshift shiftingof maxtolowervalueorlefthandsideduetosubstitution solvent phetciscalledashypsochromicshift e g maxofphenolinbasicmedia 297nm maxofphenolinacidicmedia 277nm 6 hyperchromism increaseinabsorptionintensity e duetosolvent phorsomeotherfactorscalledhyperchromiceffect 7 hypochromism decreaseinabsorptionintensityduetosubstituent solvent phetc calledhypochromiceffect 8 a1 1cm aonepercentonecentimeter istheabsorbanceofthesolutionhavingconcentration1gmper100mlofthesolution 9 molarabsorptivity istheabsorbanceofthesolutionhavingconcentrationgm mol weight 1000mlofthesolution a1 1cmxmol wt 10 10 transmittance t istheratioofit i0and transmittance t isgivenby t 100it i0 1 absorbance a isthedegreeofabsorptionoflightbyamediumthroughwhichtheenergypasses itisexpressedasthelogarithmoftheratiooflighttransmittedthroughapuresolventtotheintensityoflighttransmittedthroughthemedium itistheareaunderthecurve a logi0 ita logi0 logita 2 log tabsorptionspectrathegraphofthelightabsorptionagainstthefrequencyiscalledabsorptionspectra itischaracterizedby1 max positionofspectra2 intensityofabsorbance theamountoftheradiationabsorbedbythemolecule 1 factorsaffectingthepositionofthespectrum max a structuralfactorsi substitution placingasubstituentonachromophoremayproducechangein maxbytwomechanisms introductionofanentirelynewtransitionand orshiftingthewavelengthofexistingtransitions e g eachalkylsubstituentproduce5nmbathochromicshift ii solvent thesolventeffectarisesbecausesolvationisfrequentlydifferentforthegroundandexcitedstates ifthegroundstateissolvatedmorestronglythantheexcitedstate theenergydifferencebetweenthelevelsisincreased theincreaseinenergydifferenceisreflectedinashiftoftheabsorbancetoshorterwavelengths iii geometry e g stilbene trans stilbeneabsorbsatalongerwavelengththancis stilbeneduetostericeffects co planarityisneededforthemosteffectiveoverlapofthe orbitals thecis isomerisforcedintoanonplanarconformationduetostericeffects thecisisomeraretwistedslightlyoutofplanebystericinteractionssothatthedegreeofconjugationinthe systemisslightlylessthanthetransisomers resultingingreaterenergyforthetransitions a nonstructuralfactorsi ph e g phenolphthalein inalkalinemediumitispinkandintheacidicmediumitiscolorless i temperature temperatureprovidesmoreenergytogroundstate asaresultenergyrequiredforexcitationwillbeless sothereisbathochromicshift 2 factorsaffectingtheintensityofabsorptionofradiation i thicknessofthemedium lambert slaw whenabeamofmonochromaticlightisallowedtopassthroughatransparentmedium therateofdecreaseofintensitywiththethicknessofmediumisdirectlyproportionaltotheintensityofincidentradiation itgivesrelationshipbetweenabsorbanceandthethicknessofthemedium ii concentrationofabsorbingsolute beer slaw whenabeamofmonochromaticlightisallowedtopassthroughatransparentmedium therateofdecreaseofintensitywiththeconcentrationofabsorbingsoluteisdirectlyproportionaltotheintensityofincidentradiation itgivesrelationshipbetweenabsorbanceandtheconcentrationofthemedium a abc fundamentalequationsofspectroscopy deviationfromthebeer scurve errorsinspectrophotometricmeasurement errorsmayarisefrominstrumentaloffromchemicalfactors instrumentalerrorscanarisefromseveralsources noise fluctuationinlightsource theidealabsorbancerangeformostmeasurementisintherangeof0 2to0 8 thecalibrationcurveisrelativelylinearinthisrange otherfactorincludesspectralslitwidth ssw asslitwidthisincreased thefinestructureoftheabsorptionbandislostastheincidentlightisnomoremonochromatic generally fastscanratestendtodistortspectra alteringthepositionsofbothmaximaandminimaaswellasdiminishingpeakintensities thisintroducesbothqualitativeandquantitativeerrorsinthemeasurement anumbersofchemicalfactorsmayalsoproduceerrorsintheanalysis solute soluteinteraction e g aggregation precipitation dimerizationetc ionizationorevencomplexationoftheanalyteinsolutioncanalsoleadtoapparentdeviationfromthebeer scurve fluorescencefromabsorbingspeciesinsolutionmayalsocontributetointerference instrumentation lightsource slit monochromator sampleholder detector display lightsource sourceofelectromagneticradiation thetungstenfilamentlampisasatisfactorylightsourcefortheregion350to2000nm itconsistsofatungstenfilamentcontainedinaglassenvelope themostconvenientlightsourceforuvradiationisdischargelamp generallydeuteriumdischargelampisused itisconsistingofdeuterium filledsilicaenvelope itgivesradiationfrom185to380nm 2 slit radiationintensitycontrollingdevice enoughlightmustpassthroughthesampletoelicitameasurableresponsefromthedetector 3 monochromator wavelengthselectingdevice itconvertspolychromaticlightinmonochromaticlight lighthavingonewavelength a filters glassfiltersarepiecesofcoloredglass whichtransmitlimitedwavelengthrangesofthespectrum thecolorisproducedbyincorporatingoxidesofsuchmetals b prisms whenabeamoflightpassesthroughaprism itisbentorrefracted theamountofdeviationisdependentonthewavelength theprismismadeupofquartzforuseintheuvlight sinceglassabsorbswavelengthsshorterthanabout330nm glassprismarepreferableforthevisibleregionofthespectrum asthedispersionismuchgreaterthanthatobtainedwithquartz c grating mostmodernuvspectrophotometerusesdiffractiongratingasamonochromator itconsistingofaverylargenumberofequispacedlines 200 2000permm ruledonaglassplate theycanbeusedeitherastransmissiongratingorwhenaluminized asreflectiongrating 4 sampleholder thesampleholderisknownascuvettes cuvettesmustbetransparenttothelight sotheglasscellsareusedinthevisibleregionandquartzorsilicacellsareusedintheuvregion thecellsusedintheuvspectrophotometersareusually1cminpathlengthbutcellsareavailablefrom0 1cmto10cmormore 5 detectors radiationmeasuringdevice itisalsoknownasphotocell theyconvertradiationenergyinelectricalenergy forthedeterminationofsubstancesbyspectrophotometrictechniques precisedeterminationsofthelightintensitiesarenecessary photoelectricdetectorsaremostfrequentlyusedforthispurpose theymustbeemployedinsuchawaythattheygivearesponselinearlyproportionaltothelightinputandtheymustnotsufferfromdriftorfatigue a barrier layerphotocell itoneofthesimplestdetectors whichhastheadvantagethatitrequiresnopowersupplybutgivesacurrent whichisdirectlyproportionaltothelightintensity itisconsistsofametallicplate usuallycopperoriron uponwhichisdepositedalayerofselenium anextremelythintransparentlayerofagoodconductingmetal e g silver platinumorcopper isformedovertheseleniumtoactasoneelectrode themetallicplateactingastheother lightpassesthroughthesemitransparentsilverlayercausesreleaseofanelectron whichmigrates tothecollector theelectronaccumulatingonthecollectorresultinginapotentialdifferencebetweenthebaseandcollector whichcanbemeasuredbyalowresistancegalvanometercircuit theusefulworkingrangeofseleniumphotocellis380 780nm theirlackofsensitivitycomparedtophototubeandphotomultipliertube restrictstheirusetothecheapestcolorimetersandflamephotometers itconsistsofananodeandacathodesealedinanevacuatedglasstube whichmayhaveaquartzorsilicawindowforuvmeasurement b photoemissivetube thecathodeiscoatedwithalayeroflightsensitivematerialthatemitselectronsuponabsorptionofphotons apowersupplymaintainstheanodepositivewithrespecttothecathodesothatthephotoelectronsarecollectedattheanode thiscurrentisdirectlyproportionaltothelightintensity phototubesareavailableforuseovertheentireuv visibleregionofthespectrum butnosingletubecoverstheentirerangesatisfactorily thereforemanyinstrumentswithphototubedetectorsemployinterchangeableblueandredsensitivephototubeinordertoprovidesufficientsensitivityovertheentirespectrum c photomultipliertube itisverysensitivedetectorswithveryshortresponsetimes itcontainsaphotocathodeandaseriesofdynodes whicharealsophotosensitive ahighersuccessivepotentialismaintainedbetweeneachdynodes aphotoelectronsreleasedfromthephotocathodeisacceleratedtowardthefirstdynodebytheirvoltagedifference whereitstrikestoreleaseseveralelectrons thesecondaryelectronsarethenacceleratedtowardtheseconddynodewheretheprocessrepeats inthiswaymultiplicationoftheelectronscanbeachieved thecurrentfromphototubesandphotomultipliertubesneverfallstozero asmallresidualcurrentcalleddarkcurrentisproduced duetolongexposureofthelight 6 display readoutmeter thesignalfromthedetectorisnormallyproportionaltotheintensityoflight andafteramplificationmaybedisplayedas torafterpassingthroughalogarithmicconversioncircuitasabsorbance log1 t typesofinstruments instrumentsformeasuringtheabsorptionoflightmaybeofthesinglebeamordoublebeamtype inasinglebeaminstrument lightfromthesourcespassesthroughafilterandthenthroughthesampleandintothedetector thesignalfromthedetectorisproportionaltotheintensityofthelightbeamstrikingit tomakeameasurementofabsorbanceusingamanuallycontrolledsingle beaminstrument themonochromatorisadjustedtotherequiredwavelengthandtheappropriatelampandphotocellareselectedbymeansofswitches thefirststepistocloseashutterinthepath oradjustdarkfilter andadjust0 t thesecondstepistoopentheshutterandplacethecellcontainingonlythesolventinthelightbeamandadjustthescaleon100 t equivalentto0absorbance thethirdstepistoplacethesamplecellinthelightpathandmeasuretheintensityitoritsequivalentabsorbance indoublebeamspectrophotometer themonochromaticlightissplitbythebeamsplitterintotwoequalintensitylightbeam whicharedirectedalternativelyinrapidsuccessionthroughacellcontainingthesampleandonecontainingthesolventonly thisinstrumentmeasurestheratiooftheintensityofthebeamcomingthroughthesampleandthroughthesolvent changesintheintensityofthesourceaffectbothbeamsproportionatelysotheratiooftheirintensitiesisnotaltered therefore ahighdegreeofstabilityinthelightsourceisnotrequiredintheseinstruments differenceinthelampoutput opticalsystemthroughput anddetectorsensitivitywithwavelengthalsoaffectbothbeamsinthesameway applications 1 qualitativeanalysis theuvspectraofmostcompoundsareoflimitedvalueforqualitativeanalysisascomparedtoirandmassspectra qualitativeanalyticaluseofuvspectrahaslargelyinvolved maxandabsorptivities occasionallyincludesabsorptionminima inpharmacopoeias absorptionratioshavefounduseinidentitytests andarereferredtoasq valuesinusp 2 quantitativeanalysis uvspectroscopyisperhapsthemostwidelyusedspectroscopictechniquesforthequantitativeanalysisofchemicalsubstancesaspurematerialsandascomponentsofdosageforms a singlecomponentanalysis directanalysis essentiallyallcompoundscontainingconjugateddoublebondoraromaticrings andmanyinorganicspeciesabsorblightintheuv visibleregions inthesetechniquesthesubstancetobedeterminedisdissolvedinsuitablesolventanddilutedtotherequiredconcentrationbyappropriatedilutionsandabsorbanceismeasured indirectanalysis analysisafteradditionofsomereagent indirectmethodsarebasedontheconversionoftheanalytebyachemicalreagentthathasdifferentspectralproperties chemicalderivatizationmaybeadoptedforanyoftheseveralreasons 1 iftheanalyteabsorbsweaklyintheuvregion 2 theinterferenceformirrelevantabsorptionmaybeavoidedbyconvertingtheanalytetoaderivative whichabsorbsinthevisibleregion whereirrelevantabsorptionisnegligible 3 thistechniquecanbeusedtoimprovetheselectivityoftheassayinpresenceofotheruvradiationabsorbingsubstance 4 cost methodsofcalculatingconcentrationinsinglecomponentanalysis byusingtherelationship a abc byusingtheformula cu au as xcs byusingtheequations y mx c byusingthebeer scurvea multicomponentanalysis a simultaneousequationsmethod ifasamplecontainstwoabsorbingdrugs xandy eachofwhichabsorbsatthe maxoftheother 1and 2 itmaybepossibletodetermineboththedrugsbythesimultaneousequationsmethod criteriaforobtainingmaximumprecision belowmentionedratioshouldlieoutsidetherange0 1 2 0 a2 a1 ax2 ax1 and ay2 ay1 a2 a1 theinformationrequiredis theabsorptivitiesofxat 1and 2 ax1andax2 theabsorptivitiesofyat 1and 2 ay1anday2 theabsorbancesofthedilutedsampleat 1and 2 a1anda2letcxandcybetheconcentrationofxandyrespectivelyinthesampletheabsorbanceofthemixtureisthesumoftheindividualabsorbancesofxandyat 1a1 ax1 cx ay1 cy 1 at 2a2 ax2 cx ay2 cy 2 multiplytheequation 1 withax2and 2 withax1a1ax2 ax1cxax2 ay1cyax2 3 a2ax1 ax2cxax1 ay2cyax1 4 a1ax2 a2ax1 ay1cyax2 ay2cyax1a1ax2 a2ax1 cy ay1ax2 ay2ax1 cy a1ax2 a2ax1 ay1ax2 ay2ax1 5 samewaywecanderivecx a2ay1 a1ay2 ay1ax2 ay2ax1 6