PowerWave200微孔板分光光度计：光谱扫描功能s

UseoftheSpectralScanningCapabilitiesofthePowerWave™200MicroplateSpectrophotometerSpectralanalysisofsamplescanbeusedforanumberofdifferentrequirements,includingcontaminantdetection,peakabsorbancedetermination,andproductformation.HerewedescribesomeoftheusesofthespectralscanningcapabilitiesofthePowerWave200scanningmicroplatespectrophotometerinconjunctionwithKC4datareductionsoftware.IntroductionThespectralanalysisofsampleshasanumberofdifferentpurposes.Themostobviousisthedeterminationofthewavelengthoflightthatisabsorbedmaximallybyaparticularsolution.Similarly,absorbanceprofilescanbedeterminedinordertoascertainwavelengths,whilenotnecessarilymaximal,thatprovideadequateabsorbanceforaccuratedetermination.Contaminantsinsamplescanbedetectedbythedeviationfromknownpatternsofspectralanalysis.Inmanycasesthecontaminantcanbedeterminedbythenewspectralscan.Newlysynthesizedorunknowncompoundscanalsobeidentifiedusingaspectralanalysisofthatcompoundinsolution.Herewedescribesomeoftheapplicationsforspectralanalysisofcompoundsinsolution.MaterialsandMethodsThe96wellclearUVtransparentmicroplates,cataloguenumber3635,werepurchasedfromCostar,(Cambridge,MA).EggshadecoloryellowfoodcoloringinsolutionwasobtainedfromNationalInstitutionalFoodDistributorAssociatesInc.(Atlanta,GA).Sodiumfluorescein,methylumbelliferone(7-hydroxy-4-methyl-coumarin),andpropidiumiodidewerepurchasedfromMolecularProbes(EugeneOR),andFD&CBlueNo.1andYellowNo.5werepurchasedfromWarnerJenkinson(St.LouisMO)aspowders.BioTekInstruments,Inc.,P.O.Box998,HighlandPark,Winooski,Vermont05404-0998USACOPYRIGHT©2006TEL:[phone]FAX:[phone]OutsidetheUSA:802-655-4740E-mail:[e-mail]Ô200.Sodiumfluoresceinandpropidiumiodidesampleswereblankedonwater,whilemethylumbelliferonewasblankedonmethanol.Scanningspectrophotometricmeasure-mentsweremadeusingaPowerWave200Ôscanningmicroplatespectrophotometer(Bio-TekInstrumentsWinooski,VT)inCostarUV-transparentmicroplates.DatawascapturedusingKC4datareductionsoftwaretocontrolreaderfunction(Bio-TekInstruments,Winooski,VT).Inordertocorrectforthedifferentbackgroundabsorbanceofthemicroplateatdifferentwavelengths,theabsorbancevalueswereexportedtoMicrosoftExcelÔandtheabsorbanceateachwavelengthofawaterblankwassubtractedfromtheexperimentaldata.Thus,sampleswereblankedwithwaterateachwavelength.PeakAbsorbanceDeterminationsTheabsorptionspectraofseveralfluorescentcompoundswereexamined.Stocksolutionsof,sodiumfluorescein,propidiumiodide,werepreparedbydissolvingthepowdersindistilledwater,whilemethylumbelliferonewasdissolvedinmethanol.Forabsorbancespectrumanalysis200mlofeachsolutionwasplacedinamicroplatewellandscannedfrom200nmto700nmin1nmincrements.AsdemonstratedbyFigure1,differentcompoundswillexhibitdifferentspectrumpatternsofabsorption.Sodiumfluoresceininaqueoussolutionexhibitsapeakinabsorbancecenteredat490nm.Propidiumiodidedemonstratesthreesuchpeaks:230nm,293nmand488nm,whilemethylumbelliferoneexhibitsanabsorbancepeakat321nm.SpectralscancomparisonPurifiedgenomicherringspermDNAwasdigestedwithEcoRI(Gibco-BRL,Gaithersburg,MD)followedbyorganicphenol/chloro-form/isoamylalcoholextractionandethanolprecipitation.Aconcentratedstocksolutionwasmadewithsubsequentrehydrationtoa5mg/mlfinalconcentration.Priortospectralanalysis,theDNAstocksolutionwasfurtherdilutedwithdistilledwaterto300mg/mland150mg/ml.Followingdilution,200mlofthe300mg/mlDNAsolutionwasplacedinamicroplatewellandscannedfrom225nmto325nmin1nmincrementsusingaPowerWave200scanningmicroplatespectrophotometer.ComparisondatawasobtainedusingaSpectronicPlus1001spectrophotometer(MiltonRoy,Rochester,NY)toscanthe150mg/mlDNAsolutionoverthesamewavelengthsin1nmincrements.Figure2.ComparisonofabsorbancespectrumsproducedbythePowerWavetoaconventionalspectrophotometer.DNAinsolutionwasscannedfrom225nmto325nmin1nmincrementsusingeitheraSpectronic1001spectrophotometer(MiltonRoyCo.Rochester,NY)oraPowerWave200microplatereader(Bio-TekInstruments,Winooski,VT).Note,theDNAconcentrationusedfordeterminationswiththespectrophotometerwasapproximatelyhalfthatusedfordeterminationsusingthePowerWave200.WhenthespectraforDNAinsolutionobtainedusingthePowerWave200iscomparedtothatobtainedfromaconventionalspectro-photometer,verysimilarpeaksinabsorbanceareobserved(Figure2).DifferencesinthelightpathlengthrequirethatdifferentconcentrationsofDNAsolutionbescannedforequivalentabsorbancecomparison.Becausethelightpathlengthinacuvetteisapproximatelytwotimesthatof200mlofsolutioninaflatbottommicroplatewell(1.0cmvs.0.55cm),theconcentrationusedwiththespectrophotometerwashalfthatusedforthemicroplatereader.Althoughslightdifferencesinthemagnitudeoftheabsorbancepeakcanstillbeobservedasaresultofincompletecompensationforlightpathlength,thepeakwavelength,aswellasthegeneralshapeofthecurvewereobservedtobethesame.EffectofconcentrationonabsorbancespectraInordert