液相各檢測(cè)器介紹

LIQUIDCHROMATOGRAPHYDETECTORSAnalytesthatelutefromacolumnneedtobedetectedtoObtainachromatogram.DetectorisacriticalcomponentOfanyLCsystem.Thefollowingtypesofdetectorsareused:UV-visibledetectorFluorescencedetectorElectricalconductivitydetectorRefractiveindexdetectorMassspectraldetectorUV-VISIBLEDETECTORSSINGLEWAVELENGTHDETECTORSinglewavelengthdetectorsconsistofthefollowingComponents:AlowpressuremercurydischargelampthatemitslightIntheUVandvisibleregions.2.Afocusingquartzlens.3.Acut-offfilter(broadbandorband-pass)thatallowsSpecificwavelengthrangestobetransmittedtotheSample.4.Referenceandsamplecellsofsmallvolume(microliters).5.PhotomultiplierorphotocellstomonitortheAbsorptionoflight.6.AbsorbanceiscalculatedfromBeer’slaw:

LogIT/IO=lc=AVARIABLEWAVELENGTHDETECTORVARIABLEWAVELENGTHDETECTOR(CONTD)Variablewavelengthdetectorconsistsofthefollowingcomponents:AdeuteriumandortungstenlamptocovertheuvandVisiblespectralranges.2.CollimatingmirrorsthatfocusthesourcebeamontoAdiffractiongrating.Thediffractiongratingresolvespolychromaticbeamintoitscomponentwavelengths.ThespectralresolutionisafunctionofthenumberofLinesinthegrating.ThisarrangementallowsaspecificWavelengthtobechosenfordetectingtheanalytes.3.AsmallvolumecellintowhichtheelutingmobilephaseIsfed.4.AlensinthefrontandbackofthecelltofocusthegivenWavelengthlightintothesampleandthetransmittedlightIntothephotomultiplier.FLUORESCENCEDETECTORFLUORESCENCEDETECTORAfluorescencedetectorhasthefollowingcomponents:Apolychromaticlightsourcesuchasasaxenonlamp.2.ThislightisfocusedontoadiffractiongratingwhichAllowstheexcitationwavelengthtobechosen.3.ThechosenwavelengthlightispassedthroughtheSampleandreferencecells.4.Thelightemittedfromthesamplecelliscollectedat90ototheincidentlight.5.ThisisincidentonasecondgratingbeforebeingsentTothephotomultiplierfordetection.6.TheexitgratingenablesdetectionatachosenemissionWavelength.7.FluorescenceisinherentlymoresensitivethanAbsorptionandhasalargerlinerdynamicrange.ELECTRICALCONDUCTIVITYDETECTORElectricalconductivitydetectorisusedinionChromatography.ItmeasurestheresistanceandhencetheconductivityofthesolutionfromwhichasignalisGeneratedfordetection.ELECTRICALCONDUCTIVITYDETECTORConductanceofagivensolutionisCandisgivenby\C=1/R=a/lWhereR=resistanceinohms

=specificconductancea=cross-sectionalareaoftheelectrodel=distancebetweentheelectrodesEquivalentconductanceisdefinedas

=1000/cWherecistheconcentrationoftheelectrolyteinGramequivalents/liter.C=1/R=ca/1000lResistanceisinverselyproportionaltotheconcentration.REFRACTIVEINDEXDETECTORREFRACTIVEINDEXDETECTORTherefractiveindexdetectoristhelastchoicedetector.Itistheleastsensitivesensitivedetector.ItissensitiveTochangesinambienttemperatureandpressureandFlowrate.ItcanonlybeusedwithisocraticelutionsAndisunsuitableforgradientelutions.Itconsistsofthefollowingcomponents:Alightsourcewhichisusuallytungstenlamp.2.AnopticalmaskconfinesthebeamtotheregionoftheCell.3.AlenscollimatesthelightbeamwhichthenpassesthroughboththesampleandreferencecellstoaplaneMirror.4.ThemirrorreflectsthebeambackthroughthesampleREFRACTIVEINDEXDETECTOR(CONTD)5.ThelocationofthebeamratherthanitsintensityisDeterminedbytheangulardeflectionofthebeamcausedBytherefractiveindexdifferencebetweenthecontentsOfthetwocells.6.AsthebeamchangesthepositionoffocusonthePhotoelectriccell,theoutputchangesandtheresultingDifferencesignaliselectronicallymodifiedtoprovideaSignalproportionaltotheconcentrationofsoluteintheSamplecell.REFRACTIVEINDEXDETECTOR(CONTD)IONCHROMATOGRAPHYIonChromatographyisbasedontheexchangeofionsofLikechargebetweenthesolutionandthestationaryphase.IONCHROMATOGRAPHY(CONTD)Theequilibriainvolvedincationandanionexchangechromatographyare:IONCHROMATOGRAPHY(CONTD)WhenIONCHROMATOGRAPHY(CONTD)KdecreasesintheorderforM+ionsTl+>Ag+>Cs+>Rb+>K+>NH4+>Na+>Li+TheorderforanionsisTheseordersarebasedontheionicradiiofthevariousions.IONCHROMATOGRAPHY(CONTD)SodiumDodecylSulfateOctyltrimethylammoniumBromideIONCHROMATOGRAPHY(CONTD)SIZEEXCLUSIONCHROMATOGRAPHYItisalsoknownasgelpermeationorgelfiltrationChromatography.AsthenameimpliestheseparationIsbasedonthesizeoftheanalytemolecules.TheStationaryphasecontainsfairlylarge~10muniformSiorpolymerparticles.AnalyteandsolventmoleculesDiffuseinandoutoftheporesoftheseparticles.RetentionoftheanalytedependsontheporesizeandtheSizeofthemolecule.ItisthusimportantthattheporeSizebeintherangeofdiametersofthemoleculesBeingseparated.IfthemoleculardiameterissignificantlySmallerthantheporediametertheywillsimplybeWashedoutunretained.SIZEEXCLUSIONCHROMATOGRAPHY(CONTD)SizeexclusionisoftenappliedtolargemoleculessuchAspolymers,proteinsetc.SIZEEXCLUSIONCHROMATOGRAPHY(CONTD)Thetotalvolumeofapackedsizeexclusioncolumnis

Vt

=Vg+Vi+Vo

Vt

=totalvolume;Vg=volumeoccupiedbythesolidmatrixofthegel(stationaryphase)Vi=volumeofthesolventheldintheporesVo=freevolumeoutsidethegelparticlesTheretentionvolumeformoleculestoolargetoenterthePores=Vo

RetentionvolumeformoleculestoosmalltoberetainedInthepores=Vi+Vo

SIZEEXCLUSIONCHROMATOGRAPHY(CONTD)MoleculesofintermediatesizethatareretainedinthePoressuchthat