HPLC的方法及原理ppt课件.ppt

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1、HighPerformanceLiquidChromatography(HPLC)20030623HPLC的分类HPLC系统HPLC理论公式HPLC方法开发HPLC的定性定量分析High-performanceLiquidChromatographyEarlyliquidchromatographywascarriedoutinglasscolumnswithdiametersof1to5cmandlengthsof50to500cm.Slowflowratesleadtoverylongse

2、parationtimes.Recently,columnefficiencyhasbeenincreasedbymakingthepackingmaterialsmallerandusingveryhighpressuretoforcethemobilephasethroughthecolumn.High-performanceliquidchromatographyisthegeneraltermusedtodistinguishthenewerproceduresfromthebasicm

3、ethods.AdvantagesofHPLCReadilyadaptabletoaccuratequantitativedeterminations.Suitableforseparatingnonvolatilespecies.Canseparatethermallyfragilespecies.Veryapplicabletoseparatingsubstancesofinteresttoindustry,science,andthegeneralpublic.AminoacidsProt

4、einsNucleicacidsHydrocarbonsCarbohydratesDrugsTerpenoidsPesticidesAntibioticsSteroidsMetal-organicspeciesAvarietyofinorganicsubstances.HPLC的分类吸附色谱adsorptionchromatography分配色谱partitionchromatography离子色谱ionchromatography分子排阻色谱sizeexclusionchromatograph

5、y亲和色谱affinitychromatography吸附色谱的分离机理随着样品在固定相上的吸附能力由大到小在色谱柱上的保留由长到短分配色谱的分离机理ChromatographyStationaryPhasesrelativelypolarsurfacebulk(SiO2)xsurfacerelativelynonpolarsurfaceSilicaGelbulk(SiO2)xsurfaceDerivatizedSilicaGelWhereR=C18H37hydrocarbonchain(oct

6、adecylsilylderiv.silicaor“C18”)“normalphase”“reversedphase”Normalvs.ReversedPhaseChromatography离子色谱离子交换色谱ionexchangechromatography离子对色谱ionpairchromatography离子排斥色谱ionexclusionchromatographyＳＯ４２－ＣＯ３２－ＣＯ３２－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－＋＋＋＋＋＋＋＋＋＋＋＋ＳＯ４２－ＣＯ３２－

7、ＳＯ４２－ＣＯ３２－ＣＯ３２－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－Ｃｌ－＋＋＋＋＋＋＋＋＋＋＋＋Ｃｌ－ＨＣＯ３－ＣＯ３２－ＣＯ３２－ＣＯ３２－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－＋＋＋＋＋＋＋＋＋＋＋＋ＣＯ３２－ＳＯ４２－ＨＣＯ３－Ｃｌ－ＳＯ４２－Ｃｌ－ＨＣＯ３－ＣＯ３２－ＣＯ３２－ＨＣＯ３－ＨＣＯ３－ＣＯ３２－ＣＯ３２－ＣＯ３２－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－Temporalcourse离子交换分离机理ＣＯ３２－ＨＣＯ３－ＣＯ３２－ＨＣＯ３－ＣＯ３２－ＨＣＯ３－ＨＣ

8、Ｏ３－ＣＯ３２－ＨＣＯ３－ＣＯ３２－＋＋＋＋＋＋＋＋＋＋＋＋ＨＣＯ３－ＳＯ４２－ＣＯ３２－ＣＯ３２－ＨＣＯ３－ＨＣＯ３－ＨＣＯ３－Ｃｌ－＋＋＋＋＋＋＋＋＋＋＋＋离子对的分离机理1.生成离子对－待测离子与离子对试剂生成中性““离子对”分布于固定相与流动相之间，其分离类似传统的反相分离2.动态离子交换－离子对试剂的疏水部分吸附于固定相形成动态的离子交换表面，其分离机理类似于离子交换。3.离子间相互作用－除包括以上两种分离机理和固定相表面双电层结构的分离机理。MobilephaseTBAOH／ACN／