第十七章高效液相色谱北京化工大学

1、122.3High Performance Liquid ChromatographyHPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution. 4 5 GCHPLCInert gasesVolatile and thermally stable samplespolar solventHigh

2、Room temperatureHigh bp, thermally unstable samples included6 (1) Partition Chromatography(2) Adsorption Chromatography(3) Ion Exchange Chromatography(4) Exclusion Chromatography7（1）Partition Chromatography（2）Adsorption Chromatography（3）Ion Exchange Chromatography（4）Exclusion Chromatography8P a r t

3、i t i o n ChromatographyLiquid/Liquid Chromatography Liquid/Solid Chromatography A. Normal Phase LLCB. Reverse Phase LLCA. Normal Phase LSCB. Reverse Phase LSC9Selective retardation caused by interactions with bonded phase of stationary phase1011R - Polar group Hydrophobic groupSiSiOOOHClSiSiOOHOHCl

4、 Si(CH3)2RSiSiOOOSi(CH3)2RSi(CH3)2Rof Stationary phase OHOH13Normal Phase Partition ChromatographyReversed Phase Partition ChromatographyStationary PhasePolarNon-polarBounded group Polar groupHydrophobic groupMobile phaseNon-polarPolarPeaks sequence Polar fastestNon-polar fastest14（2）Adsorption Chro

5、matographyThe competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel15Solid adsorbent- silica gel, Al2O3 etc.Often use silica gel ( 510m).（2） Adsorption Chromatography16ion exchange resin inorganic acid or inorganic base aqueous solutionThe compet

6、ition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing)（3）Ion Exchange Chromatography17（3）Ion Exchange Chromatography18（4）Exclusion ChromatographyA mechanical sorting of molecules based on the size of the molecules in solutionSmall molecules a

7、re able to permeate more pores and are, therefore, retained longer than large molecules. 19Soft gel：Hinged glucose porous polymer，Polyacrylamide gel.u（Gel filtration Chromatography ）Semihard gel ：Polystyrene Beadu（Gel permeation Chromatography ）Super hard gel &Glass: Porous glass or Porous silic

8、a beadsu（High Performance Exclusion Chromatography）（4）Exclusion Chromatography20l Molecular weight and its distributing of macromolecular compound l Branched chain separation of different base on DNA fragment （4）Exclusion ChromatographyApplications22 232425 26Piston pumpSyringe pump Pneumatic pump 2

9、7Piston Pump Mechanism 28Syringe Pump29 30Sample Loop31 33vSingle-wavelength UV detectorv Source-Hg lampsv With spectral filter, up to 254，280，313，334，365 etc. v Wide application in biochemicalvMulti-wavelength UV detectorv Similar to photometer，Grating or PrismPMT/PD single-wavelength detectorPDA m

10、ulti-wavelength simultaneous detector，Three dimension3435Tungsten lampCoupling lensDeuterium lampSource lens(Achromat)Holmium oxidefilterSupport lensFlow cellSpectro lensGratingArrayProgrammableSlit TimeWavelengthmsecnmLIGHTSHIFT REGISTERSWITCHVIDEO LINEREADOUT CYCLEPHOTODIODECAPACITORTRANSISTORDiode Array - Details373890% MeOH0.5 mLs/mincoelute39 More sensitive than absorptiometry， preferable selectivity Source-High int