2. liquid chromatography

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2. ChromatographyChromatography basically involves theseparation of mixtures due to differences inthe distribution coefficient of sample componentsbetween 2 different phases.One of these phases is a mobile phase andthe other is a stationary phase. 3. Distribution CoefficientDefinition:Concentration of component A in stationary phase Concentration of component A in mobile phaseDifferent affinity of these 2 components to stationaryphase causes the separation. 4. Kinds of Chromatography1. Liquid Column Chromatography2. Gas Liquid Chromatography 5. Liquid Column ChromatographyA sample mixture is passed through a columnpacked with solid particles which may or may not becoated with another liquid.With the proper solvents, packing conditions, somecomponents in the sample will travel the columnmore slowly than others resulting in the desiredseparation. 6. Diagram of Simple Liquid Column Chromatography DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY Solvent(mobile or moving phase) OOOOOOOOOOOA+B+C Sample OOOOOOOOOOO(A+B+C)OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO A OOOOO OOOOOOOOOOOOOO ColumnOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOSolid Particles OOOOOOOOOOOOOOOOOOOOO (packing material- OOOOOB OOOOOOOOOOOOOO stationary phase) OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOC OOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOO Eluant (eluate) 7. Four Basic Liquid ChromatographyBasic liquid chromatography modes are named according to the mechanisminvolved:1. Liquid/Solid Chromatography (adsorption chromatography)A. Normal Phase LSCB. Reverse Phase LSC2. Liquid/Liquid Chromatography (partition chromatography)A. Normal Phase LLCB. Reverse Phase LLC3. Ion Exchange Chromatography4. Gel Permeation Chromatography (exclusion chromatography) 8. Liquid Solid ChromatographyNormal phase LSReverse phase LS +Si - O - H 30 Silica GelThe separation mechanism in LSC is based on thecompetition of the components of the mixture samplefor the active sites on an absorbent such as Silica Gel. 9. Liquid Solid ChromatographyOHHEXANE Si - OH OHCH3 CH3 CH3 - C C-CH3 CH3CH3 CH3 10. Water-Soluble Vitamins1. Niacinamide 2.PyridoxineH 3CNN HOCH 2OH CONH 2 CH 2OH3. RiboflavinCH 2OHHOCH HOCH 4. Thiamin HOCHCH 2H 3CNNO H 3C NNH 2 SCH 2CH 2OH NHClH 3CNN NCH 2CH 3 O 11. Water-Soluble Vitamins 2 3Inject 4 1Column: u Bondapak C18Solvent: MeOHSample: Water-Soluble Vitamins 0 5 10 15 20 12. Liquid-Liquid Chromatography ODPN (oxydipropionylnitrile)Normal Phase LLCReverse Phase LLCNCCH CH OCH CH CN(Normal) 3 2 2 2CH (CH ) CH (Reverse)3 2 16 3The stationary solid surface is coated with a 2nd liquid (the Stationary Phase)which is immiscible in the solvent (Mobile) phase.Partitioning of the sample between 2 phases delays or retains some componentsmore than others to effect separation. 13. Types of ChromatographyLIQUIDMOBILE PHASE Liquid-Liquid Liquid-SolidFORMATChromatography (Partition)Chromatography (Adsorption) LiquidSolidSTATIONARY PHASE Normal Phase Reverse PhaseNormal PhaseReverse Phase Mobile Phase - NonpolarMobile Phase - Polar Stationary phase - Polar Stationary phase - Nonpolar 14. Ion-Exchange ChromatographySO 3 Na + -Separation in Ion-exchange Chromatography is based on thecompetition of different ionic compounds of the sample for theactive sites on the ion-exchange resin (column-packing). 15. Mechanism of Ion-Exchange Chromatography of Amino AcidspH2- ++ SO 3NaH3N COOH Ion-exchange Resin-+ SO 3 H 3N-COO pH4.5 +Na 16. Chromatography of Amino Acids Stationary PhaseMobile Phase+ H3 N -SO3 Na+COOH + Na OH -+ SO3 H3 NCOOHExchange Resin - SO3 H3N+ COOH pH3.5OH - SO3 H 3 N+ +- +- Na COO H OH = H 2 O + Na-SO3H3 N +-+- COO HOH = H 2 O - SO3Na+pH4.5 17. Gel-Permeation ChromatographyGel-Permeation Chromatography is a mechanical sorting of moleculesbased on the size of the molecules in solution.Small molecules are able to permeate more pores and are, therefore,retained longer than large molecules. 18. Solvents Polar SolventsWater > Methanol > Acetonitrile > Ethanol >Oxydipropionitrile Non-polar Solvents N-Decane > N-Hexane > N-Pentane > Cyclohexane 19. Selecting an Operation ModeSample TypeLC ModePositional isomers LSC or LLCModerate Polarity MoleculesLSC or LLCCompounds with Similar Functionality LSC or LLCIonizable SpeciesIECCompounds with Differing SolubilityLLCMixture of Varying Sized Molecules GCC 20. Schematic Diagram of Liquid Chromatography 21. Detector1. Ultraviolet Detector 200-400nm 254 nm2. Reflective Index Detector Universal Detector 22. High Performance Liquid Chromatography 23. High Performance Liquid Chromatography 24. Retention TimeTime required for the sample to travel from the injection portthrough the column to the detector.Response D B AC 510152025 Retention Time 25. SelectivityRatio of Net Retention Time of 2 components.(Distribution Coefficient)= X2 - X0X1- X0 26. Selectivity SelectivityResponseX 2 X1X0 13 6 Retention Time 27. Resolution EquationV - V1 2 R= 1/2(W1 + W2)Response V2V1WW1 2W1 W2 Volumes 28. Resolution 29. Height Equivalent to a Theoretical PlateLength of a column necessary for the attainment of compounddistribution equilibrium measure the efficiency of the column. X 2Theoretical plates (N) = 16 ( ) Y X Y 30. Importance of Theoretical Plates (N) 31. Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate 2V24 V11 3V0W1 W2 W3 W4 V3 V4 V0 = 1.0 (Minutes) V1 = 5.0, V2 = 7.0, V3 = 11.0, V4 = 13.0W1 = 1.0, W2 =1.0, W3 = 1.0, W4 =1.0 32. Chromatogram of Orange Juice Compounds 33. General Factors Increasing Resolution Increase column length Decrease column diameter Decrease flow-rate Pack column uniformly Use uniform stationary phase (packing material) Decrease sample size Select proper stationary phase Select proper mobile phase Use proper pressure Use gradient elution 34. LC Application in Food SystemCarbohydratesAmino acids, proteinsVitamins, A, D, E, KNucleosides (purines and pyrimidines)Fatty acids, fatsAflatoxinsAntioxidantsContaminants of packaging materialsCarotenoids, chlorophyllsSaccharines