2. liquid chromatography

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2. liquid chromatography

  1. 1. CHROMATOGRAPHY
  2. 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. 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. 4. Kinds of Chromatography1. Liquid Column Chromatography2. Gas Liquid Chromatography
  5. 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. 6. Diagram of Simple Liquid Column Chromatography DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY Solvent(mobile or moving phase) OOOOOOOOOOO A+B+C Sample OOOOOOOOOOO (A+B+C) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO A OOOOO OOOO OOOOOOOOOO Column OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Solid Particles OOOOOOOOOO OOOOOOOOOOO (packing material- OOOOOB OOOO OOOOOOOOOO stationary phase) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOC OOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO Eluant (eluate)
  7. 7. Four Basic Liquid ChromatographyBasic liquid chromatography modes are named according to the mechanism involved:1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC B. Reverse Phase LSC2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC B. Reverse Phase LLC3. Ion Exchange Chromatography4. Gel Permeation Chromatography (exclusion chromatography)
  8. 8. Liquid Solid Chromatography Normal phase LS Reverse phase LS δ− δ+ Si - O - H 30 µ 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. 9. Liquid Solid Chromatography OH HEXANE Si - OH OH CH3 CH3 CH3 - C C-CH3 CH3 CH3 CH3
  10. 10. Water-Soluble Vitamins1. Niacinamide 2. Pyridoxine H 3C N N HO CH 2OH CONH 2 CH 2OH3. Riboflavin CH 2OH HOCH HOCH 4. Thiamin HOCH CH 2H 3C N N O H 3C N NH 2 S CH 2CH 2OH NH ClH 3C N N N CH 2 CH 3 O
  11. 11. Water-Soluble Vitamins 2 3Inject 4 1 Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins 0 5 10 15 20
  12. 12. Liquid-Liquid Chromatography ODPN (oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH CH OCH CH CN(Normal) 3 2 2 2 CH (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. 13. Types of Chromatography LIQUIDMOBILE PHASE Liquid-Liquid Liquid-SolidFORMAT Chromatography (Partition) Chromatography (Adsorption) Liquid SolidSTATIONARY PHASE Normal Phase Reverse Phase Normal Phase Reverse Phase Mobile Phase - Nonpolar Mobile Phase - Polar Stationary phase - Polar Stationary phase - Nonpolar
  14. 14. Ion-Exchange Chromatography SO 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. 15. Mechanism of Ion-Exchange Chromatography of Amino Acids pH2 - + + SO 3 Na H3N COOH Ion-exchange Resin - + SO 3 H 3N - COO pH4.5 + Na
  16. 16. Chromatography of Amino Acids Stationary Phase Mobile Phase + H3 N - SO3 Na+ COOH + Na OH - + SO3 H3 N COOH Exchange Resin - SO3 H3N+ COOH pH3.5 OH - SO3 H 3 N+ + - + - Na COO H OH = H 2 O + Na - SO3 H3 N + - + - COO H OH = H 2 O - SO3Na+ pH4.5
  17. 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. 18. Solvents• Polar Solvents Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile• Non-polar Solvents N-Decane > N-Hexane > N-Pentane > Cyclohexane
  19. 19. Selecting an Operation ModeSample Type LC ModePositional isomers LSC or LLCModerate Polarity Molecules LSC or LLCCompounds with Similar Functionality LSC or LLCIonizable Species IECCompounds with Differing Solubility LLCMixture of Varying Sized Molecules GCC
  20. 20. Schematic Diagram of Liquid Chromatography
  21. 21. Detector1. Ultraviolet Detector 200-400nm 254 nm2. Reflective Index Detector Universal Detector
  22. 22. High Performance Liquid Chromatography
  23. 23. High Performance Liquid Chromatography
  24. 24. Retention TimeTime required for the sample to travel from the injection portthrough the column to the detector. Response D B A C 5 10 15 20 25 Retention Time
  25. 25. SelectivityRatio of Net Retention Time of 2 components.(Distribution Coefficient) α= X2 - X0 X1 - X0
  26. 26. Selectivity– SelectivityResponse X 2 X1 X0 1 3 6 Retention Time
  27. 27. Resolution Equation V - V1 2 R= 1/2(W1 + W2)Response V2 V1 W W 1 2 W1 W2 Volumes
  28. 28. Resolution
  29. 29. Height Equivalent to a Theoretical PlateLength of a column necessary for the attainment of compounddistribution equilibrium measure the efficiency of the column. X 2 Theoretical plates (N) = 16 ( ) Y X Y
  30. 30. Importance of Theoretical Plates (N)
  31. 31. Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate 2 V2 4 V1 1 3 V0 W1 W2 W3 W4 V3 V4 V0 = 1.0 (Minutes) V1 = 5.0, V2 = 7.0, V3 = 11.0, V4 = 13.0 W1 = 1.0, W2 =1.0, W3 = 1.0, W4 =1.0
  32. 32. Chromatogram of Orange Juice Compounds
  33. 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. 34. LC Application in Food SystemCarbohydratesAmino acids, proteinsVitamins, A, D, E, KNucleosides (purines and pyrimidines)Fatty acids, fatsAflatoxinsAntioxidantsContaminants of packaging materialsCarotenoids, chlorophyllsSaccharines

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