2. liquid chromatography

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2. liquid chromatography

  1. 1. CHROMATOGRAPHY
  2. 2. Chromatography Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.
  3. 3. Definition: Different affinity of these 2 components to stationary phase causes the separation. Concentration of component A in stationary phase Concentration of component A in mobile phase Distribution Coefficient
  4. 4. Kinds of Chromatography 1. Liquid Column Chromatography 2. Gas Liquid Chromatography
  5. 5. Liquid Column Chromatography A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.
  6. 6. A + B + C OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Sample (A+B+C) Column Solid Particles (packing material- stationary phase) Eluant (eluate) DIAGRAM O F SIMPLE LIQ UID CO LUMN CHRO MATO GRAPHY A B C Solvent(mobile or moving phase) Diagram of Simple Liquid Column Chromatography
  7. 7. Basic liquid chromatography modes are named according to the mechanism involved: 1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC B. Reverse Phase LSC 2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC B. Reverse Phase LLC 3. Ion Exchange Chromatography 4. Gel Permeation Chromatography (exclusion chromatography) Four Basic Liquid Chromatography
  8. 8. Liquid Solid Chromatography 30 µ Si - O - H δ− δ+ Normal phase LS Reverse phase LS Silica Gel The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.
  9. 9. Liquid Solid Chromatography Si - OH HEXANE OH C-CH3 CH3 CH3 - C CH3 CH3 OH OH CH3 CH3
  10. 10. Water-Soluble Vitamins 1. Niacinamide 2. Pyridoxine N CONH2 N CH2OH CH2OH HO H3C 3. Riboflavin N N NH N CH2 HOCH HOCH HOCH CH2OH O OH3C H3C Cl N S N NH3C CH2 NH2 CH3 CH2CH2OH 4. Thiamin
  11. 11. Water-Soluble Vitamins 0 5 10 15 20 Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins Inject 1 2 3 4
  12. 12. Liquid-Liquid Chromatography ODPN (oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH 3 CH 2 OCH 2 CH 2 CN(Normal) CH 3 (CH 2 ) 16 CH 3 (Reverse) The 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 components more than others to effect separation.
  13. 13. MOBILE PHASE LIQUID Liquid-Liquid Chromatography (Partition) Liquid-Solid Chromatography (Adsorption) Liquid Solid Normal Phase Reverse Phase Normal Phase Reverse Phase Mobile Phase - Nonpolar Stationary phase - Polar Mobile Phase - Polar Stationary phase - Nonpolar FORMAT STATIONARY PHASE Types of Chromatography
  14. 14. Ion-Exchange Chromatography SO3 - Na+ Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).
  15. 15. Mechanism of Ion-Exchange Chromatography of Amino Acids SO3 - SO3 - Na + COO - H3N + Na + COOH H3N + pH2 pH4.5 Ion-exchange Resin
  16. 16. H3N + SO3 - SO3 - SO3 - SO3 - SO3 - SO3 - H3N + COOH OH COOH COOH H3N+ H3N + OH COO - Na + H3N + COO - Na + Na + H + OH - = H2O H + OH - = H2O Na + Na + pH3.5 Mobile PhaseStationary Phase Exchange Resin pH4.5 Chromatography of Amino Acids
  17. 17. Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules. Gel-Permeation Chromatography
  18. 18. • Polar Solvents Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile • Non-polar Solvents N-Decane > N-Hexane > N-Pentane > Cyclohexane Solvents
  19. 19. Sample Type LC Mode Positional isomers LSC or LLC Moderate Polarity Molecules LSC or LLC Compounds with Similar Functionality LSC or LLC Ionizable Species IEC Compounds with Differing Solubility LLC Mixture of Varying Sized Molecules GCC Selecting an Operation Mode
  20. 20. Schematic Diagram of Liquid Chromatography
  21. 21. Detector 1. Ultraviolet Detector 200-400nm 254 nm 2. Reflective Index Detector Universal Detector
  22. 22. High Performance Liquid Chromatography
  23. 23. High Performance Liquid Chromatography
  24. 24. Retention Time Time required for the sample to travel from the injection port through the column to the detector. Response Retention Time 5 10 15 20 25 A B C D
  25. 25. Selectivity Ratio of Net Retention Time of 2 components. (Distribution Coefficient) X2 - X0 X1 X0-α=
  26. 26. Response Retention Time X X X 1 3 6 2 1 0 – Selectivity SelectivitySelectivity
  27. 27. Resolution Equation V - V 1/2(W + W ) 2 2 1 1 R = Response Volumes W W W W V V1 1 2 2 21
  28. 28. Resolution
  29. 29. Height Equivalent to a Theoretical Plate Length of a column necessary for the attainment of compound distribution equilibrium measure the efficiency of the column. Theoretical plates (N) = 16 ( ) X Y 2 X Y
  30. 30. Importance of Theoretical Plates (N)
  31. 31. Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate 1 2 3 4V V V V W W W W 2 1 0 1 2 4 3 4 3 V 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 System Carbohydrates Amino acids, proteins Vitamins, A, D, E, K Nucleosides (purines and pyrimidines) Fatty acids, fats Aflatoxins Antioxidants Contaminants of packaging materials Carotenoids, chlorophylls Saccharines

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