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CHROMATOGRAPHY

CHROMATOGRAPHY. 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.

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CHROMATOGRAPHY

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  1. CHROMATOGRAPHY

  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. Distribution Coefficient 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

  4. Kinds of Chromatography 1. Liquid Column Chromatography 2. Gas Liquid Chromatography

  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. Diagram of Simple Liquid Column Chromatography

  7. Four Basic Liquid Chromatography 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)

  8. Liquid Solid Chromatography Normal phase LS Reverse phase LS d- d+ Si - O - H 30 m 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. Liquid Solid Chromatography OH HEXANE Si - OH OH OH CH CH 3 3 C-CH CH - C 3 3 CH 3 CH 3 CH 3

  10. Water-Soluble Vitamins

  11. Water-Soluble Vitamins

  12. NCCH CH OCH CH CN(Normal) 3 2 2 2 CH (CH ) CH (Reverse) 3 2 16 3 Liquid-Liquid Chromatography ODPN (oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC 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. Types of Chromatography LIQUID MOBILE PHASE Liquid-Solid Liquid-Liquid (Adsorption) Chromatography FORMAT Chromatography (Partition) Solid Liquid STATIONARY PHASE Reverse Phase Normal Phase Normal Phase Reverse Phase Nonpolar Mobile Phase - Mobile Phase - Polar Stationary phase - Polar Stationary phase - Nonpolar

  14. - + SO Na 3 Ion-Exchange Chromatography 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. Mechanism of Ion-Exchange Chromatography of Amino Acids pH2 - + + SO Na H N 3 3 COOH Ion-exchange Resin + - H N SO 3 3 - COO pH4.5 + Na

  16. Chromatography of Amino Acids

  17. Gel-Permeation Chromatography 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.

  18. Solvents • Polar Solvents • Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile • Non-polar Solvents • N-Decane > N-Hexane > N-Pentane > Cyclohexane

  19. Selecting an Operation Mode Sample TypeLC 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

  20. Schematic Diagram of Liquid Chromatography

  21. Detector 1.Ultraviolet Detector200-400nm 254 nm2. Reflective Index DetectorUniversal Detector

  22. High Performance Liquid Chromatography

  23. High Performance Liquid Chromatography

  24. Retention Time Time required for the sample to travel from the injection port through the column to the detector.

  25. Selectivity Ratio of Net Retention Time of 2 components. (Distribution Coefficient)

  26. Selectivity Selectivity

  27. Resolution Equation

  28. Resolution

  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.

  30. Importance of Theoretical Plates (N)

  31. Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate 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. 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 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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