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. 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. 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. 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. Liquid Solid Chromatography  Si - O - H  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. Liquid Solid Chromatography Si - OH HEXANE OH C-CH 3 CH 3 3 - C CH 3 3 OH CH 3 3

10. Water-Soluble Vitamins

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

16. Chromatography of Amino Acids

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

19. Sample TypeLC Mode Positional isomersLSC or LLC Moderate Polarity Molecules LSC or LLC Compounds with Similar FunctionalityLSC or LLC Ionizable SpeciesIEC Compounds with Differing SolubilityLLC Mixture of Varying Sized Molecules GCC Selecting an Operation Mode

20. Schematic Diagram of Liquid Chromatography

21. Detector 1.Ultraviolet Detector 200-400nm 254 nm 2.Reflective Index Detector Universal Detector

22. 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 V 0 = 1.0 (Minutes)V 1 = 5.0, V 2 = 7.0, V 3 = 11.0, V 4 = 13.0 W 1 = 1.0, W 2 =1.0, W 3 = 1.0, W 4 =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