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 ChromatographyOH
HEXANE
Si - OH OH OH CH CH 3 3 CH
- C
C-CH 3 3 CH CH 3 3 CH 3

10. Water-Soluble Vitamins

11. Water-Soluble Vitamins

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

14. Ion-Exchange ChromatographySO 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 Na H N 3
COOH
Ion-exchange Resin - + SO H N 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 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

20. Schematic Diagram of Liquid Chromatography

21. Detector
1. Ultraviolet Detector nm nm 2. Reflective Index Detector Universal 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