1. Chromatographic separation
Separating compounds based on selective
partitioning and adsorbtion
4630
1/29/11

2. CHROMATOGRAPHY
Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) 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 (Equilibrium Distribution )
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
3. Thin-layer 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
The 4 basic liquid chromatography modes are named according to the mechanism involved:
 1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC ( Polar solid phase, varying polarity liquid phase)
B. Reverse Phase LSC (Non-polar solid phase, nonpolar solvent)
 2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
 3. Ion Exchange Chromatography - Ionic interaction with resin
 4. Gel Permeation Chromatography (exclusion chromatography)
Size or rotation exclusion

8. LIQUID - SOLID -CHROMATOGRAPHY
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
See also “flash columns”

10. WATER-SOLUBLE VITAMINS

11. WATER-SOLUBLE VITAMINS

12. LIQUID-LIQUID CHROMATOGRAPHY
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

15. 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).

16. Resins Dowex - 50 Cation exchange --SO3- Dowex - 1 Anion exchange
CH2 --N+

17. MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS
Binding
Eluting

18. Chromatography of Amino Acids

21. GEL-PERMEATION CHROMATOGRAPHY
Aka. Size exclusion chromatography
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.

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

23. SELECTING AN OPERATING 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

24. Schematic Diagram of Liquid Chromatography

25. Detectors
1. Ultraviolet Detector light absorbtion -sensitive nm nm 2. Reflective Index Detector Universal Detector- weak . Useful for sugars and alcohols Fluorimetric detector degree excite, detect extremely sensitive
Light absorbtion or scattering

26. High Performance Liquid Chromatography
HPLC

27. High Performance Liquid Chromatography

28. Chromatogram of Organic Compounds from Fermented Cabbage

29. Chromatogram of Orange Juice Compounds

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

31. SELECTIVITY (a) Ratio of Net Retention Time of 2 components.
(Equilibrium Distribution Coefficient)

32. Selectivity
Selectivity

33. RESOLUTION EQUATION

34. HEIGHT EQUIVALENT TO A THEORETICAL PLATE
Length of a column necessary for the attainment of compound distribution equilibrium (measure the efficiency of the column).

35. RESOLUTION

37. EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATE
V0 = 1.02(Minutes) V1 = V2 = V3 = V4 = 9.14
W1 = 1.0 (Minutes) W2 =1.0 W3 = 1.0 W4 =1.0

38. GENERAL FACTORS INCREASING RESOLUTION
1. Increase column length
2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution

39. Thin layer chromatography
Simple, requires little equipment

40. Rf - retention factor

41. Test various solvent systems to separate
Mimics Liq-Liq

42. TLC detection
UV shadow or react with
a colorimetric compound