CHROMATOGRAPHY

Definition: Chromatography basically involves the separation due to differences in the equilibrium distribution of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.

Distribution Coefficient =

Different affinity of these 2 components to stationary phase causes the separation.

Kinds of Chromatography

1. Liquid Column Chromatography

2. Gas Liquid Chromatography

3. Paper Chromatography

4. Thin-layer Chromatography

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.

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

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)

LIQUID SOLID CHROMATOGRAPHY

T

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.

LIQUID SOLID CHROMATOGRAPHY

WATER-SOLUBLE VITAMINS

1. Niacinamide

2. Pyridoxine

3. Riboflavin

LIQUID-LIQUID CHROMATOGRAPHY

In 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.

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).

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

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.

SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

SELECTING AN OPERATING MODE

Sample Type LC Mode

Positional isomers LSC or LLC

Moderate Polarity Molecules LSC or LLC

Compounds with Similar Functionality LS or LLC

Ionizable Species IEC

Compounds with Differing Solubility LLC

Mixture of Varying Sized Molecules GCC

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

SELECTIVITY (a)

Ratio of Net Retention Time of 2 components.

(Equilibrium Distribution Coefficient)

RESOLUTION EQUATION

HEIGHT EQUIVALENT TO A THEORETICAL PLATE

Length of a column necessary for the attainment of compound distribution equilibrium (measure the efficiency of the column).

THE IMPORTANCE OF N

EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATE

V0 = 1.02 V1 = 4.92 V2 = 6.59 V3 = 8.17 V4 = 9.14

W1 = 1.0 W2 =1.0 W3 = 1.0 W4 =1.0

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