Size Exclusion Chromatography: Theory

THEORY

The efficiency and ability of a gel to slow down the movement of various substances downwards in a packed column with the respective gel entirely depends on the molecular size of the substance vis-a-vis to the pore sizes prevailing within the gel matrix. Evidently, a substance with high molecular weight is unable to diffuse into the pores of the gel and thereby moves down the column more rapidly through the channels between the grains of the gel. On the contrary, a substance having molecular size distinctly smaller than the largest pores of the gel shall naturally penetrate the pores and move with a slower pace down the column. In this manner the substances having molecular size greater than the pores shall undergo exclusion thereby affecting their elution from the column into the space immediately ahead of the relatively small molecular weight compo-nents. In other words, the substances are found to be eluted from the column strictly in order of the decreasing molecular size.

The liquid phase which is absorbed by the synthetic polymer granules (e.g., Sephadex) is mostly avail-able in a wide range as solvent for solute molecules in contact with the gel. It has been observed that the actual distribution of the solute in between the inside and outside of the respective gel granules is nothing but a criterion of the available space. However, the underlying distribution coefficient occurring between the granu-lar and interstitial aqueous phases is found to be independent of three major factors, namely :

(a) pH

(b) Ionic strength, and

(c) Concentration of the solvent.

1. DISTRIBUTION COEFFICIENT (K_D)

The distribution coefficient is defined by the underlying expression :

where,  V_R = Retention volume for the component of interest,

V_O = Retention volume for a non-retained component (or exclusion volume), and

V_T = Retention volume for a component that has full access to all the pores of the support (or total permeation volume).

2. PERFORMANCE

The column performance may be determined from the number of theoretical plates per metre (n), calculated by the help of the following expression :

where, V_R = Retention volume* for the component of interest,

W_h = Width of the peak of interest at half peak height, measured in the same units as V_R.

3. MATERIALS

There are usually three types of materials that are employed in the size-exclusion chromatography for pharmaceutical substances which have been discussed briefly as under :

(a) Agarose FC

Presentation : Swollen beads 60 to 140 µm in diameter, available as a 4% suspension in water,

Applications : (1) It is used for the separation of proteins having molecular weights ranging from 6 × 10 ⁴ to 2 × 10 ⁷, and

It is employed for the separation of polysaccharides having molecular weights varying from 3 × 10 ³ to 5 × 10 ⁶.

(b) Agarose FC, Cross-linked

Presentation : Prepared from agarose by reaction with 2, 3-dibromopropan-1-ol in strongly alkaline enviroment. It occurs as swollen beads 60 to 140 μ m in diameter and is available as a 4% suspension in water.

Applications : (1) It is employed for the separation of proteins having molecular weights ranging between 6 × 10 ⁴ to 20 × 10 ⁶, and

It is used for the separation of polysaccharides having molecular weights varying between 3 × 10 ³ to

5 × 10 ⁶.

(c) Silica Gel FC

Presentation : It is very finely divided power having an average particle size 10 μ m with a very hydrophilic surface. It has an average pore diameter of about 30 nm. It is fairly compatible with aqueous solutions of pH 2 to 8 and also with various organic solvents.

Applications : It is employed for the separation of proteins having molecular weights ranging from 1 × 10 ³ to 3 × 10 ⁵.