Size Exclusion Chromatography

SIZE EXCLUSION CHROMATOGRAPHY

INTRODUCTION

The size-exclusion chromatography (or gel-chromatography) is a means of separation which is exclusively dependent on the exchange of solute molecules between the solvent of the mobile-phase and the same solvent within the pores of the column-packing material. In reality, it is the pore-size-range of the packing material that solely determines the molecular-size-range within which a particular separation can take place effectively.

The timely adoption of the cross-linked dextran gels (i.e., Sephadex) in late-fifties as a packing mate-rial for column chromatography opened an altogether new horizon of chromatographic separation whereby substances, in general, undergo separation more or less as per their molecular size.

In actual practice, the inert gels of dextran (I)-a polyglucose or other types of polymers, for instance : agarose and polyacrylamides, wherein the macromolecules invariably are cross-linked to afford a reasonably porous 3D-structure*, served as the stationary phases in size-exclusion chromatography.

The salient features of ‘gels’ are enumerated below :

(i)          The extent or degree of cross-linking and obviously the sizes of the pores within the body of the gels are rigidly monitored and controlled during the course of manufacture,

(ii)       Mostly the gels are hydrophilic in nature and evidently they swell-up in contact with water,

(iii)     Gels having a large degree of cross-linking and a relatively large pore size usually need a larger volume of water in order to fill up the pores available within the gel-structure in comparison to the tightly linked gels, as could be seen in Table 31.1.

(iv)      Buffered aqueous solutions normally serve as mobile phases in size-exclusion chromatography. However, highly modified gel polymers are also available commercially (e.g., Sephadex-LX) that exclusively make use of organic solvents