SIZE EXCLUSION CHROMATOGRAPHY
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
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