In gel filtration the resolution is a function of column length (the longer the better). However, one drawback is related to the maximum sample volume which can be loaded. The larger the volume of sample loaded, the more the overlap between separated peaks. Generally speaking, the sample size one can load is limited to about 3-5% of the total column volume . Thus, gel filtration is best saved for the end stages of a purification , when the sample can be readily concentrated to a small volume. Gel filtration can also be used to remove salts from the sample, due to its ability to separate "small" from "large" components. Finally, gel filtration can be among the most "gentle" purification methods due to the lack of chemical interaction with the resin.

Mechanism of Size Exclusion Chromatography:

Size exclusion (also known as gel filtration chromatography) is a case of liquid-liquid partition chromatography, in which the solute molecules are get distributed in between two liquid phases, (i) liquid in the gel pores and (ii) liquid outside the gel.

The size exclusion may be explained by Steric Exclusion Mechanism. As the gel particles contains range of pore sizes, small molecules can enter in large number of pores while the large molecules will get small number of pores into which they can enter. Thus the different fractions of total pore volume are accessible to molecules of different sizes. Thus, molecules with different sizes will differ in distribution coefficient between these two liquid phases [ As the small molecules can enter in more pores while larger molecules can enter in pores only larger than the molecular size]

The total volume (V_t) of a column packed with a gel that has been swelled by solvent is given by

V _t = V_g + V_l + V_o

Where V_g is the volume occupied by the solid matrix of gel, V_i is the volume of solvent held in the pores or interstices and V_o is the free volume outside the gel particles. When mixing or diffusion occurs, the diffusion equilibrium and the retention volume (V_R) of the given species is given by

V_R = V_(int.) + K_dV_(int.)

where distribution coefficient (K_d) is given by K_d = Vi_(acc) / V_(total)

where Vi_(acc) is the accessible pore volume. V_(total) is the total pore volume and V_(int.) is the interstitial volume.