Steric Exclusion Chromatography
Exploitation of cosolvent exclusion with adsorptive chromatography methods is fairly straight forward. As mentioned previously for ion exchange and hydroxyapatite, the sample to be fractionated can be applied to the column, and then PEG or other excluded agents were introduced to impose size selectivity during the elution. This creates an implication that it might be possible to use PEG in combination with a nonadsorptive chromatography surface to achieve size-based separations of biomolecules, with the smallest species eluting first and larger species eluting later in proportion to their relative size. This would be a welcome addition to the field because there is no rapid high-capacity chromatography method capable of high-resolution size-based fractionation. However, there are 2 major barriers. The first barrier is that the sample must be mixed with preferentially excluded agent before it is introduced into the column. The problem is that a concentration of excluded agent sufficient to achieve sample binding causes precipitation in the sample and clogs the column on contact. This particular problem has been overcome in the field of HIC by the technique of sample loading by in-line dilution. High-salt buffer is loaded continuously through one line, while sample is loaded continuously through another. They meet and mix immediately before entering the column. Depending on the chromatography system and valve setup, precolumn residence time of the protein in the high-salt buffer can be as brief as seconds, so there is insufficient time for the precipitates to form before column contact. The second barrier is more restrictive. Most of the binding surfaces (i.e., functional ligand) in columns packed with porous particles resides within the particle pores. Proteins enter those pores by diffusion. Diffusion is slow. Because the viscosity of PEG reduces diffusivity in direct proportion, flow rate must be reduced to compensate. This creates in-column residence times of several minutes and the protein-PEG mixture precipitates in the void space between particles.58 These 2 restrictions effectively prevent loading proteins at high concentrations of PEG on nonadsorptive columns packed with porous particles.
