Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized protein coats 24% or less of the sorbent surface. It was found that columns on which proteins were immobilized still behaved as a cation-exchange chromatography sorbents, but their selectivity was different from the parent weak cation-exchange column. This was interpreted to mean that in addition to the normal electrostatic interactions expected in ion- exchange chromatography, protein analytes interact with immobilized protein on the sorbent surface. Anionic proteins were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It is concluded that these protein-protein interactions occur after proteins are captured by the primary interaction mechanism of the column, in this case, electrostatic interaction. Protein-protein interaction is a secondary, lateral process. These lateral interactions were observed between 4% and 24% surface saturation. The significance of this observation is that in preparative chromatography and the case of "fouled" columns, strongly adsorbed proteins could alter the elution characteristics of sample proteins being target for analysis or purification.