A Theoretical Examination of Adsorption Processes in Preparative Liquid Chromatography of Proteins

Abstract

This article examines retention in preparative chromatography in terms of the stoichiometric displacement model. This model is based on an equilibrium in which proteins and small displacing agents compete for ligands on the surface of chromatographic sorbents. Both the equilibrium constant for this process and the three‐dimensional structure of the macromolecule being separated are shown to be major determinants of retention in the analytical (linear) mode.A theoretical application of this model to preparative (nonlinear) separations shows a high probability that there will be perturbations of the chromatographic distribution coefficient. The activity coefficients of proteins, molecular orientation of proteins toward the sorbent surface, and adsorption kinetics are both load and sample dependent. In addition, displacement effects by other macromolecular species are predicted at high proteins concentrations. This theoretical analysis concludes that mathematical modeling of the retention process under nonlinear conditions will be more sample specific and require significantly more data on column operating conditions, interfering sample components, sample load, and the chemical nature of the stationary phase.