Abstract
Intraparticle transport of proteins usually represents the principal resistance controlling their uptake in preparative separations. In ion-exchange chromatography two limiting models are commonly used to describe such uptake: pore diffusion, in which only free protein in the pore lumen contributes to transport, and homogeneous diffusion, in which the transport flux is determined by the gradient in the total protein concentration, free or adsorbed. Several studies have noted a transition from pore to homogeneous diffusion with increasing ionic strength in some systems, and here we investigate the mechanistic basis for this transition. The studies were performed on a set of custom-synthesized methacrylate-based strong cation exchangers differing in ligand density into which uptake of two proteins was examined using confocal microscopy and frontal loading experiments. We find that the transition in uptake mechanism occurs in all cases studied, and generally coincides with an optimum in the dynamic binding capacity at moderately high flow rates. The transition appears to occur when protein-surface attraction is weakened sufficiently, and this is correlated with the isocratic retention factor k' for the system of interest: the transition occurs in the vicinity of k' approximately 3000. This result, which may indicate that adsorption is sufficiently weak to allow the protein to diffuse along or near the surface, provides a predictive basis for optimizing preparative separations using only isocratic retention data.