High-performance liquid chromatography of amino acids, peptides and proteins : CIII. Mass transfer resistances in ion-exchange and dye-affinity chromatography of proteins

Abstract

Adsorption equilibria and rate kinetics have been investigated for the binding of several proteins, with different molecular geometrics, to several ion-exchange and dye-affinity chromatographic resins with varying pore size and protein accessibilities. The pore geometry was shown to play a significant role in the protein capacity and loadability of both the ion-exchange and dye-affinity resins. For example the Fractogel HW75—Cibacron Blue F3GA affinity sorbent had the greatest capacity for the small protein, lysozyme, compared to the other Fractogel HW-Cibacron Blue F3GA sorbents, and similarly, the ion-exchange resins, such as DEAE-Fractogel 65, bound more human serum albumin (HSA), as opposed to the larger protein, ferritin. The apparent diffusion of protein from the bulk phase to the ligands/ionic sites was calculated to be considerably restricted when the pore to protein size ratio was small, as is the case of DEAE Fractogel 65/ferritin system, and the dye-affinity Fractogel HW55/HSA system. In these circumstances, pore diffusivity was calculated to be up to 100-fold smaller than bulk diffusivity.