High-performance liquid chromatography of amino acids, peptides and proteins : XCV. Thermodynamic and kinetic investigations on rigid and soft affinity gels with varying particle and pore sizes: Comparison of thermodynamic parameters and the adsorption behaviour of proteins evaluated from bath and frontal analysis experiments

Abstract

The thermodynamic constants, associated with the interaction of three proteins with triazine dye affinity sorbents, have been derived from bath and frontal analysis experiments. In cases where mass-transfer restrictions are very high, calculation of the thermodynamic constants directly from frontal analysis experiments could not be achieved. In such cases, a portion of the adsorbate was always present in the effluent, a situation which has its effect as the split peak phenomenon. With Fractogel-based triazine dye affinity sorbents none of the test proteins applied in frontal analysis were adsorbed. A similar behaviour was observed for a Cellufine sorbent during the adsorption of human serum albumin and the Blue Sepharose CL6B sorbent during the adsorption of alcohol dehydrogenase, which displayed much slower apparent adsorption kinetics than observed in the bath experiments. These phenomena were shown to be associated with changes in the gel structure, caused in part by the column packing procedure. Silica-based sorbents performed better in the adsorption of lysozyme in the column mode than soft-gel affinity sorbents, as was evident in the higher capacities and steeper breakthrough curves. At high protein concentrations (feedstock concentration > 0.2 mg/ml) breakthrough curves obtained with small- and large-particle-size sorbents, but of constant pore size, were found to be identical. This finding demonstrates that the use of small-particle-size sorbents ( e.g. particle diameter, d p ⩽ 5 μm) for the preparative isolation of proteins may not be justified when operating in the overload mode. With other higher-molecular-weight proteins and the silica-based sorbent systems examined, the small-particle-size sorbents ( d p = 5 μm) displayed less symmetrical shapes of their breakthrough curves than the larger-particle-size and soft-gel sorbents. This behaviour was further exacerbated when non-porous glass or silica-based sorbents were utilized. These non-porous affinity sorbents displayed nearly rectangular breakthrough shapes at the onset of the adsorption process, but comparatively slow adsorption kinetics became evident as saturation was approached. This phenomenon has been attributed to surface rearrangement and/or reorientation of the adsorbed proteins, particularly with sorbents of high ligand densities.