Influence of pore and particle size on the frontal uptake of proteins : Implications for preparative anion-exchange chromatography

Abstract

Several silica-based anion-exchange packings were synthesized with nominal pore sizes of 250, 500 and 1000 Å in 10-, 20- and 50-μm particles. The static (“equilibrium”) adsorption capacities for bovene serum albumin (mol.wt. 69 000), α-lactalbumin (17 500) and ferritin (440 000) were first measured using bulk material. The media were then packed into columns for frontal uptake experiments to measure adsorption from a flowing mobile phase. In general, frontal uptake was inversely related to both flow-rate and particle size. However, the magnitude of these relationships was strongly dependent on the pore to protein diameter ratio. More specifically, the uptake of bovine serum albumin was significantly more sensitive to linear velocity and particle size than α-lactalbumin. A mathematical model of the chromatographic process was used to calculate radial adsorption profiles across the chromatographic particle during frontal uptake. It was shown that restricted intraparticle diffusion due to insufficient pore size causes incomplete utilization of internal surface area. Under such conditions, protein is only bound within a finite shell on the outermost side of the particle; therefore, the effective loadability of the packing is greatly reduced. These data suggest that a pore size of at least 500 Å will be required for the preparative chromatography of proteins with a molecular weight higher than ca. 1000 000. This observation is especially evident when using particle sizes greater than 10 μm.