High-performance liquid chromatography of amino acids, peptides and proteins. CIII. Mass transfer resistances in ion-exchange and dye-affinity chromatography of proteins.
Adsorption equilibria and rate kinetics have been investigated for the binding of several proteins, with different molecular geometries, 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.
Johnston
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Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Hearn
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MT
D000327:Adsorption; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents; D007475:Ion Exchange Resins; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D004396:Coloring Agents; D007475:Ion Exchange Resins; D011506:Proteins