Extended DLVO calculations expose the role of the structural nature of the adsorbent beads during chromatography.

Protein adsorption onto hydrophobic interaction chromatography supports was studied by a surface-thermodynamics approach. To gather relevant experimental information, contact angle measurements and zeta potential determinations were performed on three different commercial adsorbent beads, Phenyl Sepharose 6 Fast Flow, Toyopearl Phenyl 650-C and Source 15 Phenyl, having soft to rigid backbone structure. Similar information was obtained for a collection of model proteins, lysozyme, bovine serum albumin (BSA), polygalacturonase, aminopeptidase, chymosin, aspartic protease, beta-galactosidase, human immunoglobulin G, and lactoferrin, were evaluated in the hydrated and in the dehydrated state. Based on the mentioned experimental data, calculations were performed to obtain the (interfacial) energy versus distance profiles of nine individual (model) proteins on (commercial) beads of three different types. All of these beads harbored the phenyl-ligand onto a matrix of differentiated chemical nature. Extended Derjaguin, Landau, Verwey, and Overbeek (DLVO) calculations were correlated with actual chromatographic behavior. Typical chromatography conditions were employed. The population of model proteins utilized in this study could be segregated into two groups, according to the minimum values observed for the resulting interaction energy pockets and the corresponding retention volumes (or times) during chromatography. Moreover, trends were also identified as a function of the type of adsorbent bead under consideration. This has revealed the influence of the physicochemical nature of the bead structure on the adsorption process and consequently, on the expected separation behavior.