Patch controlled protein adsorption in mixed-mode chromatography with benzylamine as functional ligand

The adsorption mechanisms of bovine serum albumin (BSA) on mixed-mode adsorbents with benzylamine as the functional ligand were discussed in terms of the isotherm adsorption behavior and chromatographic retention under various conditions. The adsorbents showed the salt-tolerant and pH-dependent properties for protein binding. The results indicated that the electrostatic interactions might be an important contributor to the protein adsorption under the conditions of electrostatic attractive protein–ligand interactions. While there are some amounts of electrostatic repulsion protein–ligand interactions, it seems that the adsorption process is patch controlled, and the specific salt concentration to result in a minimum adsorption capacity could be found. Accordingly the isocratic retention factors have a minimum value under a certain salt concentration, and showed the typical “U” shape curve as the function of salt concentration. In addition, the total number of released water molecules during protein adsorption was analyzed using the preferential interaction theory, and was found to be consistent with the estimated hydrophobic contact area between the bound protein and adsorbent surface.