Effect of Structural Properties of Both Proteins and Stationary Phases to Adsorption Behavior of Proteins in Hydrophobic Interaction Chromatography

A series of specially designed agarose-based HIC adsorbents were examined using human immunoglobulin G (IgG) and human serum albumin (HSA) as model proteins, to investigate the effects of structural properties of both proteins and stationary phases on the adsorption behavior in HIC. The adsorbents examined differ from each other in terms of ligand density, spacer arm and ligand type. Dynamic binding capacity (DBC) was determined to reflect the retention of proteins on adsorbents. The results showed that the adsorption behaviors of IgG and HSA on hydrophobic adsorbents were in different manner. HSA tended to be retained in a HIC adsorbent with high local hydrophobicity within a small area, and their interaction was strictly salt-dependent; whereas IgG was sensitive to spatial accessibility of hydrophobic ligands rather than ligand type. This manner of adsorption enabled IgG to be adsorbed on adsorbents with relatively high ligand density in absence of water-structuring salt. The results were ascribed to the different structural flexibility, hydrophobic surface distribution and size of IgG and HSA, two proteins with different types of structure.