Sucrose dependence of solute retention on human serum albumin stationary phase: hydrophobic effect and surface tension considerations.

In a chromatographic system using human serum albumin (HSA) as a stationary phase, D,L dansyl amino acids as solutes, and sucrose as a mobile-phase modifier, a study on the surface tension effect of sugar on compound retention was carried out by varying the salting-out agent concentration c and the column temperature T. The thermodynamic parameters for solute transfer from the mobile to the stationary phase were determined from linear van't Hoff plots. An enthalpy-entropy compensation study revealed that the type of interaction between solute and HSA was independent of the molecular structure of the dansyl amino acids and the mobile-phase composition. An analysis of the experimental variations in the retention factor and the enantioselectivity values with c was performed using a theoretical model. It was shown that the decrease in solute retention accompanying the sucrose concentration increase was principally governed by a structural rearrangement of the binding cavity due to the increased surface tension effects. The cavity apolar residues were assumed to fold out of contact with the medium in order to reduce the surface area accessible to sucrose molecules, thus implying a restriction of the curvature radius of the cavity. Such behavior caused a decrease in the hydrophobic interaction for ligand binding on HSA explaining the observed thermodynamic parameter trends over the sucrose concentration range.