Study of protein-ligand binding effects by direct chromatographic on-column injection

The direct zonal on-column injection method was applied to a high-performance liquid chromatographic study of pollutant-protein binding interactions in solution. The protein and the protein-ligand complex are excluded on the basis of the size from the diol support, and the free ligand penetrates into the pores and is more retained. The pattern of the ligand elution profile depends on the protein-ligand dissociation constant. This effect was quantitatively analysed by developing a numerical simulation algorithm in which the column is divided into slices of given thickness. The column length, flow-rate and shape of the injection signal are given as input parameters. A global dispersion coefficient accounts for peak broadening. A rapid equilibrium is assumed with the hypothesis that a monovalent ligand interacts with a single binding site on the protein. The interaction of bovine serum albumin with pentachlorophenol was studied, and an apparent dissociation constant for the protein-ligand complex was determined by fitting the theoretical profile to the experimental one. The effect of the acetonitrile content in the solvent was studied. An important decrease of the dissociation constant is observed that affects the chromatographic elution pattern.