Preparative reversed-phase high performance liquid chromatography of insulin variants: Characterization of adsorption isotherms by frontal analysis

Adsorption isotherms for the binding of bovine and porcine insulins onto the reverse-phase HPLC adsorbent Whatman BioPrep C-4 (20 mu m particle diameter, 150 Angstrom pore size) have been measured by frontal analysis. The characteristics of the adsorption of both proteins were accurately described by Langmuir isotherms and values of the maximum adsorption capacities and the dissociation constants of the adsorbed complexes were measured. These values were consistent with measurements of retention factors by isocratic pulse chromatography which indicated that porcine insulin was adsorbed more strongly. The composition of the mobile phase had a significant effect on the strength of the interaction, and the. Langmuir isotherm parameters and the associated retention,factors varied with the concentration of the organic modifier, acetonitrile, present in the mobile phase. Examination of the competitive nature of the adsorption by two-component frontal analysis confirmed these conclusions from the single component studies and, at high concentrations, the more strongly adsorbed porcine insulin was observed to displace the more weakly adsorbed bovine variant. The concentrations of each insulin variant in the column effluent were determined by rapid chromatographic monitoring of collected fractions.