Angiotensin I retention behavior on Butyl-Sepharose under linear loading chromatographic conditions

Adsorption behavior of angiotensin I on a commercial Butyl-Sepharose support has been studied in function of temperature and ammonium sulphate concentration. Under isocratic elution conditions and at the higher salt concentrations, a characteristic of the chromatographic performance of angiotensin I was the broadness of the corresponding peak and in most of the cases the appearance of two peaks. These results have been interpreted in terms of on-column cis-trans isomerization of angiotensin I (a proline containing polypeptide) followed by its “re-conformation” after the interaction with the support. It has been proposed that the peak splitting phenomenon, a combined effect between temperature, salt concentration in the mobile phase and the ligand, is caused by slow kinetics of isomerization that is on the same time scale as the chromatographic separation. Salt concentration and temperature promote the conversion of the trans form of angiotensin I into its cis form, which has a bigger hydrophobic surface area, in the presence of Butyl-Sepharose. The retention of the cis form of angiotensin I increases with the increase in salt concentration in the mobile phase and seems to be little affected by temperature.