Effect of mobile phase components on the separation of polypeptides using carbon dioxide-based mobile phases

The effect of mobile phase modifier and additive on the chromatographic pro perties of various small polypeptides was explored under subcritical condit ions, A polymeric column was used to separate various enkephalin analogs, b radykinin, and oxytocin using a carbon dioxide-based mobile phase with eith er an ethanol or a 2-methoxy-ethanol modifier. The role of the modifier was found to be secondary to that of the mobile phase additive. As progressive ly stronger acidic mobile phase additives were used, the peak profiles of t he various polypeptides improved and retention decreased, Heptadecafluorooc tanesulfonic acid was found to be the most useful additive for these types of solutes under near-critical conditions, while the potassium salt of hept adecafluorooctanesulfonic acid failed to elute any of the polypeptides. At low temperatures, pressure gradients with a carbon dioxide/ethanol/heptadec afluorooctanesulfonic acid ternary mobile phase produced reasonably good pe ak profiles with the polymeric column.