Silica versus polymer-based stationary phases for reversed-phase high-performance liquid chromatographic analyses of rat insulin biosynthesis : A comparison of resolution and recovery

Abstract

Because of the problems caused by the irreversible binding of insulins and proinsulins to several silica-based reversed-phase columns, the use of polymeric reversed-phase columns was investigated for the analysis of rat islet polypeptides involved in insulin biosynthesis. No irreversible binding of insulins and proinsulins was observed for the polymeric reversed-phase columns, probably due to the absence of silanol groups. The six polypeptides involved in insulin biosynthesis in rat islets were equally well resolved in shallow trifluoroacetic acid-acetonitrile gradients on the silica-based Nucleosil 300-5C 4 column (45°C), the polymer-based Asahipak C4P-50 (25 and 45°C), and ODP-50 columns (45°C). In shallow triethylammonium phosphate-acetonitrile gradients (25°C) satisfactory resolution of the two rat proinsulins was only obtained on the polymer-based Asahipak C4P-50 and C8P-50 columns. Increasing the separation temperature to 45°C improved the separation of the two insulins and the two proinsulins in all cases. The shifts in retention times for the individual islet polypeptides observed in relation to the increased separation temperature were found to be different for the silica C 4 and the polymer C 4 columns. Recoveries of rat islet polypeptides were comparably high from the silica- and the polymer-based C 4 columns and linear load-response curves were obtained in the microgram to picogram mass range of both columns.