Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.