Evaluation of octadecyl-bonded alumina for separations of proteins and peptides by reversed-phase high-performance liquid chromatography

Abstract

A recently-developed octadecyl-bonded alumina (ODA) stationary phase was evaluated for the separation of peptides and proteins by reversed phase high performance liquid chromatography. Using standard water-acetonitrile mobile phase gradients containing 0.1 % trifluoroacetic acid, the average peak capacity obtained for the separation of a mixture of ribonuclease a, cytochrome c, lysozyme and carbonic anhydrase on an ODA column are similar to that obtained on a widely used octadecylsilane (ODS) column. However, overall chromatographic resolution of the components of this mixture on ODA is inferior to that obtained on ODS. Cytochrome c peak areas were found to be ∼50% smaller on the ODA column than on ODS. On the other hand, both peak capacities and resolutions of octapeptide mixtures were found to be generally superior on the ODA column, and peak areas for a representative octapeptide were found to be virtually identical for both ODA and ODS columns. The differences in the results obtained on the ODA and ODS columns for these separations are attributed to the smaller pore size and unique fused-microplatelet shape of the ODA particles. Comparisons of the separations of the tryptic digest of cytochrome c on the ODS and ODA columns demonstrate that the ODA phase is potentially as useful as ODS for peptide mapping applications.