Comparison of polybutadiene-coated alumina and octadecyl-bonded silica for separations of proteins and peptides by reversed-phase high-performance liquid chromatography

Abstract

The chromatographic retention and separations of proteins and peptides on a novel polybutadiene-coated alumina (PBDA) high-performance liquid chromatographic stationary phase are compared to those obtained on a widely-used polymeric octadecylsilane (ODS) phase. Using acetonitrile-water mobile phase gradients containing 0.1% trifluoroacetic acid, the average peak capacities (which are inversely proportional to average peak widths) and peak resolutions obtained for chromatograms of mixtures of ribonuclease A, cytochrome c, lysozyme and carbonic anhydrase are five times lower on a column packed with PBDA than on one packed with ODS. Irreversible adsorption causes increases in column backpressure during successive analyses of protein solutions on PBDA phases and 50% reductions in protein peak areas on the PBDA phase compared to ODS. In contrast to those results, peak capacities, resolutions and peaks areas for synthetic octapeptides on the PBDA and ODS phases are more comparable to each other. Chromatographic capacity factors of 31 low-molecular-weight organic compounds on PBDA and ODS columns are shown to correlate well. The critical concentrations of organic modifier required to elute proteins and octapeptides from PBDA columns are lower than that required for ODS, but still correlate linearly with corresponding values from ODS columns. It is concluded from these results that the retentions of peptides, proteins and smaller molecules on both the PBDA and ODS phases are governed by similar hydrophobic interaction mechanism, while peak broadening due to mass transfer resistance increases more rapidly with solute size on the PBDA stationary phase than it does on ODS. The increase in solute mass transfer resistance with solute size on the PBDA column is attributed to solute interactions with the uniquely-shaped PBDA particles.