Potential of silica monolithic columns in peptide separations

The objective of the work described here was to evaluate the efficacy of silica monolith supports in high-speed reversed-phase liquid chromatography (RPLC) of peptides. This was done using a commercial Chromolith column with an octadecylsilane stationary phase and a tryptic digest of cytochrome c. Columns (100 mm×4.6 mm) were operated at mobile phase velocities ranging from 1 ml/min (2.0 mm/s) to 10 ml/min (25 mm/s). There was little noticeable change over this flow rate range in either resolution, peak elution volume, or analyte concentration in collected fractions. It was concluded that capillary columns in this silica monolith format would be particularly valuable in peptide separations for proteomics. There was, however, a small, but perceptible contamination of peaks at high mobile phase velocity with earlier eluting analytes. Based on the fact that peak shape did not change at high mobile phase velocity, it is suggested that this phenomena might be due to the presence of peptide conformers in structural equilibrium on the sorbent surface. When elution rate exceeds the rate of conformer interchange, conformers could elute as broadened or even separate peaks.