Rapid peptide mapping by high-performance liquid chromatography

Short columns, packed with pellicular sorbents made of 2-μm fluid-impervious silica microspheres, were used at elevated column temperatures for rapid peptide mapping by high-performance liquid chromatography (HPLC). Enzymic digests of various proteins were chromatographed by gradient elution. In many cases the time of analysis was 10 min or less. In order to increase the retention particularly, that of short, polar peptides under such conditions, 1 mM octyl sodium sulfate or 5 mM hexyl sodium sulfate were added to the starting eluent. The length of the 4.6 mm I.D. columns was 30 or 75 mm, the sample load was in the range of 10–1000 pmoles. Highest analytical sensitivity was obtained at a flow-rate of 0.5 ml/min and room temperature, whereas for rapid analysis flow-rates of up to 2 ml/min were used at 80°C. This temperature allowed the use of relatively high flow velocities of the mobile phase without significant loss in efficiency. The method was highly reproducible, as shown by the results obtained by automated analysis of cyanogen bromide fragments of lysozyme at high speed. The quality of the rapid peptide maps compares favorably with that of maps obtained by standard reversed-phase HPLC methods, which require much longer analysis times.