Fast, high-efficiency peptide separations on a 50-μm reversed-phase silica monolith in a nanoLC–MS set-up

Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-μm reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC–MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETPmin = 5–10 μm at 2.4 mm/s). Flow rates up to 1.95 μL/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-μm monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min.