Comments on the Implementation of a Simple Peak Capacity Optimization Procedure and Comparison of Poly(styrene–divinylbenzene) and RP-18 Silica Monolithic Capillary Columns of 5-cm for the Analysis of Protein Digests

Abstract

For the analysis of protein digests, the peak capacity in reversed-phase liquid chromatography is of paramount importance. A univariate method to maximize the peak capacity as developed by Wang et al. (Anal Chem 78:3406–3416, 20) has been applied and tested for a monolithic RP-18 silica capillary column. In their method, using model peptides representing a bovine serum albumin digest, the gradient time and temperature are kept constant while the flow rate and eluent strength are varied. Despite our criticism on the fixed starting conditions, a long gradient time leading to an unnecessary long analysis time and a high temperature leading to possible degradation products in the chromatogram, and the peak capacity as the only optimization parameter this fast and simple optimization strategy turns out to be applicable to capillary monolithic columns. Furthermore, the influence of the peak capacity on a second optimization parameter, the MS protein identification score, is examined. The procedure is also used to enhance the performance of two popular types of monolithic capillary LC columns (silica-C18 and poly(styrene–divinylbenzene)) of the same length for the analysis of protein digests. Comparison of both columns show that the calculated chromatographic parameters, like productivity and peak capacity, and identification score for both columns are about the same. For a more complicated nine-protein digest the performance of the silica monolith is slightly better.