High-resolution separations of tryptic digest mixtures using core–shell particulate columns operated at 1200 bar

Abstract

Combining two recent advances in instrumentation and column technology (ultra-high-pressure LC instruments and core–shell particles), the current peak-capacity generation limits in one-dimensional LC have been explored for the case of tryptic digest separations. To operate as close as possible to the Knox and Saleem limit of the particles, and hence to operate the 2.6μm core–shell particles at their kinetic optimum, the separations were conducted in a coupled column systems at 1200bar. Using coupled columns with a total length of 450mm at 1200bar and applying 40 and 120min gradients (tG/t0=17 and 52, respectively), peak capacities of nc=480 and 760 were measured. The kinetic performance was further improved by coupling six 150mm long columns and applying 1200bar, yielding a flow rate close to the optimum of the van Deemter curve while scaling the gradient volume. At tG/t0=52 a peptide separation yielding a peak capacity of 1360 was achieved, applying a 480min gradient. The observed increase of peak capacity with column length agrees well with the theoretical expectations based on the linear solvent strength (LSS) model.