Maximizing the Peak Production Rate in Off-line Comprehensive Two-dimensional Liquid Chromatography with Mass Spectrometry Detection

In the past decade high performance liquid chromatography (HPLC) has emerged as a technique for the separation of complex proteomic samples because of its outstanding chromatographic resolving power,1–3 the possibilities to automate the analysis (ease-of-use and outstanding reproducibility)4 and its compatibility with mass-spectrometric (MS) detection using electrospray (ES) interfacing.5–6

A good performance criterion for the gradient separation of peptides is peak capacity, which is defined as the maximum number of peaks that can be separated with a resolution of 1 and elute in the applied gradient window.7 The maximum peak capacity obtained in one-dimensional (1D) LC is mainly determined by the column technology (column length and particle size) and the duration of the gradient. The maximum allowable column length is determined by the permeability of the chromatographic bed and the maximum pressure drop of the HPLC instrumentation.8–9 Whereas fast separations are typically obtained on short columns packed with small (<2 μm) silica particles, high-efficiency 1D-LC separations are obtained on long columns packed with a slightly larger particle size applying long gradients.10–11