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

Whereas 1D-LC allows the separation of hundreds of analytes, multidimensional separation approaches have the potential to separate thousands of components.^12–13 Of all the 2D-LC techniques, off-line 2D-LC (or LC×LC) offers the most flexibility in terms of separation modes and LC conditions, because matching eluents, flow-rates and transfer volumes are less critical than in on-line 2D-LC.¹⁴ Moreover, off-line 2D-LC allows re-analysis of samples when partial injection of the fractions is applied. Because peptides can be pre-concentrated and desalted between the two dimensions, external band-broadening due to the second-dimension injection volume must be minimized.¹⁵ Consequently, for proteomics separations a large (1 mm) i.d. first-dimension column is typically applied, providing high sample capacity, and a small (75 μm) i.d. column is applied as second-dimension column, providing high mass sensitivity and easy coupling with MS.

where ¹n_c is the peak capacity obtained in the first dimension and ²n_c the peak capacity obtained in the second dimension.

This article describes an optimization strategy to obtain the best possible performance in the shortest analysis time — called the peak production rate — for comprehensive off-line two-dimensional LC.