Recent advances in capillary ultrahigh pressure liquid chromatography.

In the twenty years since its initial demonstration, capillary ultrahigh pressure liquid chromatography (UHPLC) has proven to be one of most powerful separation techniques for the analysis of complex mixtures. This review focuses on the most recent advances made since 2010 towards increasing the performance of such separations. Improvements in capillary column preparation techniques that have led to columns with unprecedented performance are described. New stationary phases and phase supports that have been reported over the past decade are detailed, with a focus on their use in capillary formats. A discussion on the instrument developments that have been required to ensure that extra-column effects do not diminish the intrinsic efficiency of these columns during analysis is also included. Finally, the impact of these capillary UHPLC topics on the field of proteomics and ways in which capillary UHPLC may continue to be applied to the separation of complex samples are addressed.

Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2µm particles.

Commercial chromatographic instrumentation for bottom-up proteomics is often inadequate to resolve the number of peptides in many samples. This has inspired a number of complex approaches to increase peak capacity, including various multidimensional approaches, and reliance on advancements in mass spectrometry. One-dimensional reversed phase separations are limited by the pressure capabilities of commercial instruments and prevent the realization of greater separation power in terms of speed and resolution inherent to smaller sorbents and ultrahigh pressure liquid chromatography. Many applications with complex samples could benefit from the increased separation performance of long capillary columns packed with sub-2µm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45kpsi. The system consists of a commercially available capillary liquid chromatography instrument, for sample management and gradient creation, and is modified with a storage loop and isolated pneumatic amplifier pump for elevated separation pressure. The system's performance is assessed with a complex peptide mixture and a range of microcapillary columns packed with sub-2µm C18 particles.

Slurry concentration effects on the bed morphology and separation efficiency of capillaries packed with sub-2 µm particles.

Transcolumn dispersion limitations on the separation efficiency of chromatographic columns suggest the need for packing methods that increase bed homogeneity and minimize potential wall effects. Here we address the influence of the slurry concentration in the slurry packing process on the resulting morphology and separation efficiency of ultrahigh-pressure liquid chromatography capillary columns.30­75 µm i.d. capillaries were packed with fully porous 0.9, 1.7, and 1.9 µm bridged-ethyl hybrid particles and 1.9 µm Kinetex core­shell particles. Capillaries prepared with higher slurry concentrations(20­100 mg/mL) showed higher separation efficiencies than those prepared using a low slurry con-centration (2­3 mg/mL). The effect is explained by an analysis of transcolumn bed heterogeneities in three-dimensional reconstructions acquired from the packed capillaries using confocal laser scanning microscopy. The three-dimensional analysis of porosity distributions and local particle size illustrates that beds packed with higher slurry concentrations suppress particle size segregation, however, at the expense of a larger amount of packing voids. In core­shell packings, where only few packing voids were found, the higher slurry concentration allowed for an additional densification of the bed's wall region, as revealed by a radial analysis of the mean particle distances. Overall, wall effects are attenuated in packed columns prepared with both wide and narrow particle size distributions, which will allow for improved chromatographic performance.