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-2 µm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45 kpsi. 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-2 µm C18 particles.

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Method qualification is a key step in the development of routine analytical monitoring of pharmaceutical products. However, when relying on published monographs that describe longer method times based on older HPLC column and instrument technology, this can delay the overall analysis process for generated drug products. In this study, high-throughput ultrahigh pressure liquid chromatography techniques were implemented to decrease the amount of time needed to complete a 24-run sequence to identify linearity, recovery, and repeatability for both drug assay and impurity analysis to 16 min. Multiple experimental parameters were tested to identify a range of experimental settings that could be used for the sequence while still maintaining this fast analysis time. The full sequence was replicated on a different system and with different columns, further demonstrating its robustness.