Application Note

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Proteomics laboratories have traditionally relied on nano-high performance liquid chromatography (HPLC) for their analyses. However, this technique is not powerful enough to meet the separation requirement of proteomics applications, considerably reducing the number of protein and peptide identifications that can be performed. Increasing the analytical power is very important for both life science and research purposes, especially discovery proteomics. One solution is to equip conventional HPLC instruments with thinner and longer columns, thereby enhancing analytical capabilities. This configuration does not incur any additional sample loss since the internal volume of the column remains unchanged, but pressure limitations can quickly become a problem.

Nano-ultra high performance liquid chromatography (UHPLC) technology has emerged as a viable alternative to conventional HPLC, pushing the limits of feasible column designs through higher operational pressure. The technique offers increased pressure rating capabilities, while also allowing scientists to investigate the effectiveness of different column dimensions and solid phase materials and select the most appropriate ones for their application. Nano-UHPLC can be used in conjunction with mass spectrometry systems, enabling an improved number of peptide and protein identifications to be achieved and enhancing laboratory productivity. In particular, online nano-UHPLC allows longer, smaller ID columns improving chromatographic performance.

Nevertheless, migrating from nano-HPLC to nano-UHPLC and coupling directly to a mass spectrometer requires alterations to the set-up to allow for the increase in pressure. It is also necessary to systematically evaluate the experimental parameters, including column dimensions (length and inner diameter), flow rate, beads/solid phase material and bead size, and how they influence the chromatographic performance of the system. It is also important to take into careful consideration that using a high pressure technology can result in increased internal connection leaks, while manufacturing small ID columns can be a rather challenging task. State-of-the-art online nano-UHPLC technology has been designed to effectively address these challenges.