A New Parallel High-Pressure Packing System Enables Rapid Multiplexed Production of Capillary Columns

Reversed-phase HPLC is the most commonly applied peptide-separation technique in MS-based proteomics. Particle-packed capillary columns are predominantly used in nanoflow HPLC systems. Despite being the broadly applied standard for many years, capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column packing with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as the driving fluid, our system replaces the traditional setup of helium-pressured packing bombs. By using 10× multiplexing, we have reduced the production time to just under 2 min for several 50 cm columns with 1.9-µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters and lengths packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station. [Display omitted] •We present a newly designed high-pressure packing station for capillary columns.•Detailed part lists and manufacturing instructions included.•40 to 800 times faster packing of capillary columns for high-performance proteomics.•Columns produced with the presented setup have state-of-the-art quality metrics. Capillary columns in nanoflow application are a basis of high-performance LC-MS–based proteomics but are difficult or expensive to procure. We present a new high-pressure packing station for capillary columns (1000–3000 bar) together with detailed instructions for reproducing our setup. Compared with previous publications, this produces capillary columns 40 to 800 times faster at undiminished chromatographic performance.