Nano- and Capillary LC Separations Using Micro-Pillar Array Columns for Maximal Efficiency and Robustness

As an alternative to the conventional packed bed nano LC columns that are frequently used in bottom-up proteomics research, a breakthrough in column manufacturing recently led to the introduction of silicon-based micromachined nano LC chip columns known as micro pillar array columns (μPAC™). These columns have an inherent high permeability and an unprecedented low ‘on-column’ dispersion due to the perfect order of the separation bed .Due to the perfect order, the peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions). Sample components remain therefore much more concentrated during separation resulting in unprecedented separation performance. The freestanding nature of the pillars also leads to much lower backpressure allowing a high operational flow rate flexibility with exceptional peak capacities. In addition, the monolithic nature of the beds makes them extremely rugged and guarantees an unprecedented longevity of the columns. Moreover, it also guarantees run-to-run time repeatability that is about an order of magnitude better than packed bed columns, thus facilitating and accelerating protein identification. After the introduction of a 200 cm long column, which is ideally suited to perform comprehensive proteome research, a 50 cm long μPAC™ column is now available which can be used in a more routine research setting. With an internal volume of 3 μL, this column is perfectly suited to perform high throughput analyses with shorter gradient solvent times (30, 60 and 90 minute gradients) and it can be used over a wide range of flow rates, between 100 and 2000 nL/min. Recently performed experiments with 500 ng of HeLa cell digest indicate that an increase in protein identifications up to 50% and a gain of 70% in peptide identifications can be achieved when comparing the 50 cm μPAC™ column to the current state-of-the-art packed bed columns. In the recent months, we have also been developing a column that is deeper and wider to meet the requirements of capillary LC flow rate. New data on these columns will be presented at the conference.