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Next to the development of dedicated low-input sample preparation methods, which aim at reducing sample losses prior to analysis, changes to the chromatographic support material have recently been identified key to the sensitive profiling of low-input proteomics samples by nano-HPLC combined with ESI-MS/MS. While superficially porous stationary phases have been demonstrated to hold the great potential of high chromatographic separation power and to further reduce on-column losses, their expedient integration into standard proteomics nano-flow HPLC systems has long suffered from the intrinsically low loading capacity. However, especially in the context of low-input proteomics, which aims at analyzing protein amounts in the nano-to pico-gram-range, the capacity of these novel reversed-phase (RP) HPLC support materials is no longer a limiting factor.
μPAC™ column technology for single cell proteomics PharmaFluidics The micro-Chip Chromatography Company PharmaFluidics’ μPAC™ (micro Pillar Array Column) technology is a novel and innovative approach to a chromatographic support structure and builds upon micromachining chromatographic separation beds into silicon, with exceptional properties that result in excellent chromatographic performance with high resolution and high sensitivity. The inherent high permeability and low ‘on-column’ dispersion obtained by the perfect order of the separation bed makes µPAC™ based chromatography unique in its kind. The peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions) and therefore components remain much more concentrated during separation resulting in unprecedented separation performance (1). The freestanding nature of the pillars also leads to much lower backpressure allowing a high operational flow rate flexibility with exceptional peak capacities (2). Apart from an improved efficiency, pillar array columns can also be designed with substantially lower flow resistances compared to packed bed columns. The distance between the pillars can be independently controlled from the pillar size, enabling the fabrication of columns over a range of external porosities. The result is a top performing nano LC column that is very robust and is much less prone to sample related column failure. In addition, μPAC™ columns will results in an unprecedented column-to-column reproducibility since all columns will be virtually identical due to the use of the same mask for the micro-machined fabrication production.
Lastly, PharmaFluidics offers also a μPAC™ Trapping column to support the use of 50 and 200 cm long analytical μPAC™ columns. This micromachined trapping column was developed with an identical stationary phase support morphology as the analytical μPAC™ columns to ensure optimal chromatographic performance. Effectively desalting and preconcentrating the analytes of interest onto the trap column offers a large flexibility regarding sample preparation. The unique design of these trapping columns allows loading of relative high volume samples in a bidirectional way at high flow rates (10-20 μl/min) without harming the separation bed integrity.
Overall the gain in sensitivity due to improved peak width and peak capacities provided by these new types of μPAC™ chromatographic columns, makes them extremely attractive for ultrasensitive proteomics applications such as single cell proteomics.
(1) W. De Malsche, H. Gardeniers, G. Desmet, Experimental Study of Porous Silicon Shell Pillars under Retentive Conditions, Anal. Chem. 80 (2008) 5391-5400.
(2) W. De Malsche, J. Op De Beeck, S. De Bruyne, H. Gardeniers, G. Desmet, Realization of 1 × 10E6 Theoretical Plates in Liquid Chromatography Using Very Long Pillar Array Columns,