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The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 μm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low-complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV-4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.

HPLC microchips are investigated experimentally with respect to packing density, pressure drop-flow rate relation, hydraulic permeability, and separation efficiency. The prototype microchips provide minimal dead volume, on-chip UV detection, and a 75 mm long separation channel with a ca. 50 microm x 75 microm trapezoidal cross-section. A custom-built stainless-steel holder allowed to adopt optimized packing conditions. Separation channels were slurry-packed with 3, 5, and 10 microm-sized spherical, porous C8-silica particles. Differences in interparticle porosity, permeability, and plate height data are analyzed and consistently explained by different microchannel-to-particle size (particle-aspect) ratios and particle size distributions.