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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.

The average interparticle voidage or porosity (εinter) in cylindrical capillaries is studied in dependence on the column diameter (dc) to particle diameter (dp) ratio for 5 < dc/dp< 50. Using optimized slurry and packing solvents, high pressure and ultrasonication, 5 μm‐sized porous C18‐silica particles were slurry‐packed into fused‐silica capillaries having ids from 30 to 250 μm. Packing densities are assessed by a polystyrene standard which is size‐excluded from the intraparticle pore space of the packings. Fordc/dp> 35 densely packed beds are realized (εinter= 0.36–0.37), while for decreasing aspect ratios an exponential increase in εinter is observed reaching εinter ∼︁ 0.47 at dc/dp= 5. This behaviour is ascribed to a combination of the geometrical wall effect operating in the direct vicinity of the column wall, caused by the inability of the particles to form a dense packing against the hard surface of the column wall, and particle characteristics like the size distribution, shape and surface roughness. Results are compared with the literature data to address also the importance of absolute particle size in studying structure–transport relations in packed beds in dependence on the aspect ratio dc/dp.