Computational Fluid Dynamics-Guided Slurry Packing of Capillary Columns for High-Efficiency NanoLC−MS Proteomics

Packed capillary columns serve as the core separation unit in mass spectrometry-based proteomics. However, fine particles, particle aggregates, and broad particle size distribution inherent to commercial sub-2 μm C18 packing materials can induce column bed heterogeneity, which ultimately limits separation performance. This work proposes a computational fluid dynamics (CFD)-guided packing control strategy: a classification operation window is constructed based on the particle size-dependent centrifugal transport model established in a glycerol-water binary system. Following centrifugal classification of commercial C18 particles, target capillary columns are fabricated via slurry packing. After classification, the particle size distribution of the packing material is significantly narrowed: the coefficient of variation decreases from 28.5% to 10.4%, and the D90/D10 ratio reduces from 2.48 to 1.30, verifying the effective removal of fine particles. Compared with capillary columns prepared from unclassified raw packing, columns fabricated with classified packing exhibit a lower column pressure drop and substantially improved mass transfer kinetics. In nanoLC-MS/MS analyses, the average protein signal intensity for bovine serum albumin (BSA) increases by 122%, and the number of identifiable proteins in human colorectal adenocarcinoma cell (DLD-1) lysates rises from 6763 to 7379. Given that the stationary phase chemical properties remain unchanged before and after classification, the performance improvement is attributed to enhanced mass transfer efficiency in the column bed after the removal of fine and irregular particles. The results obtained in this study confirm that CFD-guided particle population control is an effective strategy for improving the performance of slurry-packed capillary columns and provides a generalizable packing material approach for the optimization of nanoLC-MS-based proteomic separation.