Comprehensive Optimization of Packing Parameters for Hydraulic‐Packed Capillary Columns

ABSTRACT Rationale The performance of the capillary column directly impacts the separation efficiency of complex sample in liquid chromatography–mass spectrometry–based proteomics studies. The hydraulic packing system offers an effective solution by reducing packing time and expediting the preparation process of column preparation. However, its operational complexity and strict parameter regulation requirements hinder efficient application. Methods This study developed an innovative device for the hydraulic capillary column packing, followed by a systematic optimization and evaluation of crucial parameters such as packing pressure, magnetic stirring rate, concentration of solid‐phase material, and choice of packing solvent in the capillary column packing process. Results The comprehensive evaluation of the prepared capillary columns showed that the hydraulic packing method enabled rapid and large‐scale preparation, while also demonstrating exceptional stability and reproducibility. Conclusions The proposed hydraulic capillary column packing strategy in this study holds theoretical and practical implications for advancing the technology of column preparation, enhancing separation efficiency, and expanding the depth of analysis in proteomics research.