Preparation of phenylboronic acid-silica hybrid monolithic column with one-pot approach for capillary liquid chromatography of biomolecules

A phenylboronic acid-silica hybrid monolithic column for capillary liquid chromatography (cLC) was prepared through one-pot process by using 4-vinylphenylboronic acid (VPBA) and alkoxysilanes simultaneously. The effects of the molar ratio of tetramethyloxysilane/γ-methacryloxypropyltrimethoxysilane (TMOS/γ-MAPS), amount of VPBA, and the volume of diethylene glycol (DEG) on the morphologies, permeabilities and pore properties of the prepared VPBA-silica hybrid monolithic columns were studied in detail. A relatively uniform monolithic structure with high porosity was obtained with optimized ingredients. A series of cis-diol-containing compounds, alkylbenzenes, amides, and anilines were utilized to evaluate the retention behaviors of the VPBA-silica hybrid monolithic column. The result demonstrated that the prepared VPBA-silica hybrid monolithic column exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange apart from the expected affinity interaction. The run-to-run, column-to-column and batch-to-batch reproducibility of the VPBA-silica hybrid monolith were satisfactory with the relative standard deviations (RSDs) less than 1.63% (n = 5), 2.02% (n = 3) and 2.90% (n = 5), respectively, indicating the effectiveness and practicability of the proposed method. In addition, the VPBA-silica hybrid monolithic column was further applied to the separation of proteins and tryptic digest of bovine serum albumin (BSA), respectively. The successful applications suggested the potential of the VPBA-silica hybrid monolith in proteome analysis.