One-pot preparation of glutathione–silica hybrid monolith for mixed-mode capillary liquid chromatography based on “thiol-ene” click chemistry

•A GSH–silica hybrid monolith was prepared via thiol-ene click reaction in one-pot process.•The obtained hybrid monolith showed a mixed-mode chromatographic behavior.•It can be applied to the separation of a series of small molecules and protein tryptic digests.

A novel glutathione (GSH)–silica hybrid monolithic column synthesized via a combination of thiol-ene click reaction and one-pot process was described, where thiol-end GSH organic monomer and 2,2-azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethyloxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) and then introduced into a fused-silica capillary for simultaneous polycondensation and “thiol-ene” click reaction to form the GSH–silica hybrid monolith. The effects of the molar ratio of TMOS/γ-MAPS, the amount of GSH, and the volume of porogen on the morphology, permeability and pore properties of the prepared GSH–silica hybrid monoliths were studied in detail. A uniform monolithic network with high porosity was obtained. A series of test compounds including alkylbenzenes, amides, and anilines were used to evaluate the retention behaviors of the GSH–silica hybrid monolithic column. The results demonstrated that the prepared GSH–silica hybrid monolith exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange interaction. The run-to-run, column-to-column and batch-to-batch reproducibilities of the GSH–silica hybrid monolith for phenols’ retention were satisfactory with the relative standard deviations (RSDs) less than 1.3% (n = 5), 2.6% (n = 3) and 3.2% (n = 3), respectively, indicating the effectiveness and practicability of the proposed method. In addition, the GSH–silica hybrid monolith was applied to the separation of nucleotides, peptides and protein tryptic digests, respectively. The successful applications suggested the potential of the GSH–silica hybrid monolith in complex sample analysis.