Boronic acid-fumed silica nanoparticles incorporated large surface area monoliths for protein separation by nano-liquid chromatography.

In this study, a novel boronic acid-fumed silica nanoparticles incorporated hybrid monolithic stationary phase for glycoprotein separation by nano-liquid chromatography was developed. The stationary phase was prepared in 100 μm capillary by an in situ copolymerization of methacryloyl-fumed silica nanoparticle (MFSNP), 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent composed of cyclohexanol and dodecanol. The preparation of the monolith was optimized by investigating the ratio of functional monomer to crosslinker and the effect of MFSNP content. The resulting monolithic column was functionalized with 3-aminophenylboronic acid (3-APBA). The column morphology, pore size, and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photo electron spectroscopy, and BET equation by means of the nitrogen adsorption-desorption isotherm, respectively. Good permeability stability and column efficiency were observed on the monolith with nano-flow. The results also indicated that the MFSNP content was very important for final preparation of the monolithic stationary phase. The monolith with MFSNP could achieve better separation than one without MFSNP. The chromatographic performance of the monolith with respect to hydrophobic/affinity interactions was evaluated by the separation of alkylbenzene derivatives, proteins, and glycoprotein, respectively. The column efficiencies for alkylbenzenes on the hybrid monolithic column reached to 15,600-25,000 plates/m at the velocity of 1.2 mm/s in nano-liquid chromatography.