Preparation and evaluation of poly(4-vinylphenylboronic acid-co-pentaerythritol triacrylate) monolithic column for capillary liquid chromatography of small molecules and proteins

A mixed-mode monolithic stationary phase was prepared for capillary liquid chromatography (cLC) by in situ copolymerization of 4-vinylphenylboronic acid (VPBA) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of ethylene glycol/cyclohexanol. The monomer of VPBA functioned as ion-exchange sites, hydrophilic ligands, hydrophobic groups and affinity sites, while PETA was introduced as a hydrophilic crosslinker. The resultant monoliths with different column properties (e.g. morphology, permeability and selectivity) were optimized by adjusting the ratio of VPBA to PETA and the composition of porogenic solvent. The results showed that the selectivity of the monoliths increased with increasing content of VPBA in the polymerization mixture. A series of alkylbenzenes, amides, and anilines were used to evaluate the column performance in terms of hydrophobic, hydrophilic and cation-exchange interactions. At an optimized flow rate of 50 μL/min (corresponding to 0.265 mm/s), the monolith exhibited high column efficiencies of 43,000–100,000 plates/m for alkylbenzenes. Good repeatability was obtained with relative standard deviation (RSD) of retention factor (k) less than 0.65% for run-to-run (n = 5) and less than 2.49% for column-to-column (n = 5). In addition, the poly(VPBA-co-PETA) monolithic column was applied to the separation of phenols, nucleobases, and proteins, respectively. These successful applications demonstrate the purposed monoliths are promising for cLC separation of small molecules and proteins.