Polymeric cation-exchange monolithic columns containing phosphoric acid functional groups for capillary liquid chromatography of peptides and proteins

Abstract

Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-μm i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269 mg/mL were measured for the PAHEMA–PEGDA and BMEP–PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15 min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA–PEGDA monolith. The BMEP–PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP–PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.