This dissertation focuses on the preparation of polymeric monolithic capillaries for ion exchange chromatography of peptides and proteins, since polymeric monoliths have shown promise for providing improved protein separations. Characteristics of monolithic columns include low back pressure, simplicity of fabrication and biocompatibility. Preparation of strong and weak cation-exchange monolithic stationary phases in 75 μm I.D. capillaries by direct in situ copolymerization was achieved using various functional monomers including sulfopropyl methacrylate, phosphoric acid 2-hydroxyethyl methacrylate, bis[2-(methacryloyloxy)ethyl] phosphate and 2-carboxyethyl acrylate with polyethylene glycol diacrylate and other PEG materials. The resulting monoliths provided excellent ion exchange capillary LC of peptides and proteins with good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64%) reproducibilities. Narrow peaks were obtained and peak capacities of over 20 were achieved. Dynamic binding capacities of over 30 mg/mL of column volume for lysozyme were measured. A single monomer was used to synthesize a phosphoric acid containing monolith to improve its stability and reproducibility. The monolith was synthesized from only BMEP in 75 μm I.D. UV transparent fused-silica capillaries by photo-initiated polymerization. A dynamic binding capacity (lysozyme) of approximately 70 mg/mL of column volume was measured. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. Good reproducibilities were achieved. Zwitterionic monolithic columns based on photo-initiated copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betain and poly(ethylene glycol) diacrylate were prepared in 75 μm I.D. fused silica capillaries for hydrophilic interaction chromatography. Inverse size exclusion chromatography was used to characterize the pore structure of the resulting monolith. A typical hydrophilic interaction chromatography mechanism was observed when the organic content in the mobile phase was higher than 60%. Good separations of amides, phenols, and benzoic acids were achieved. The effects of mobile phase pH, salt concentration, and organic modifier content on retention were investigated.
College and Department
Physical and Mathematical Sciences; Chemistry and Biochemistry
BYU ScholarsArchive Citation
Chen, Xin, "Development of Monolithic Stationary phases for Cation-Exchange Capillary Liquid Chromatography of Peptides and Proteins" (2011). Theses and Dissertations. 2467.
monolith, ion exchange, hydrophilic interaction, liquid chromatography