Monolithic poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) capillary columns as novel styrene stationary phases for biopolymer separation

Novel monolithic capillary supports (200 μm I.D.) were prepared by polymerisation of methylstyrene (MS) and 1,2-bis(p-vinylphenyl)ethane (BVPE) as a crosslinker in the presence of inert diluents (porogens). These polymeric reversed-phases (MS/BVPE) showed excellent mechanical stability and minimised swelling in organic solvents. The chromatographic potential of monolithic MS/BVPE as a stationary phase for micro-high-performance liquid chromatography (μ-HPLC) was investigated by the separation of proteins and peptides applying reversed-phase (RP) and nucleic acids applying ion-pair reversed-phase (IP-RP) conditions. The permeability and chromatographic efficiency of the capillary columns were found to be highly influenced by the total monomer to porogen content as well as by the microporogen nature and its ratio. In the course of these optimisation studies, monoliths covering a broad permeability range were fabricated. The application of volumetric flow rates up to 200 μl/min allowed swift resolution of proteins, while smaller biomolecules were successfully separated at a higher overall porosity. A protein test mixture containing ribonuclease A, cytochrome c, α-lactalbumin, β-lactoglobulin B and ovalbumin was thus baseline separated in 35 s, a homologous series of phosphorylated oligothymidylic acids [d(pT)12–18] in less than 2 min.