Comparison between monolithic conventional size, microbore and capillary poly( p-methylstyrene- co-1,2-bis( p-vinylphenyl)ethane) high-performance liquid chromatography columns : Synthesis, application, long-term stability and reproducibility

Abstract

Novel monolithic supports (MS/BVPE) were prepared by thermally initiated free radical copolymerisation of p-methylstyrene (MS) and 1,2-bis( p-vinylphenyl)ethane (BVPE). The polymer was synthesised in fused silica capillaries (80 mm × 0.2 mm and 80 mm × 0.53 mm) and in borosilicate glass columns (90 mm × 1.0 mm and 90 mm × 3.0 mm) to yield different HPLC column designs. A comparison of those column dimensions regarding morphology as well as separation efficiency and applicability in bioanalysis is presented. The efficiency towards proteins as well as oligonucleotides was found to be considerably improved with decreasing column I.D. While a 5-protein mixture was baseline separated on all investigated column designs, the separation of small biomolecules like oligonucleotides or peptides on microbore and conventional size glass columns was strongly restricted in terms of resolution due to extensive peak broadening or the occurrence of peak asymmetry. Monolithic MS/BVPE capillary columns up to 0.53 mm I.D., however, proved to be applicable to the fractionation of the whole spectrum of biopolymers, including proteins, peptides, oligonucleotides as well as double-stranded DNA fragments. Due to the fact that reliable chromatography makes great demand on the robustness of the stationary phase, monolithic MS/BVPE capillaries were subjected to a comprehensive reproducibility study including run-to-run as well as batch-to-batch reproducibility.