Hydrophobic, Pellicular, Monolithic Capillary Columns Based on Cross-Linked Polynorbornene for Biopolymer Separations

Abstract

Monolithic capillary columns were prepared by transition metal-catalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene inside a silanized 200-μm-i.d. fused-silica capillary using a mixture of toluene and 2-propanol as porogen and Cl₂(PCy₃)₂Ru(CHPh) as initiator. The synthesized columns allowed the rapid and highly efficient separation of single- and double-stranded nucleic acids by ion-pair reversed-phase high-performance liquid chromatography and of proteins by reversed-phase high-performance liquid chromatography. Compared to 3-mm-i.d. analytical columns synthesized from an identical polymerization mixture, a considerable improvement in the peak widths at half-height of oligonucleotides in the order of 60−80% was obtained. Significant differences in morphology between the capillary column, where the surface of the monolith was rather soft and rugulose, and the analytical column, where the surface was very sharp and smooth, were observed, most probably due to differences in polymerization kinetics. The synthesized monoliths were successfully applied to the separation of the diastereomers of phosphorothioate oligodeoxynucleotides. To confirm the identity of the eluting compounds on the basis of their intact molecular masses, the chromatographic separation system was on-line hyphenated to electrospray ionization mass spectrometry.