Ring-opening metathesis polymerization-derived monolithic anion exchangers for the fast separation of double-stranded DNA fragments

Abstract

Ring-opening metathesis polymerization- (ROMP) derived monoliths were prepared from 5-norborn-2-enemethyl bromide (NBE-CH 2Br) and tris(5-norborn-2-enemethoxy)methylsilane ((NBE-CH 2O) 3SiCH 3) within the confines of surface-silanized borosilicate columns (100 × 3 mm I.D.), applying Grubbs’ first generation benzylidene-type catalyst [RuCl 2(PCy 3) 2(CHPh)]. Monoliths were converted into weak anion exchangers via reaction with diethyl amine. The resulting monolithic anion exchangers demonstrated a very good potential for the anion-exchange separation of nucleic acids applying a phosphate buffer (0.05 mol/L, pH 7) and NaCl (1.0 mol/L) as a gradient former. Fast and efficient separations, indicated by sharp and highly symmetric analyte peaks, were established. Except for the 267 and 298 base pair fragments, the eleven fragments of a ds-pUC18 DNA Hae III digest were baseline separated within ∼8 min. Nineteen fragments of a ds-pBR322 Hae III digest were separated within ∼12 min. There, only the 192 and 213 base pair fragments and the 458, 504 and 540 base pair fragments coeluted. A ds-pUC18 DNA Hae III digest was used as a control analyte in evaluating the influence of organic additives on the mobile phase such as methanol and acetonitrile on nucleic acid separation. Methanol, and even better, acetonitrile improved the separation efficiency and shortened the analysis time.