Control of oligonucleotide retention on a pH-stabilized strong anion exchange column

Abstract

Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alkaline conditions. In this article, we report the use of an anion exchange column prepared from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and composition. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5′ or 3′ terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column’s hydrophobic interactions when CH 3CN is used with NaClO 4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base composition-dependent) separations.