Separation of oligonucleotides, nucleotides, and nucleosides on columns of polystyrene anion-exchangers with solvent systems containing ethanol

Abstract

A study of the absorption of oligonucleotides by polystyrene anion-exchangers has shown that a large component of the total binding forces consists of noionic interactions and that these interactions can be substantially reduced in the ion-exchange chromatography of these molecules by adding ethanol to the eluting solvents. This technique permits the separation of oligonucleotide species at pH values near neutrality and with solvent systems containing gradients of relatively low concentrations of chloride ion. Chromatography of oligonucleotides with solvents containing 40% ethanol yields reproducible retention volumes that are dependent on their chain lengths and, to some extent, on their base compositions and the presence of 3′-terminal or 5′-terminal phosphate groups in the molecules. The flexibility in the use of solvent systems with three variable factors: pH, ethanol concentration, and gradient of salt concentration, can also be exploited in the separation of mixtures of nucleosides and nucleotides, and this observation has permitted the development of simple procedures for the characterization of ribo- and deoxyribooligonucleotides with respect to their base compositions, their chain lengths, and the identity of their 3′- and 5′-terminal nucleosides.