Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances.

•Generic ion-exchange method for analytical and preparative separation of nucleotides.•Chromatographic resolution of over 20 nucleotides in a single experimental run.•The use of a cost-effective resin available in bulk format for preparative separation is demonstrated.•Aqueous ammonium bicarbonate-based eluent profile allows convenient drying process.•Fully implemented for preparative separation of nucleotides in multicomponent reaction mixtures.