Optimization of the separation of the Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 × 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10–100 mg.