Combined pH gradient and anion-exchange high-performance liquid chromatographic separation of oligodeoxyribonucleotides

A novel method of elution using a pH gradient to separate small thymidine- and guanosine-containing oligonucleotides on a Pharmacia Mono Q HR anion-exchange column is described. The method is based on the alkaline titration of ring protons of the thymine and guanine base moieties and results in excellent separations of di-, tri- and tetranucleotides that either are not resolved in salt gradients near neutral pH or require long elution times when salt gradient elution is used with strongly alkaline eluents.