EFFICIENT SEPARATION OF NATURAL RIBONUCLEOTIDES BY LOW-PRESSURE ANION-EXCHANGE CHROMATOGRAPHY

Chromatographic conditions for the separation of twelve purine and pyrimidine ribonucleotides (mono-, di- and triphosphates) by ion-exchange chromatography were investigated. Two types of anion exchangers with tertiary or quaternary ammonium functionalities (DEAE and QAE types) were compared. Parameters examined included pH of the mobile phase, elution buffer composition and flow-rate. Excellent resolution of all compounds in 50 min was achieved with a strongly basic anion exchanger in the formate form using a linear gradient of ammonium formate with constant formic acid concentration in the mobile phase. The proposed method utilizes (1) a strongly basic anion exchanger based on a macroporous hydrophilic organic polymer, (2) a volatile elution buffer system which permits one-run purification of nucleotides (no further column operation is required, e.g., desalting) and (3) low-pressure liquid chromatographic equipment with simple column preparation. The method was applied to the purification of [U-C-14]adenosine-5'-diphosphate and other radiolabelled nucleotides and their analogues in order to obtain highly radiochemically pure compounds.