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

Chromatographic conditions for the separation of twelve purine and pyr imidine 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) w ere compared. Parameters examined included pH of the mobile phase, elu tion buffer composition and flow-rate. Excellent resolution of all com pounds in 50 min was achieved with a strongly basic anion exchanger in the formate form using a linear gradient of ammonium formate with con stant formic acid concentration in the mobile phase. The proposed meth od utilizes (1) a strongly basic anion exchanger based on a macroporou s hydrophilic organic polymer, (2) a volatile elution buffer system wh ich permits one-run purification of nucleotides (no further column ope ration is required, e.g., desalting) and (3) low-pressure liquid chrom atographic equipment with simple column preparation. The method was ap plied to the purification of [U-C-14]adenosine-5'-diphosphate and othe r radiolabelled nucleotides and their analogues in order to obtain hig hly radiochemically pure compounds.