Analytical Techniques for Cell Fractions. Iii. Nucleotides and Related Compounds.

The methods described in this paper are devised for the separation of the common nucleotides, nucleosides, and purine and pyrimidine bases on one column at one pH with the compounds quantitated by a sensitive automatic technique. Following the classical work of Cohn and co-workers (l-6)) nucleotides have been generally separated on anion-exchange columns at acid pH’s, nucleosides on anion-exchange columns at alkaline pH’s, and purine and pyrimidine bases either on anion exchangers or on cationexchange columns in the acid range (l-7). Separation, though incomplete, of mixtures of bases, nucleosides, and nucleotides on a single anionexchange column (2) has been achieved by using buffers ranging from pH 10.2 to 2.75. The observation that the pyrimidine nucleotides were much less strongly adsorbed, per unit charge on the molecule, than the purine nucleotides (2) suggested that both nucleotide solubility and interaction with the resin matrix through nonionic forces (in addition to the expected ionic effects) may be responsible for the elution patterns observed. The possibility was considered, therefore, that bases, nucleosides, and nucleotides might be eluted in the same relative order from the same column, providing the proper conditions could be found. Brief reports of this work have appeared elsewhere (8, 9).