Analysis of Nucleosides on Columns of Fractionated

Recent investigations carried out in several laboratories have demonstrated that columns of Sephadex G-25 and G-10 can be used for separating nucleosidesl 5 . The s~parations reported so far were obtained on large amounts of material (at least 300 nmoles of each nucleoside) and required over 10 h. We report here a new method allowing the separation in 2.5 h of the four common nucleosides, of either series, at the level of 1-10 nmoles. This new procedure, involving chromatography of nucleosides at pH lOA on fractionated, Sephadex G-I0 beads, has already been extensively tested in our6 as well as in other laboratories79 . A new experimental set-up and a rapid and precise calculation method, requiring 15-20 min per analysis, have been developed in connection with the new procedure. The present method has been largely inspired by an improved procedure for the separation of nucleosides on columns of Bio Gel P-2, also developed in our laboratoryl0. The two methods have comparable merits, at least as far as the separation of the four common nucleosides is concerned. The order of elution of nucleosides trom the two columns is not the same, however; the choice of one method over the other may therefore depend upon the nucleoside composition of the mixture to be analyzed. In addition it is likely that one method has distinct advantages over the other when dealing with the separation of other nucleosides. As in the case of P-2 columns, nucleotides are much less retarded than nucleosides. This permits analyses of nucleosides in the presence of nucleotides and chain length determinations.