Joseph X. Khym

The co-use of retention time and bandwidth measurements in evaluations of nucleotide pools by ion-exchange chromatography.

Abstract In our studies involving the metabolic functions of free nucleotides in mammalian cells, we have found that certain less common nucleoside phosphates and nonnucleotide components, such as the nucleosides and bases, coelute with nucleotides such as AMP, GMP, CDP, ADP, etc. in our high-resolution anion-exchange procedure. This type of interference was discovered by the rigorous comparison of both retention times and bandwidths of solute bands from unknown mixtures and authentic compounds which had been chromatographed under identical conditions. To eliminate this interference, we carry out a fractionation of acid-soluble components by chromatography on polyacrylamide-gel columns. This preliminary step allows nucleotides to be separated as a group from bases and nucleosides. Examples of such combined chromatography are given, and the use of this technique in obtaining valid quantitations for nucleotide pools is demonstrated.