Digestion to nucleotides followed by gradient or stepwise elution from anion-exchange columns has been a preferred method for the base analysis of nucleic acids (l-6).
In this communication we describe a system for the separation of nucleotides on a cation-exchange column that is particularly advantageous for base analysis of nucleic acids. Previous applications of cationexchange columns to nucleotide separations have been reported (7-9). The separation of 5'-deoxynucleotides using a system similar to ours was demonstrated in unpublished experiments of C. F. Crampton and J. L. Rodeheaver which were mentioned by Cohn (10).
Cohn and Uziel (11) have recently developed a system that employs digestion to the nucleoside level followed by elution from a cationexchange column with a single solvent. This' system permits immediate reuse of columns for sequential analyses and eliminates spectrophotometric problems caused by a changing solvent. Peaks are sharp, so that nucleosides emerge at high concentration. Using a sensitive continuousflow spectrophotometer, Cohn was able to obtain quantitative base compositions on nanomole quantities of nucleoside mixtures in one hour.
The system we have developed extends all these advantages to nucleotide separation, and thus eliminates the need to convert nucleotides to nuclosides and affords the opportunity for quantitation by radiophosphorous as well as by spectrophotometry.
Our system co-chromatographs the 2'-and 3'-ribonucleotide isomers so, that alkaline hydrolyzates of RNA give sharp peaks. The system operates at room temperature and ordinary pressure, and the use of a volatile buffer facilitates recovery of purified nucleotides by evaporation. no