Anion‐Exclusion and Anion‐Exchange Chromatography of Nucleotides

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  • RSRam P. Singhal
    Ram P. Singhal
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Abstract
Anion‐exclusion chromatography, previously shown to resolve nucleosides, has been applied to separate nucleotides on “cation‐exchange” columns at acid pH values. The study of pH and ionic effects indicates that these separations occur by anion repulsion, not by cation exchange. Cation exchange, previously held reponsible for these separations, is inoperative since nucleotides at acid pH values bear net negative charges, as do the fixed anions of the resin. The separations can be explained by anion‐exclusion chromatography) which involves two factors: repulsion between identically charged solute and resin ions, and partition between aqueous eluant and organic resin matrix. The results indicate that separations by anion exclusion are as good as or better than those by anion‐exchange chromatography.Anion‐exchange chromatography has also been reinvestigated to develop systems that can be used to separate the four major nucleotides in single eluants and to separate most nucleosides, nucleotides and other RNA hydrolysis products in one analysis.The ion‐exclusion separations are compared with those of anion‐exchange chromatography. The advantages and disadvantages of both methods are discussed. A typical example of the analysis of a nucleoside‐nucleotide mixture derived from the total hydrolysis of tRNAs for the assay of the base comparison and for the analysis of terminal end groups is described.

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