Use of Reverse Phase Ion Pair Chromatography to Fractionate and Purify DNA Fragments and Monomeric Components of RNA.

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Abstract
A study of the separation of oligonucleotides using reverse phase ion pair chromatography is reported. The work includes a comparison of several countercations (triethylammonium, tetraethylammonium, tetrabutylammonium and tetrapentylammonium) as possible candidates for the formation of ion pairs with oligonucleotides in order to obtain an optimum separation on a C18 reverse phase support, using high performance liquid chromatography. The effect of the countercation concentration was also explored. It is shown that 0.0075 M tetrabutylammonium sulfate (pH 6.9) brings about a satisfactory separation up to a hexadecanucleotide DNA sequence. The following mixtures are clearly resolvable into 2 groups using tetrapentylammonium phosphate (6.25 .times. 10-4 M) and a linear gradient of acetonitrile: Four ribonucleosides and their 2'',3''-cyclic phosphates: 4 ribonucleosides and their 5''-monophosphates; 4 ribonucleosides and their 2''- and 3''-monophosphates.