Reverse-phase ion-pair chromatography of polyvalent ions using oligonucleotides as model substances

Abstract

The retention of oligonucleotides of uridylic and adenylic acids as model substances was investigated employing the reverese-phase ion-pair chromatography technique and using LiChrosorb RP-18 and tetra- n-alkylammonium compounds. The retention sequence is controlled by the charge of the oligonucleotides, thus determining the number of ion pairs formed. The chain length of the n-alkyl group of the ion pair reagent and the content of organic solvent in the hydroorganic eluant also contribute to retention. A semiquantitative model was developed to describe the dependence of k′ on the ion pair concentration in a certain range. A comparison of retention in the reverse-phase ion-pair and ion-exchange chromatographic techniques established distinct differences in the dependence of log k′ on the charge of the oligonucleotide. Hydrophobic interactions also appear to make a larger contribution to retention in reverse-phase ion-pair chromatography than in ion-exchange chromatography.