Zwitterion-Pair chromatography of nucleotides and related species

Abstract

A new form of ion-pair chromatography is established and characterised, in which a zwitterion-pairing agent (11-amino undecanoic acid, C11AA, at up to 2 m M concentration in eluent) is used to enhance the retention of nucleotides in reversed-phase high-performance liquid chromatography. Excellent separations of these compounds (giving up to 5000 plates for a 100-mm column) are obtained within the pH range 4 to 6 (maintained by 75 m M phosphate buffer), with retention at pH 4 being up to twenty times that at pH 6. At fixed pH retention increases directly with the amount of C11AA adsorbed by the stationary phase. It is proposed that the enhancement of retention by addition of C11AA and its pH dependence arises from the formation of quadrupolar ion pairs between C11AA and the nucleotides. The order of elution, in general, is monophosphates (least retained), diphosphates, triphosphates. However, these groups can either be separated entirely from each other, or made to interleave by suitable adjustment of pH and C11AA concentration. Deoxy- and cyclic-nucleotides are more strongly retained than their parent compounds. The method is flexible and has great potential for further development.