Comparative performance of ion exchange and ion-paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples.

We have compared anion exchange chromatography on APS-Hypersil (4.6 x 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 x 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange systems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.