Comparison of high-performance ion-exchange and ion-pair liquid chromatographic methods for the separation of tumour cell nucleotides
Kremmer, T.; Boldizsár, M.; Holczinger, L.
Journal of Chromatography 415(1): 53-63
1987
ISSN/ISBN: 0021-9673 PMID: 3584363 Document Number: 304644
Ion-exchange liquid chromatography (IELC) on a novel anion-exchanger, Polyanion SI HR 5/5, and the ion-pair technique (IPLC) using Hypersil ODS and/or MinoRPC reversed phases with tetrabutylammonium phosphate as pairing agent were compared for the separation of nucleotides. Modifications to the concentration gradient in IELC in the range 0.01-0.3 M ammonium phosphate resulted in the simultaneous separation of twelve to fourteen biologically important nucleotides. IPLC studies revealed that the capacity factors and resolution of nucleotides were more sensitive to the ionic strength than the methanol content. It was concluded that a well controlled ion concentration (0.08-0.09M sodium chloride) should be maintained in the mobile phase and a linear methanol gradient ranging from 0 to 20% (v/v) was suitable for optimal resolution. Separations of four nucleosides and twelve nucleotides were further improved using a mixed-type reversed-phase column (C2/C18, MinoRPC). Using these complementary methods, it was possible to reveal the metabolic changes induced by different drug treatments (cyclophosphamide, DL-.alpha.-difluoromethylornithine) in the nucleotide pool of P388 leukaemia cells.