Effect of pH, Temperature, and Ionic Strength on Reversed-Phase Ion-Pair High Performance Liquid Chroma-Tography of Purine Nucleotide Monophosphate

Abstract

Ion-pair reversed phase high performance liquid chromatography on Zorbax ODS was applied to the separation of the purine nucleotide monophosphates, GMP, IMP, XMP, AMP and ZMP. Three parameters (pH, ionic strength and temperature) which greatly effect ionic equilibrium were studied as to their effect on retention and resolution of the purine nucleotide monophosphates (PuMPs). GMP and IMP essentially co-elute at pH 4 - 6.25 but GMP elutes earlier than IMP as the pH is lowered from 4.0 to 2.25. Lowering the pH 6.25 to 2.25 lowers ḱ of those PuMPs (GMP, XMP, AMP and ZMP) with a pKa between 2 and 4 but not the ḱ of IMP which does not have a pKa in the region.

Column temperature was varied from 15[ddot] to 45[ddot] in 5[ddot] increments for several pH values from 2.25 to 6. The effect of column temperature was similar for all pHs checked. As temperature was elevated above 25[ddot], there was a decrease in ḱ which resulted in a deterioration in resolution and as the temperature was decreased to 15[ddot] there was an increase in ḱ and a concomitant improvement in resolution, but there was no resolvement of peaks which co-elute at 25[ddot]. Similarly, an increase in ionic strength decreased ḱ and decreasing ionic strength increased ḱ. Good resolution of the selected PuMPs is obtained between pH 2.25 and 3 with 20 mM kPO 4. 5mM tetrabutylammonium hydroxide and 3.5% acetonitrite. Some improvement in resolution is observed below 25[ddot] but there is an increase in backpressure. Lowering the phosphate concentration below 20 mM leads to large ḱ with very broad peaks.