An insight into the phenomena involved in a multiple-function stationary phase for the capillary electrochromatographic separation of 2'-, 3'-, and 5'-monophosphorylated nucleoside isomers

The electrochromatographic separations of 2'-, 3'- and 5'-monophosphates of adenosine, guanosine, cytidine, and uridine were carried out with an open-tubular capillary column which was wall-coated with a highly selective reagent, 28-membered macrocyclic polyamine, 4, 8, 12, 18, 22, 26-hexaaza-1,15-dioxacyclooctaeicosane (28]ane-N6O2). The effects of pH, composition and concentration of background electrolyte (BGE), applied voltage, column length, and the additive of the BGE, such as metal ions, borate, beta-cyclodextrin and organic solvent on the separation of these monophosphorylated nucleotide isomers were investigated. The results suggested that the interactions between analytes and the bonded groups on the wall predominantly comprise anion coordination and anion exchange in addition to the electrophoresis. A well-resolved electrochromatogram was obtained with the capillary column of 100 cm (75 cm effective length) x 75 microm inside diameter (ID), citrate buffer (20 mM, pH 3.99), applied voltage of -22 kV and detection at 254 nm. Column efficiency was found with the average theoretical plate numbers of 119,500/m and a low detection limit of 0.01 microM level could be achieved for the separation of these isomers.