A 28-membered macrocyclic polyamine, 4,8,12,18,22,26-hexaaza-1,15-dioxacyclooctaeicosane ([28]ane-N 6 O 2 ) with two dipropylenetriamine moieties bound together by diethylether unit was able to form polyprotonated, highly charged species in a wide pH region. It was covalently bonded on the inner surface of the fused silica capillary. The capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. With the phosphate buffer (30 mM) and an applied voltage of -15 kV, the influence of buffer pH and temperature on the interaction between nucleotides and the bonded phase were investigated. The results indicated that a coordination reaction occurs between the analytes and the bonded phase, and this is followed by cleavage of the terminal phosphate. As a result we found three peaks for each nucleoside triphosphate and two peaks for the diphosphates after samples were injected at 35 • C, while only two peaks for the triphosphates and no splitting for the diphosphates at 20 • C were indicated. In other words, a significant effect of temperature on the hydrolysis was observed, and the bonded phase showed a preference for the binding of nucleoside triphosphate over nucleoside diphosphate.