Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated using two 15 cm, 2.1. mm I.D. ZIC-pHILIC columns coupled in series, using simple LC-UV instrumentation. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 minutes using a flow rate of 200 µL/minute. In complex cell culture samples dNTP/NTPs could be selectively separated in 70 minutes using a flow rate of 100 µL/minute.