HILIC and temperature: explanation of inverse Van´t Hoff plots

Hydrophilic Interaction Chromatography (HILIC) of nucleoside triphosphates with temperature as a separation parameter

Elin Johnsen, Steven Ray Wilson, Ingvild Odsbu, Andreas Krapp, Helle Malerod, Kirsten Skarstad and Elsa Lundanes
Journal of Chromatography A, (ahead of print):
doi:10.1016/j.chroma.2011.01.066

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. 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 with a flow rate of 200 μL/minute. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 70 minutes using a flow rate of 100 μL/minute.