Temperature effects on retention in reversed-phase liquid chromatography of nucleosides and their bases using water as the mobile phase

Using elevated temperature, a number of hydrophobic organic compounds were separated rapidly by reversed-phase liquid chromatography (RP–LC) using a conventional octadecylsilane (ODS) stationary phase and pure water as the mobile phase. Thirteen nucleosides and their bases were used as representative hydrophobic analytes and two types of polymeric ODS-packed columns were chosen as representative reversed-phase columns. The experimental results showed that the partitioning behaviour of the hydrophobic analytes between the stationary and mobile phases was extremely dependent on the temperature of the columns. By elevating the temperature of the columns or by using temperature programming methods, the analytes which were bound by the stationary phase could be released to the mobile phase without need for any organic solvent in the mobile phase. That is, the separation could be achieved with pure water as the mobile phase. The observed effects were attributed to two effects. First, the elevated temperature enhanced the mobility of the bonded stationary phase, resulting in reduced hydrophobic interactions between the analytes and the stationary phase. Second, elevating the temperature enhanced the water solubility of the analytes, thereby increasing their transfer to the mobile phase. This technique can be employed in order to reduce or even eliminate the use of organic solvents in some RP–LC separations.