Hydrophilic Interaction Chromatography (HILIC) is a liquid chromatographic separation mechanism commonly used for polar biological molecules. While nucleotides are very polar analytes, only a few studies have been conducted on their separation using HILIC. Herein, the use of enhanced-fluidity liquid chromatography (EFLC) for the separation of nucleosides and nucleotides under HILIC conditions is investigated. Enhanced-fluidity liquid chromatography involves using common mobile phases with the addition of substantial proportions of a dissolved gas which provides enhanced mobile phase diffusivity and lower viscosity. The impact of varying mobile phase composition: buffer composition, type of base, salt used, salt concentration and mole fraction of CO 2 was studied to provide optimized HILIC separations. Each of these parameters plays a key role in the retention of the analytes, which demonstrates the complexity of the retention mechanism in HILIC. The tailing of phosphorylated compounds was overcome with the use of phosphate buffer and the addition of a strong base; efficiency and peak asymmetry were compared with the addition of either triethylamine (TEA), 1,4-diazabicyclo

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