Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides

Hydrophilic interaction chromatography (HILIC) is a liquid chromatographic separation mechanism commonly used for polar biological molecules. The use of enhanced-fluidity liquid chromatography (EFLC) with mixtures of methanol/water/carbon dioxide is compared to acetonitrile/water mobile phases for the separation of nucleosides and nucleotides under HILIC conditions. Enhanced-fluidity liquid chromatography involves using common mobile phases with the addition of substantial proportions of a dissolved gas which provides greater mobile phase diffusivity and lower viscosity. The impact of varying several experimental parameters, including temperature, addition of base, salt, and CO₂ 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 salts and the addition of a strong base; efficiency and peak asymmetry were compared with the addition of either triethylamine (TEA), 1,4-diazabicyclo 2.2.2] octane (DABCO) or 1,5-diazabicyclo 4.3.0] non-5-ene (DBN). DBN and DABCO both led to increased efficiency and lower peak asymmetry; DBN provided the best results. Sodium chloride and carbon dioxide were added to enhance the selectivity between the analytes, giving a successful isocratic separation of nucleosides and nucleotides within 8 min. The retention mechanism involved in EFL-HILIC was explored by varying the temperature and the mole fraction of CO₂. These studies showed that partitioning was the dominant mechanism. The thermodynamics study confirmed that the solvent strength is maintained in EFLC and that a change in entropy was mainly responsible for the improved selectivity. The selectivity using methanol/water/carbon dioxide varied greatly compared to that obtained with acetonitrile/water. Finally while this study highlights the optimization of EFL-HILIC for the separation of nucleosides and nucleotides under isocratic conditions, this is also an example of the broad range of polarities of compounds that EFL-HILIC can separate.