In HILIC, small variations of the elution conditions often result in differences in the retention greater than the ones observed in RPLC. Therefore, particular attention has to be paid to the selection and the preparation of the eluents.
It is important to know that, generally, the impact on the retention in HILIC is observed for modification of the chromatographic parameters in the following order.
organic solvent content > salt content ~ pH > column temperature
On this basis, the first and most important parameter to optimize is the organic solvent content. As already emphasized, the mobile phase in HILIC is constituted by a mixture of water– water miscible organic solvent, such as in RPLC. However, the elution strength for common HILIC solvents follows the order:
methanol > ethanol > isopropanol > tetrahydrofuran > acetonitrile
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