Hydrophilic interaction liquid chromatography (HILIC) is a chromatographic mode used mainly for the separation of polar compounds that are too hydrophilic to be retained by a typical alkyl reversed phase column.

The Stationary Phase

In HILIC, the stationary phase is always a polar packing material. The type of interactions between the analyte and the stationary phase are mainly polar interactions, such as ionic, ion-dipole, or hydrogen-bonding. There is also a partition between the water-enriched layer in the surface of the stationary phase and the acetonitrile-rich mobile phase (figure 1).

There are a wide variety of stationary phases that work in HILIC mode: unbonded silica (figure 2), diol bonded phases (figure 3), amino phases (figure 4), and pentafluorophenyl phases, among others.

The Mobile Phase

Mobile phases in HILIC usually contain a high percentage of organic solvent, commonly acetonitrile, and a low percentage of aqueous buffer. Methanol is not recommended in HILIC as it does not allow the formation of the bilayer.

The aqueous mobile phase under HILIC conditions acts as the strong solvent. So if a gradient is to be considered, the amount of water should be gradually raised in order to increase the mobile phase strength. The buffer selection is especially critical in HILIC: the concentration and pH will extremely affect the selectivity (figure 3). 

Ammonium acetate and ammonium formate are very popular buffers in HILIC as they are fully compatible with MS detection and soluble in a mobile phase with a high percentage of acetonitrile.

The Injection Solvent

HILIC is very sensitive to the injection solvent: ideally the sample should be dissolved in the same solvent as starting mobile phase conditions. Sometimes it is also possible to get good results by dissolving the sample in a weaker solvent than the starting mobile phase conditions (i.e. a lower percentage of water).

Conclusion

HILIC is a high pressure liquid chromatography (HPLC) mode that combines the use of a polar column with a mobile phase composed of a high percentage of organic solvent and a low percentage of aqueous buffer, where the water phase acts as the strong solvent. This technique is of great interest for the analysis of very polar compounds that do not show good retention in reversed phase.

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Related resources:

• HILIC Method Development Guidelines
• Guidelines for HILIC Method Development
• Increasing LC-MS MS Sensitivity with Luna HILIC
• Optimization Tips for HILIC Analysis of Basic Compounds