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What is HILIC?

Hydrophilic Interaction Liquid Chromatography

HILIC or Hydrophilic Interaction Liquid Chromatography is a high-performance liquid chromatographic (HPLC) technique for separation of polar and hydrophilic compounds. Originally the separation technique was called "Hydrophilic-Interaction Chromatography", and occasionally the expression "Aqueous Normal Phase" has also been used.

To put it simple one can say that HILIC is a normal-phase type of separation but uses reversed-phase type eluents.

Thus, in HILIC one has:

  • A column with a hydrophilic stationary phase
  • An eluent with water, buffer and a high concentration of water-miscible organic solvent.

A typical HILIC application uses acetonitrile at a concentration between 50-95% in an aqueous buffer such as ammonium formate, ammonium acetate or their acids, which have high solubility in organic solvents. HILIC can be used with many detection techniques, but when combined with electro spray ionisation mass spectrometry (ESI-MS) for example, HILIC will also enable higher sensitivities.

Reversed Elution Order Compared to RPLC

The elution order in HILIC is roughly the reverse of that in RPLC (Reversed-Phase Liquid Chromatography). A compound that elutes in the void volume on a RPLC column typically has high retention in HILIC, and vice versa, see Figure 1.

Compounds such as, acids, bases, ions, sugars, and other charged and neutral hydrophilic compounds that are troublesome to separate in RPLC, are much easier to separate in HILIC due to the different separation selectivity. Some compounds are possible to separate by more than one chromatographic technique, and HILIC does in that respect partly overlap both with RPLC, NPLC (Normal Phase Liquid Chromatography) and IC (Ion Chromatography). Figure 2 illustrates the overlap between these different chromatographic techniques.





For separation of
polar and hydrophilic compounds.


Figure 1. Comparison of peptide separations in HILIC and RPLC chromatography.


Figure 2. HILIC fills a gap in the chromatographic toolbox, but also partly overlap with the RPLC, NPLC and IC separation techniques.