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HILIC Mechanism

Partitioning Into a Hydrophilic Environment

Present HILIC theory dictates that HILIC retention is caused by a partitioning of the injected analyte solute molecules between the mobile phase eluent and a water-enriched layer in the hydrophilic HILIC stationary phase, see Figure 9. The more hydrophilic the analyte is, the more is the partitioning equilibrium shifted towards the immobilized water layer in the stationary phase, and thereby, the more is the analyte retained.

Mechanism Still Debated

Although it is well established that a hydrophilic surface holds water when exposed to mixtures of organic solvent and water, the HILIC partitioning theory is only based on circumstantial evidence. There are studies pointing towards a more multimodal separation mechanism, involving hydrogen bonding as well as dipole-dipole interactions as important contributions.

Adjusting the Retention

The retention and selectivity in HILIC is affected by adjusting the eluent by varying the fraction (and type) of organic solvent, the concentration (and type) of buffer, and the pH. Retention increases with increasing fraction of organic solvent. The pH affects retention since an ionized molecule is more hydrophilic and is stronger retained in HILIC, compared to its neutral state. The effect of column temperature in HILIC separations is often rather small though (typically less than in RPLC), but ultimately this depends on the nature of the retained molecule.

More information on the aspects of the HILIC mechanism and the effect of different solvents and buffer types on the separation can be found in the hardcopy booklet A Practical Guide to HILIC, available free of charge from SeQuant.






For separation of
polar and hydrophilic compounds.


Figure 9. The HILIC partitioning process.


Figure #. Important factors for adjusting the retention in HILIC.