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Hydrophilic interaction chromatography (HILIC)

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Hydrophilic interaction chromatography often abbreviated as HILIC is a special form of normal-phase chromatography, designed explicitly for polar compounds. Polar compounds exhibit a strong affinity for water therefore the name hydrophilic interaction is given to this chromatographic type. Hydrophilic interaction chromatography was performed for the first time in 1975. However, the acronym HILIC was coined in 1990 by a scientist,  Andrew J. Alpert from the USA.

What is hydrophilic interaction chromatography

Hydrophilic interaction chromatography is a type of liquid column chromatography. In hydrophilic interaction chromatography (HILIC), the analytes interact with and get retained onto a hydrophilic stationary phase. It is a special variant of normal-phase chromatography but still, HILIC is different from normal-phase because it employs a water-miscible mobile phase. As opposed to that, the traditional normal-phase chromatography is based on pure organic solvents as mobile phases such as n-hexane.

Therefore, hydrophilic interaction chromatography is a unique chromatographic type. Its stationary phase is the same as that used for normal-phase while the mobile phase eluent required for HILIC resembles the mobile phases of reverse-phase chromatography. It also allows the separation of charged substances  (ions) just like ion-exchange chromatography because charged substances are polar in nature.

HILIC combines the features of three major liquid column chromatographic techniques. Image idea inspired by springer article.

What is stationary phase in HILIC

Un-derivatized, bare silica gel is commonly used as the stationary phase in hydrophilic interaction chromatography. The hydroxyl (OH) functional groups on silica provide a large surface area for retention of highly polar, water-soluble compounds from the solute mixture. Silica gel chemically treated with additional functional groups such as a cyano (R-CN), an imide (R2NH), amine (RNH2), or an amide (R-CONH2) functional group can also be used as a stationary phase in HILIC. Polymer-based stationary phase materials can also be used.

What is the mobile phase in HILIC

Popular mobile phase choices for HILIC include solvents that are water-compatible such as acetone, acetonitrile, methanol, tetrahydrofuran, etc. The proportion of the aqueous solvent in a mobile phase composition is progressively increased during HILIC to facilitate analyte separation.

The eluotropic series given below arrange solvents in an increasing order of HILIC elution strength.

Aqueous buffers such as an ammonium salt buffer or a trifluoroacetic acid buffer can also be employed as mobile phases for hydrophilic interaction chromatography. The buffer’s pH can be manipulated to change its ionic strength and facilitate its role as a mobile phase in hydrophilic interaction chromatography.

How to perform hydrophilic interaction chromatography

Step I: Column packing

The column is packed with a highly polar stationary phase. Commercially packed stainless-steel columns used for high-performance liquid chromatography (HPLC) can be modified according to the requirements of a normal-phase chromatography.

Step II: Column pre-wetting

The pure, aqueous-based mobile phase is passed through the column. The polar stationary phase strongly adsorbs a layer of water from the mobile phase which then becomes a part of the stationary phase. This water-rich layer immobilized onto the stationary phase strongly controls the analyte retention mechanism.

Step III : Analyte separation

The sample mixture is then loaded onto the column. Analyte molecules are separated based on a liquid-liquid partitioning between the mobile phase solvent and the water-rich layer immobilized onto the stationary phase. Multiple interaction mechanisms are often involved in solute retention on a HILIC stationary phase. Electrostatic forces of attraction, ion-dipole interactions, ion exchange,  and hydrogen bonding however dominate the HILIC retention mechanism.

Step IV: Elution

Elution is carried out by increasing the concentration of the polar solvent in the mobile phase. As the mobile phase gets more hydrophilic, it will strongly attract the hydrophilic analyte components, overcoming their force of attraction with the stationary phase.

Conversely, a highly polar mobile phase may compete with the analyte itself for the active polar sites present on the stationary phase. In this way, the polar solvent molecules get adsorbed onto the stationary phase, replacing analyte molecules. These analyte molecules are consequently eluted out of the column. 

Least polar components elute out first followed by moderately polar. Extremely polar components are retained onto the stationary phase for the longest time and are eluted out the last.

Step V: Detection

HPLC performed in HILIC mode can be coupled to a mass spectrometer (MS) detector for the detection and identification of the end products recovered. Other detectors used for liquid chromatography such as an ultraviolet (UV) or a fluorescence detector also support chromatographic analysis via HILIC.

Applications of hydrophilic interaction chromatography

Biomedical industry

  • Hydrophilic interaction chromatography is important for the separation of polar molecules such as carbohydrates, proteins, glycoproteins, antibiotics, and nucleic acids from complex biological sample mixtures.
  • HILIC finds important applications in the biomedical field for pesticide analysis.

Pharmaceutical industry

  • HILIC can also be applied for the separation of organic acidic, basic, and, other polar drugs in the pharmaceutical industry.

Food industry

  • In the food industry, hydrophilic interaction chromatography can help detect small polar compounds such as contaminants, biotoxins, biogenic amines, etc.
  • Natural extracts such as flavonoids present in foodstuff can also be analyzed via HILIC.

Environmental analysis

  • HPLC applied in HILIC mode can also help detect polar impurities and pollutants present in environmental water.

Conclusion

In conclusion, hydrophilic interaction chromatography (HILIC) holds a particular significance for the isolation, separation and, enrichment of highly polar analyte molecules, otherwise difficult to separate through reverse-phase HPLC.

HILIC can be applied as a complementary technique in conjunction with other chromatographic techniques to achieve a higher separation efficiency.

Check out this video for a visual interpretation of the chemistry behind hydrophilic interaction chromatography.

Additional readings that you may find relevant to the topic discussed in this article:

  1. High-performance liquid chromatography (HPLC)
  2. Hydrophobic interaction chromatography (HIC)

References

 1. Buszewski, B. and S. Noga (2012). “Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique.” Analytical and bioanalytical chemistry 402(1): 231-247.

 2. Erkmen, C., W. H. Gebrehiwot and B. Uslu (2021). “Hydrophilic Interaction Liquid Chromatography (HILIC): Latest Applications in the Pharmaceutical Researches.” Current pharmaceutical analysis 17(3): 316-345.

3. Gama, M. R. and C. B. G. Bottoli (2018) Hydrophilic Interaction Liquid Chromatography of Small Molecules. Encyclopedia of Analytical Chemistry: 1-23.

4. Sentkowska, A., M. Biesaga and K. Pyrzynska (2016). “Application of Hydrophilic Interaction Liquid Chromatography for the Quantification of Flavonoids in Genista tinctoria Extract.” Journal of Analytical Methods in Chemistry: 3789348.

 

Hydrophilic interaction chromatography (HILIC)

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