Hydrophilic interaction chromatography

See also: Chromatography

Short description: Type of chromatography

Analyte's separated by HILIC vs Reverse phase Chromatography (RPC), using increasingly organic and aqueous mobile phases respectively. Analytes organized by lipophilicity.

Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) is a type of liquid chromatography that uses a hydrophilic stationary phase and a high-organic mobile phase for the separation of analytes by polarity.[1][2] While it is not as popular as some other types of liquid chromatography, the number of scientific publications using HILIC have greatly increased since the early 2000s.[3] HILIC is similar to reverse phase chromatography in its mobile phase composition, and also to normal phase chromatography, with its polar stationary phase.[4][5] It also has overlap with ion exchange chromatography.[4] Sometimes, HILIC is considered to be a hybrid of these techniques.[6]

HILIC was named in 1990 by Andrew Alpert, who described it as a type of liquid-liquid partition chromatography.[2] He suggested that analytes elute in order of increasing polarity,[2] a conclusion supported by review and re-evaluation of published data.[7] The mechanism for HILIC is still not entirely understood, but it is thought to rely on analytes partitioning between the organic-rich mobile phase and a water-enriched layer that forms of the surface of the polar stationary phase, in a liquid-liquid extraction system.[2][5] More polar analytes will have stronger interactions with the water-enriched layer and with the column itself, therefore being retained on the column for longer.[3]

## Contents

* 1 Stationary Phase
* 2 Mobile phase
* 3 Additives
* 4 Choice of pH
* 5 ERLIC
* 5.1 Cationic ERLIC
* 5.2 Anionic ERLIC
* 6 Applications
* 7 See also
* 8 References

## Stationary Phase

One of the key factors influencing HILIC separations is the chemical nature of the stationary phase that is packed into the column.[8] Stationary phases on HILIC columns not only provide physical support for the water layer which analytes separate into, but also interact with the analytes through hydrogen bonding and electrostatic interactions, affecting their retention and therefore the mechanism of separation.[3][5]

Typical HILIC stationary phases are polar, made of classical bare silica or silica gels modified with various polar groups.[1][9] Some commonly used stationary phases include bare silica, or silica chemically bonded to amino-,[10] amide-,[11] cyano-, or diol\- groups.[12][9] Ion exchanger groups, both cationic and anionic {{citation needed|date=November 2025} 6">Lardeux, Honorine; Guillarme, Davy; D'Atri, Valentina (2023-02-08). "Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization". Journal of Chromatography A 1690. doi:10.1016/j.chroma.2023.463785. ISSN 0021-9673. PMID 36641941. </ref> groups are also commonly used.[9]

While most HILIC phases are polar, there have also been exceptions where non-polar bonded silicas are used with extremely high organic solvent composition. In this case, interactions are affected by exposed patches of silica in between the bonded ligands on the support.[13]

## Mobile phase

The mobile phase, or the liquid phase that runs across the column during separation, for HILIC is typically composed of a high amount of water-miscible, polar organic solvent and a low amount of water.[6] Typically, acetonitrile ("MeCN", also designated as "ACN") is used for the organic solvent, though other aprotic water-miscible solvents, such as alcohols at higher concentration, tetrahydrofuran, or dioxane, can also be used.[3]

As with other methods of chromatography, the mobile phase can be delivered isocratically or with a gradient starting at high-organic progressing towards increasing aqueous content.[3] If using a mobile phase gradient, the mobile phase will progressively increases in polar-aqueous content, causing increasingly polar analytes to be eluted.[2][14]

## Additives

Mobile phase pH and electrostatic interactions, as well as analyte polarity, are regulated by the addition of ionic additives.[3] These additives improve separation efficiency, including more symmetric peaks, less peak tailing, and better recovery from the stationary phase.[3][15] Ammonium acetate and ammonium formate are commonly used, as they have good solubility in high organic.[16]

When considering additive addition, compatibility with detectors is important to consider. HILIC is often used with a mass spectrometry (MS), which cannot handle non-volatile salts like sodium perchlorate, which may suppress ion signal in the instrument, though it may increase mobile phase polarity and assist with elution in HILIC.[3][17]

## Choice of pH

With surface chemistries that are weakly ionic, the choice of pH can affect the ionic nature of the column chemistry. Properly adjusted, the pH can be set to reduce the selectivity toward functional groups with the same charge as the column, or enhance it for oppositely charged functional groups.{{citation needed|date=November 2025} the polarity of the solutes. However, for column surface chemistries that are strongly ionic, and thus resistant to pH values in the mid-range of the pH scale (pH 3.5–8.5), these separations will be reflective of the polarity of the analytes alone.[citation needed] When used with mass spectrometry, p nd thus detection.[18]

## ERLIC

ERLIC (electrostatic repulsion interaction chromatography) is a type of HILIC that relies on electrostatic interactions, coined by Alpert in 2008.[19] The ionic stationary phase in ERLIC is chosen to have a similar charge to the analyte(s) so that the analyte is repelled by the stationary phase but also retained by the aqueous layer, allowing for enhanced interaction of the remaining polar, oppositely-charged functional groups of the analyte.[20][19] Electrostatic effects have an order of magnitude stronger chemical potential than neutral polar effects. These opposing effects can, in some cases, enable isocratic separations, with the mobile phase held constant instead of delivered at a gradient.[20] ERLIC can be used to reduce retention of more polar functional groups and minimize the influence of common ionic groups within a set of analytes. [citation needed]

### Cationic ERLIC

A negatively charged cation exchange column can be used for ERLIC separations to reduce the influence of anionic (negatively charged) groups on analyte retention. For example, reducing the influence the phosphates of nucleotides or of phosphonyl antibiotic mixtures; or sialic acid groups of modified carbohydrates, to allow separation based more on the basic and/or neutral functional groups of these molecules. Modifying the polarity of a weakly ionic group (e.g. carboxyl) on the surface is easily accomplished by adjusting the pH to be within two pH units of that group's pKa.[20mM buffer), pH 9.2 mobile phase on a polymeric, zwitterionic, betaine-sulfonate surface to separate phosphonyl antibiotic mixtures (each containing a phosphate group). [citation needed] This enhances the influence of the groups over its surface chemistry, slightly diminished (by pH), quaternary amine. These analytes will show a reduced retention on the column eluting earlier, and in higher amounts of organic solvent, than if a neutral polar HILIC surface were used. [citation needed] This also increases their detection sensitivity by negative ion mass sp mber 2025)">citation needed]

### Anionic ERLIC

Similarly, a positively charged anion exchange column can be used to reduce the influence of cationic (positively charged) functional groups on the retention time of analytes. For example, when selectively isolating phosphorylated peptides or sulfated polysaccharide molecules, use of a pH between 1 and 2 pH units reduces the polarity of two of the three ionizable oxygens of the phosphate group, and thus allows easy desorption from the (oppositely charged) surface chemistry. Negatively charged carboxyl groups in the analyte will be protonated at this low pH, and thus also contribute less to the polarity and therefore separation of the analyte {{citation needed|date=November 2025} umb|332x332px|Comparing HILIC, normal phase, and reversed phase chromatography in terms of sensitivity for electrospray ionization mass spectrometry and analyte polarity. ]]

## Applications

HILIC can be applied in many fields including proteomics,[20] metabolomics,[21] medical studies,[22][23] and agricultural/ food studies,[24] among others. It can be used to separate proteins and peptides, nucleosides, amino acids, sacharides, carbohydrates, and other small, polar, ionizable compounds.[25] HILIC is especially common in metabolomic studies, both for targeted and untargeted approaches, given its ability to retain polar analytes that are poorly suited for traditional reverse-phased columns.[21][2] This separation technique is also particularly suitable for glycosylation analysis[26] and quality assurance of glycoproteins and glycoforms in biologic medical products.[27] For the detection of polar compounds with the use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can offer a ten fold increase in sensitivity over reversed-phase chromatography because the organic solvent is much more volatile.[28] HILIC is considered orthogonal reverse phase chromatography, and these two are increasingly being combined in studies for more comprehensive coverage.[29][30]

## See also

* Chromatography
* High performance liquid chromatography
* Liquid chromatography mass spectrometry
* Reversed phase chromatography
* Ion exchange chromatography
* Partition chromatography

## References

1. ↑ 1.0 1.1 Jandera, Pavel (2011). "Stationary and mobile phases in hydrophilic interaction chromatography: a review". Analytica Chimica Acta 692 (1): 1–25. doi:10.1016/j.aca.2011.02.047. ISSN 0003-2670. PMID 21501708. Bibcode: 2011AcAC..692....1J. https://www.sciencedirect.com/science/article/pii/S0003267011002728.
2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 Alpert, Andrew J. (1990). "Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds". Journal of Chromatography 499: 177-196. doi:10.1016/S0021-9673(00)96972-3. PMID 2324207. Bibcode: 1990JChA..499..177A.
3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 Buszewski, Bogusław; Noga, Sylwia (January 2012). "Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique" (in en). Analytical and Bioanalytical Chemistry 402 (1): 231–247. doi:10.1007/s00216-011-5308-5. ISSN 1618-2642. PMID 21879300. Bibcode: 2012ABiCh.402..231B.
4. ↑ 4.0 4.1 Sheng, Qianying; Liu, Meiyan; Lan, Minbo; Qing, Guangyan (2023-08-01). "Hydrophilic interaction liquid chromatography promotes the development of bio-separation and bio-analytical chemistry". TrAC Trends in Analytical Chemistry 165. doi:10.1016/j.trac.2023.117148. ISSN 0165-9936. https://www.sciencedirect.com/science/article/pii/S0165993623002352.
5. ↑ 5.0 5.1 5.2 Redón, Lídia; Subirats, Xavier; Rosés, Martí (2021-10-25). "Volume and composition of semi-adsorbed stationary phases in hydrophilic interaction liquid chromatography. Comparison of water adsorption in common stationary phases and eluents". Journal of Chromatography A 1656. doi:10.1016/j.chroma.2021.462543. ISSN 0021-9673. PMID 34571282.
6. ↑ 6.0 6.1 Kohler, Isabelle; Verhoeven, Michel; Haselberg, Rob; Gargano, Andrea F. G. (2022-04-01). "Hydrophilic interaction chromatography – mass spectrometry for metabolomics and proteomics: state-of-the-art and current trends". Microchemical Journal 175. doi:10.1016/j.microc.2021.106986. ISSN 0026-265X. https://www.sciencedirect.com/science/article/pii/S0026265X21010729.
7. ↑ Petrus Hemström and Knut Irgum (2006). "Review: Hydrophilic Interaction Chromatography". J. Sep. Sci. 29 (12): 1784-1821. doi:10.1002/jssc.200600199. PMID 16970185.
8. ↑ Kumar, Abhinav; Heaton, James C.; McCalley, David V. (2013-02-08). "Practical investigation of the factors that affect the selectivity in hydrophilic interaction chromatography". Journal of Chromatography A 1276: 33–46. doi:10.1016/j.chroma.2012.12.037. ISSN 0021-9673. https://www.sciencedirect.com/science/article/pii/S002196731201895X.
9. ↑ 9.0 9.1 9.2 Qiao, Lizhen; Shi, Xianzhe; Xu, Guowang (2016-07-01). "Recent advances in development and characterization of stationary phases for hydrophilic interaction chromatography". TrAC Trends in Analytical Chemistry. Theory and Practice of Chromatography, Dedicated to the Life and Work of Georges Guiochon 81: 23–33. doi:10.1016/j.trac.2016.03.021. ISSN 0165-9936. https://www.sciencedirect.com/science/article/pii/S0165993616000388.
10. ↑ Shaw, P. E.; Wilson, C. W. (1982). "Separation of Sorbitol and Mannoheptulose from Fructose, Glucose and Sucrose on Reversed-Phase and Amine-Modified HPLC Columns" (in en). Journal of Chromatographic Science 20 (5): 209–212. doi:10.1093/chromsci/20.5.209. ISSN 0021-9665. https://academic.oup.com/chromsci/article-lookup/doi/10.1093/chromsci/20.5.209.
11. ↑ Koh, Dong-wan; Park, Jae-woong; Lim, Jung-hoon; Yea, Myeong-Jai; Bang, Dae-young (2018). "A rapid method for simultaneous quantification of 13 sugars and sugar alcohols in food products by UPLC-ELSD". Food Chemistry 240: 694–700. doi:10.1016/j.foodchem.2017.07.142. ISSN 0308-8146. PMID 28946331. https://www.sciencedirect.com/science/article/pii/S0308814617312967.
12. ↑ Cite error: Invalid `<ref>` tag; no text was provided for refs named `:6`
13. ↑ Bij, Klaas E.; Horváth, Csaba; Melander, Wayne R.; Nahum, Avi (1981-01-09). "Surface silanols in silica-bonded hydrocarbonaceous stationary phases: II. Irregular retention behavior and effect of silanol masking". Journal of Chromatography A 203: 65–84. doi:10.1016/S0021-9673(00)80282-4. ISSN 0021-9673. https://www.sciencedirect.com/science/article/pii/S0021967300802824.
14. ↑ McCalley, David V. (November 2017). "Understanding and manipulating the separation in hydrophilic interaction liquid chromatography" (in en). Journal of Chromatography A 1523: 49–71. doi:10.1016/j.chroma.2017.06.026. PMID 28668366. https://linkinghub.elsevier.com/retrieve/pii/S0021967317308762.
15. ↑ Tengattini, Sara; Massolini, Gabriella; Rinaldi, Francesca; Calleri, Enrica; Temporini, Caterina (2024-05-01). "Hydrophilic interaction liquid chromatography (HILIC) for the analysis of intact proteins and glycoproteins". TrAC Trends in Analytical Chemistry 174. doi:10.1016/j.trac.2024.117702. ISSN 0165-9936.
16. ↑ Moldoveanu, Serban C.; David, Victor (2017), "Solvents, Buffers, and Additives Used in the Mobile Phase" (in en), Selection of the HPLC Method in Chemical Analysis (Elsevier): pp. 393–450, doi:10.1016/b978-0-12-803684-6.00013-5, ISBN 978-0-12-803684-6, https://linkinghub.elsevier.com/retrieve/pii/B9780128036846000135, retrieved 2025-12-05
17. ↑ Tóth, Gábor; Vékey, Károly; Sugár, Simon; Kovalszky, Ilona; Drahos, László; Turiák, Lilla (2020-05-24). "Salt gradient chromatographic separation of chondroitin sulfate disaccharides". Journal of Chromatography A 1619. doi:10.1016/j.chroma.2020.460979. ISSN 0021-9673. PMID 32093904.
18. ↑ Khrisanfova, Anna; Smagina, Maria; Maksimov, Grigorii; Tsizin, Grigorii; Shpigun, Oleg; Chernobrovkina, Alla (2025-09-13). "Evaluating independent effect of mobile phase components on retention mechanism of ionizable analytes in hydrophilic interaction liquid chromatography". Journal of Chromatography A 1758. doi:10.1016/j.chroma.2025.466201. ISSN 0021-9673. https://www.sciencedirect.com/science/article/pii/S0021967325005473.
19. ↑ 19.0 19.1 Alpert, Andrew J. (January 2008). "Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides". Anal. Chem. 80 (1): 62-76. doi:10.1021/ac070997p. PMID 18027909. Bibcode: 2008AnaCh..80...62A.
20. ↑ 20.0 20.1 20.2 Boersema, Paul J.; Mohammed, Shabaz; Heck, Albert J. R. (May 2008). "Hydrophilic interaction liquid chromatography (HILIC) in proteomics" (in en). Analytical and Bioanalytical Chemistry 391 (1): 151–159. doi:10.1007/s00216-008-1865-7. ISSN 1618-2642. PMID 18264818.
21. ↑ 21.0 21.1 Tang, Dao-Quan; Zou, Ll; Yin, Xiao-Xing; Ong, Choon Nam (September 2016). "HILIC-MS for metabolomics: An attractive and complementary approach to RPLC-MS" (in en). Mass Spectrometry Reviews 35 (5): 574–600. doi:10.1002/mas.21445. ISSN 0277-7037. PMID 25284160. Bibcode: 2016MSRv...35..574T. https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/mas.21445.
22. ↑ Serafimov, Kristian; Tischlarik, Johanna Ruth; Lämmerhofer, Michael (2025-04-15). "Targeted and untargeted urinary metabolomics of alkaptonuria patients using ultra high-performance liquid chromatography-tandem mass spectrometry". Journal of Pharmaceutical and Biomedical Analysis 256. doi:10.1016/j.jpba.2025.116684. ISSN 0731-7085. PMID 39842076. https://www.sciencedirect.com/science/article/pii/S0731708525000251.
23. ↑ Deng, Sisi; Kim, Wooyong; Cheng, Kefan; Yang, Qianlu; Singh, Yogesh; Bae, Gyuntae; Bézière, Nicolas; Mager, Lukas et al. (2025-05-13). "Identification and impact of microbiota-derived metabolites in ascites of ovarian and gastrointestinal cancer" (in en). Cancer & Metabolism 13 (1): 21. doi:10.1186/s40170-025-00391-5. ISSN 2049-3002. PMID 40361187.
24. ↑ Wilkie, Daisy; White, Brad; Heidari, Golnaz; Naffa, Rafea; Peddie, Gaile; Rowlands, Gareth J.; Plieger, Paul G. (2025-09-08). "Methods for Untargeted Analysis of Milk Metabolites: Influence of Extraction Method and Optimization of Separation" (in en). Metabolites 15 (9): 597. doi:10.3390/metabo15090597. ISSN 2218-1989. PMID 41002981.
25. ↑ Gama, Mariana R.; Bottoli, Carla B.G. (2018), "Hydrophilic Interaction Liquid Chromatography of Small Molecules" (in en), Encyclopedia of Analytical Chemistry (John Wiley & Sons, Ltd): pp. 1–23, doi:10.1002/9780470027318.a9385.pub2, ISBN 978-0-470-02731-8, https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470027318.a9385.pub2, retrieved 2025-11-30
26. ↑ Ahn, Joomi; Bones, Jonathan; Yu, Ying Qing; Rudd, Pauline M.; Gilar, Martin (2010-02-01). "Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 μm sorbent". Journal of Chromatography B 878 (3–4): 403–408. doi:10.1016/j.jchromb.2009.12.013. PMID 20036624.
27. ↑ Glycosylation analysis by hydrophilic interaction chromatography (HILIC) – N-Glyco mapping of the ZP-domain of murine TGFR-3 (Application Note TOSOH Biosciences). Retrieved May 23, 2013.
28. ↑ Eric S. Grumbach (October 2004). "Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity". LCGC Magazine. http://www.lcgcmag.com/lcgc/issue/issueDetail.jsp?id=4734. Retrieved 2008-07-14.
29. ↑ Boersema, Paul J.; Mohammed, Shabaz; Heck, Albert J. R. (2008-02-09). "Hydrophilic interaction liquid chromatography (HILIC) in proteomics" (in en). Analytical and Bioanalytical Chemistry 391 (1): 151–159. doi:10.1007/s00216-008-1865-7. ISSN 1618-2642. PMID 18264818. PMC 2324128. http://link.springer.com/10.1007/s00216-008-1865-7.
30. ↑ Wang, Xiande; Li, Weiyong; Rasmussen, Henrik T. (2005-08-12). "Orthogonal method development using hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography for the determination of pharmaceuticals and impurities" (in en). Journal of Chromatography A 1083 (1-2): 58–62. doi:10.1016/j.chroma.2005.05.082. https://linkinghub.elsevier.com/retrieve/pii/S0021967305011520.

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