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Hydrophilic interaction chromatography

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See also: Chromatography

Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC)[1] 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.[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.[7] He suggested that analytes elute in order of increasing polarity,[7] a conclusion supported by review and re-evaluation of published data.[8] 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.[7][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]

HILIC Partition Technique Useful Range


Stationary Phase

One of the key factors influencing HILIC separations is the chemical nature of the stationary phase that is packed into the column.[2][9] 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.[2][10] Some commonly used stationary phases include bare silica, or silica chemically bonded to amino-,[4] amide-,[11] cyano-, or diol- groups.[9][10] Ion exchanger groups, both cationic [citation needed]and anionic [citation needed], as well as zwitterionic[3][12] groups are also commonly used. [10]

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. [7] [14]

All ions partition into the stationary phase to some degree, so an occasional "wash" with water is required to ensure a reproducible stationary phase.[citation needed]

Additives

Mobile phase pH and electrostatic interactions, as well as analyte polarity, are regulated by the addition of ionic additives, commonly ammonium acetate and ammonium formate, to the mobile phase. [3] These additives improve separation efficiency, including more symmetric peaks, less peak tailing, and better recovery from the stationary phase. [3][15]

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][16]

Choice of pH

With surface chemistries that are weakly ionic, the choice of pH can affect the ionic nature of the column chemistry. [citation needed] 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] Similarly, the choice of pH affects 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]

Applications

HILIC can be applied in many fields including proteomics,[17] metabolomics,[18] medical studies,[19] [20] and agricultural/ food studies,[21] among others. It can be used to separate proteins and peptides, nucleosides, amino acids, sacharides, carbohydrates, and other small, polar, ionizable compounds. [22] 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. [23][18] Metabolite sample preparation techniques for biological samples is relatively simple for HILIC applications, also contributing to HILIC's increased use. [22] This separation technique is also particularly suitable for glycosylation analysis[24] and quality assurance of glycoproteins and glycoforms in biologic medical products.[25] 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.[26]

ERLIC

ERLIC (electrostatic repulsion interaction chromatography) is a type of HILIC that relies on electrostatic interactions, coined by Alpert in 2008. [27] 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. [17][27] Electrostatic effects have an order of magnitude stronger chemical potential than neutral polar effects.[citation needed] These opposing effects can, in some cases, enable isocratic separations, with the mobile phase held constant instead of delivered at a gradient.[17] 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. [citation needed] 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.[citation needed] For strongly ionic functional groups of the surface (i.e. sulfates or phosphates), lower amount of buffer can be used so the residual charge is not completely ion paired. An example of this would be the use of a 12.5mM (rather than the recommended >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 column's sulfonic acid functional 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 spectrometry.[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. [citation needed] 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]

References

  1. ^ Jandera, Pavel (2011). "Stationary and mobile phases in hydrophilic interaction chromatography: a review". Analytica Chimica Acta. 692 (1): 1–25. Bibcode:2011AcAC..692....1J. doi:10.1016/j.aca.2011.02.047. ISSN 0003-2670. PMID 21501708.
  2. ^ a b c Jandera, Pavel (2011). "Stationary and mobile phases in hydrophilic interaction chromatography: a review". Analytica Chimica Acta. 692 (1): 1–25. Bibcode:2011AcAC..692....1J. doi:10.1016/j.aca.2011.02.047. ISSN 0003-2670. PMID 21501708.
  3. ^ a b c d e f g h i Buszewski, Bogusław; Noga, Sylwia (January 2012). "Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique". Analytical and Bioanalytical Chemistry. 402 (1): 231–247. Bibcode:2012ABiCh.402..231B. doi:10.1007/s00216-011-5308-5. ISSN 1618-2642. PMC 3249561. PMID 21879300.
  4. ^ a b c 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 117148. doi:10.1016/j.trac.2023.117148. ISSN 0165-9936. Cite error: The named reference ":3" was defined multiple times with different content (see the help page).
  5. ^ a b c 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 462543. doi:10.1016/j.chroma.2021.462543. hdl:2445/183349. ISSN 0021-9673. PMID 34571282.
  6. ^ a b 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 106986. doi:10.1016/j.microc.2021.106986. ISSN 0026-265X.
  7. ^ a b c d Alpert, Andrew J. (1990). "Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds". Journal of Chromatography. 499: 177–196. Bibcode:1990JChA..499..177A. doi:10.1016/S0021-9673(00)96972-3. PMID 2324207.
  8. ^ Petrus Hemström and Knut Irgum (2006). "Review: Hydrophilic Interaction Chromatography". J. Sep. Sci. 29 (12): 1784–1821. doi:10.1002/jssc.200600199. PMID 16970185.
  9. ^ a b 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. PMID 23332781.
  10. ^ a b c 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.
  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.
  12. ^ 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 463785. doi:10.1016/j.chroma.2023.463785. ISSN 0021-9673. PMID 36641941.
  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.
  14. ^ McCalley, David V. (November 2017). "Understanding and manipulating the separation in hydrophilic interaction liquid chromatography". Journal of Chromatography A. 1523: 49–71. doi:10.1016/j.chroma.2017.06.026. PMID 28668366.
  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 117702. doi:10.1016/j.trac.2024.117702. ISSN 0165-9936.
  16. ^ 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 460979. doi:10.1016/j.chroma.2020.460979. ISSN 0021-9673. PMID 32093904.
  17. ^ a b c Boersema, Paul J.; Mohammed, Shabaz; Heck, Albert J. R. (May 2008). "Hydrophilic interaction liquid chromatography (HILIC) in proteomics". Analytical and Bioanalytical Chemistry. 391 (1): 151–159. doi:10.1007/s00216-008-1865-7. ISSN 1618-2642. PMC 2324128. PMID 18264818.
  18. ^ a b 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". Mass Spectrometry Reviews. 35 (5): 574–600. doi:10.1002/mas.21445. ISSN 0277-7037.
  19. ^ 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: 116684. doi:10.1016/j.jpba.2025.116684. ISSN 0731-7085.{{cite journal}}: CS1 maint: article number as page number (link)
  20. ^ Deng, Sisi; Kim, Wooyong; Cheng, Kefan; Yang, Qianlu; Singh, Yogesh; Bae, Gyuntae; Bézière, Nicolas; Mager, Lukas; Kommoss, Stefan; Sprengel, Jannik; Trautwein, Christoph (2025-05-13). "Identification and impact of microbiota-derived metabolites in ascites of ovarian and gastrointestinal cancer". Cancer & Metabolism. 13 (1): 21. doi:10.1186/s40170-025-00391-5. ISSN 2049-3002. PMC 12076955. PMID 40361187.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  21. ^ 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". Metabolites. 15 (9): 597. doi:10.3390/metabo15090597. ISSN 2218-1989. PMC 12471616. PMID 41002981.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  22. ^ a b Cite error: The named reference :12 was invoked but never defined (see the help page).
  23. ^ Cite error: The named reference alpert2 was invoked but never defined (see the help page).
  24. ^ 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.
  25. ^ 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.
  26. ^ Eric S. Grumbach; et al. (October 2004). "Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity". LCGC Magazine. Archived from the original on 2007-08-06. Retrieved 2008-07-14.
  27. ^ a b 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. Bibcode:2008AnaCh..80...62A. doi:10.1021/ac070997p. PMID 18027909.
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