Hydrophilic interaction chromatography

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Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) [1] is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. [2] He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data. [3]

Contents

HILIC Partition Technique Useful Range HILIC Partition Method Graphic.png
HILIC Partition Technique Useful Range

Surface

Any polar chromatographic surface can be used for HILIC separations. Even non-polar bonded silicas have been used with extremely high organic solvent composition, thanks to the exposed patches of silica in between the bonded ligands on the support, which can affect the interactions. [4] With that exception, HILIC phases can be grouped into five categories of neutral polar or ionic surfaces: [5]

Mobile phase

A typical mobile phase for HILIC chromatography includes acetonitrile ("MeCN", also designated as "ACN") with a small amount of water. However, any aprotic solvent miscible with water (e.g. THF or dioxane) can be used. Alcohols can also be used, however, their concentration must be higher to achieve the same degree of retention for an analyte relative to an aprotic solvent–water combination. See also Aqueous normal phase chromatography.

It is commonly believed that in HILIC, the mobile phase forms a water-rich layer on the surface of the polar stationary phase vs. the water-deficient mobile phase, creating a liquid/liquid extraction system. The analyte is distributed between these two layers. However, HILIC is more than just simple partitioning and includes hydrogen donor interactions between neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention. This distinguishes HILIC as a mechanism distinct from ion exchange chromatography. The more polar compounds will have a stronger interaction with the stationary aqueous layer than the less polar compounds. Thus, a separation based on a compound's polarity and degree of solvation takes place.

Additives

Ionic additives, such as ammonium acetate and ammonium formate, are usually used to control the mobile phase pH and ion strength. In HILIC they can also contribute to the polarity of the analyte, resulting in differential changes in retention. For extremely polar analytes (e.g. aminoglycoside antibiotics (gentamicin) or adenosine triphosphate), higher concentrations of buffer (c. 100 mM) are required to ensure that the analyte will be in a single ionic form. Otherwise, asymmetric peak shape, chromatographic tailing, and/or poor recovery from the stationary phase will be observed. For the separation of neutral polar analytes (e.g. carbohydrates), no buffer is necessary.

Other salts, such as 100–300 mM sodium perchlorate, that are soluble in high-organic solvent mixtures (c. 70–90% acetonitrile), can be used to increase the mobile phase polarity to affect elution These salts are not volatile, so this technique is less useful with a mass spectrometer as the detector. Usually a gradient (to increasing amounts of water) is enough to promote elution.

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

Applications

The HILIC mode of separation is used extensively for separation of some biomolecules, organic and some inorganic molecules [10] by differences in polarity. Its utility has increased due to the simplified sample preparation for biological samples, when analyzing for metabolites, since the metabolic process generally results in the addition of polar groups to enhance elimination from the cellular tissue. This separation technique is also particularly suitable for glycosylation analysis [11] and quality assurance of glycoproteins and glycoforms in biologic medical products. [12] 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 [10] because the organic solvent is much more volatile.

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. 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, and thus might be easier to understand when doing methods development.

ERLIC

In 2008, Alpert coined the term, ERLIC [13] (electrostatic repulsion hydrophilic interaction chromatography), for HILIC separations where an ionic column surface chemistry is used to repel a common ionic polar group on an analyte or within a set of analytes, to facilitate separation by the remaining polar groups. Electrostatic effects have an order of magnitude stronger chemical potential than neutral polar effects. This allows one to minimize the influence of a common, ionic group within a set of analyte molecules; or to reduce the degree of retention from these more polar functional groups, even enabling isocratic separations in lieu of a gradient in some situations. His subsequent publication further described orientation effects [14] which others have also called ion-pair normal phase [15] or e-HILIC, reflecting retention mechanisms sensitive to a particular ionic portion of the analyte, either attractive or repulsive. ERLIC (eHILIC) separations need not be isocratic, but the net effect is the reduction of the attraction of a particularly strong polar group, which then requires less strong elution conditions, and the enhanced interaction of the remaining polar (opposite charged ionic, or non-ionic) functional groups of the analyte(s).Based on the ERLIC column invented by Andrew Alpert, a new peptide mapping methodology was developed with unique properties of separation of asparagine deamidation and isomerization. This unique properties would be very beneficial for future mass spectrometry based multi-attributes monitoring in biologics quality control. [16]

Cationic eHILIC

For example, one could use a cation exchange (negatively charged) surface chemistry for ERLIC separations to reduce the influence on retention of anionic (negatively charged) groups (the phosphates of nucleotides or of phosphonyl antibiotic mixtures; or sialic acid groups of modified carbohydrates) to now 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. For strongly ionic functional groups of the surface (i.e. sulfates or phosphates) one could instead use a lower amount of buffer 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). This enhances the influence of the column's sulfonic acid functional groups of its surface chemistry over its, slightly diminished (by pH), quaternary amine. Commensurate with this, 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. This also increases their detection sensitivity by negative ion mass spectrometry.

Anionic eHILIC

By analogy to the above, one can use an anion exchange (positively charged) column surface chemistry to reduce the influence on retention of cationic (positively charged) functional groups for a set of analytes, such as when selectively isolating phosphorylated peptides or sulfated polysaccharide molecules. Use of a pH between 1 and 2 pH units will reduce the polarity of two of the three ionizable oxygens of the phosphate group, and thus will allow easy desorption from the (oppositely charged) surface chemistry. It will also reduce the influence of negatively charged carboxyls in the analytes, since they will be protonated at this low a pH value, and thus contribute less overall polarity to the molecule. Any common, positively charged amino groups will be repelled from the column surface chemistry and thus these conditions enhance the role of the phosphate's polarity (as well as other neutral polar groups) in the separation.

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions.

<span class="mw-page-title-main">Gas chromatography</span> Type of chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Ion chromatography</span> Separates ions and polar molecules

Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.

<span class="mw-page-title-main">Liquid chromatography–mass spectrometry</span> Analytical chemistry technique

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography – MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS has also begun to be used in clinical applications.

<span class="mw-page-title-main">Solid-phase extraction</span> Process to separate compounds by properties

Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.

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Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.

Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC); however, SFC typically utilizes carbon dioxide as the mobile phase. Therefore, the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.

Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.

Aqueous normal-phase chromatography (ANP) is a chromatographic technique that involves the mobile phase compositions and polarities between reversed-phase chromatography (RP) and normal-phase chromatography (NP), while the stationary phases are polar.

<span class="mw-page-title-main">Elution</span> Extraction of a material by washing with a solvent

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.

<span class="mw-page-title-main">Countercurrent chromatography</span>

Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force and is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.

<span class="mw-page-title-main">Capillary electrochromatography</span> Method of separating components of a mixture via electro-osmosis

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<span class="mw-page-title-main">Glowmatography</span>

Glowmatography is a laboratory technique for the separation of dyes present in solutions contained in glow sticks. The chemical components of such solutions can be chromatographically separated into polar and nonpolar components. Developed as a laboratory class experiment, it can be used to demonstrate chemistry concepts of polarity, chemical kinetics, and chemiluminescence.

Chiral analysis refers to the quantification of component enantiomers of racemic drug substances or pharmaceutical compounds. Other synonyms commonly used include enantiomer analysis, enantiomeric analysis, and enantioselective analysis. Chiral analysis includes all analytical procedures focused on the characterization of the properties of chiral drugs. Chiral analysis is usually performed with chiral separation methods where the enantiomers are separated on an analytical scale and simultaneously assayed for each enantiomer.

References

  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.
  2. ↑ 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.
  3. ↑ Petrus Hemström and Knut Irgum (2006). "Review: Hydrophilic Interaction Chromatography". J. Sep. Sci. 29 (12): 1784–1821. doi:10.1002/jssc.200600199. PMID   16970185.
  4. ↑ 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.
  5. ↑ 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 . ISSN   0021-9673. PMID   34571282.
  6. ↑ 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". Journal of Chromatographic Science. 20 (5): 209–212. doi:10.1093/chromsci/20.5.209. ISSN   0021-9665.
  7. ↑ 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.
  8. ↑ Boguslaw Buszewski and Sylwia Noga (2012). "Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique". Anal. Bioanal. Chem. 402 (1): 231–247. doi:10.1007/s00216-011-5308-5. PMC   3249561 . PMID   21879300.
  9. ↑ 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.
  10. 1 2 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.
  11. ↑ 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.
  12. ↑ 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.
  13. ↑ 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.
  14. ↑ Alpert, Andrew J.; et al. (June 2010). "Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography". Anal. Chem. 82 (12): 5253–5259. doi:10.1021/ac100651k. PMC   2884984 . PMID   20481592.
  15. ↑ Ding, W.; et al. (September 2009). "Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-Phase LC and MS". Molecular & Cellular Proteomics. 8 (9): 2170–2185. doi:10.1074/mcp.M900088-MCP200. PMC   2742440 . PMID   19525481.
  16. ↑ Zhen, J., Kim, J., Zhou, Y., Gaidamauskas, E., Subramanian, S., & Feng, P. (2018, October). Antibody characterization using novel ERLIC-MS/MS-based peptide mapping. In MAbs (Vol. 10, No. 7, pp. 951-959). Taylor & Francis.
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