Hydrophilic interaction liquid chromatography

Hydrophilic interaction liquid chromatography

HILIC (HydrophILic Interaction Chromatography or Hydrophilic Interaction LIquid Chromatography) is a version of normal phase liquid chromatography. The name was suggested by Dr. Andrew Alpert in his 1990 paper on the subjectcite journal | last = Alpert | first = Andrew J. | title = Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds | journal = Journal of Chromatography | volume = 499 | year = 1990 | pages = 177–196 | doi = 10.1016/S0021-9673(00)96972-3 ] . He described the chromatographic mechanism for it as liquid-liquid partition chromatography.

Any polar chromatographic surface can be used for HILIC separations. Even nonpolar bonded silicas have been used with extremely high organic solvent composition, when the silica used for the chromatographic media was particularly polar. With that exception, HILIC phases can be grouped into five categories of neutral polar or ionic surfaces:
*simple unbonded silica silanol or diol bonded phases
*amino or anionic bonded phases
*amide bonded phases
*cationic bonded phases
*zwitterionic bonded phases.

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.

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 (ca. 100mM) are required to assure 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.

Use of other salts such as 100-300mM sodium perchlorate, which are soluble in high-organic solvent mixtures (ca. 70% acetonitrile), can be used to increase the mobile phase polarity to effect 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.

The HILIC mode of separation is used intensively for separation of some biomolecules by differences in polarity differences, organic and some inorganic moleculescite journal | journal = LCGC Magazine | year = 2004 | month = October | title = Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity | author = Eric S. Grumbach et al. | URL = http://www.lcgcmag.com/lcgc/issue/issueDetail.jsp?id=4734 | accessdate = 2008-07-14 ] . 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. Additionally, with the use of mass spectrometry as a chromatographic detector, HILIC offers a tenfold increase in sensitivity over reversed-phase chromatography because the organic solvent is much more volatile.

In 2008, Alpert coined the term, ERLIC cite journal | author = Alpert, Andrew J. | title = Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides | journal = Anal. Chem. | month = January | volume = 80 | year = 2008 | pages = 62–76 | doi = 10.1021/ac070997p ] (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. This allows one to minimize the influence of the common, ionic group within the set of molecules; or to reduce the degree of retention from these more polar functional groups, enabling isocratic separations.

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 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.

For example, one could use a cation exchange surface (negatively charged) chemistry for ERLIC separations to reduce the influence of the anionic (negatively charged) groups (phosphates of nucleotides or of phosphonyl antibiotic mixtures, or sialic acid groups of modified carbohydrates) to allow discrimination based on the basic and/or neutral functional groups of these molecules. Alternatively, one could use a pH 9.2 mobile phase on a polymeric, zwitterionic, betain-sulphonate surface to enhance the influence of its sulphonic acid functional group over the, now diminished, quarternary amine of this surface chemistry to separate the phosphonyl antibiotic mixtures. Commensurate with this, these analytes will show a reduced retention on the column chemistry and will elute earlier and in higher amounts of organic solvent than if a neutral polar HILIC surface were used. This then increases their detection sensitivity by mass spectrometry.

Analogously, one could use an anion exchange column surface (positively charged) chemistry to reduce the influence of cationic (positively charged) functional groups on a set of analytes, as when selectively isolating phosphorylated peptide 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. The common, positively charged amino group will be repelled from the column surface chemistry and thus these conditions will enhance the role of the phosphate's polarity (as well as other neutral polar groups) in the separation.


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