- 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 silicasilanol or diol bonded phases
*amino oranionic bonded phases
*amide bonded phases
*cationic bonded phases
*zwitterionic bonded phases.A typical
mobile phase for HILIC chromatography includesacetonitrile ("MeCN", also designated as "ACN") with a small amount of water. However, any aprotic solvent miscible with water (e.g.THF ordioxane ) can be used. Alcohols can also be used, however, their concentration must be higher to achieve the same degree of retention for ananalyte relative to an aprotic solvent - water combination. See alsoAqueous 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-deficientmobile phase , creating a liquid/liquid extraction system. Theanalyte 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 fromion exchange chromatography . The morepolar compound s will have a stronger interaction with the stationaryaqueous layer than the lesspolar compound s. 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 themobile 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
biomolecule s 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.
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