Hydrophilic interaction liquid chromatography
HILIC (HydrophILic Interaction Chromatography or Hydrophilic Interaction LIquid Chromatography) is a version of normal phase liquid chromatography a name suggested by Dr. Andrew Alpert in his 1990 paper on the subject (J. Chromatogr. 499 (1990) 177) where he first proposed the chromatographic mechanism for it as one of 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 as an 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 water miscible aprotic solvent (e.g. THF or dioxane) can be used. Use of alcohols is also possible, however, their concentration will need be higher in order 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. 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 chromatography of neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention, thus separating it 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 (gentamycin), or ATP) higher concentrations of buffer (ca. 100mM) will be required to assure 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 neutral polar analyte separations (e.g carbohydrates) no buffer is necessary.
Use of other salts such as 100-300mM sodium perchlorate, which are soluble in high organic solvents (ca. 70% acetonitrile), is permissible for increasing the polarity of the mobile phase to effect elution, although this technique is less useful if one is relying on a mass spectrometer as a universal detector, since these salts are not volatile. Usually a gradient to increasing amounts of water suffices to promote elution.
All ions to some degree will partition into the stationary phase, so an occasional water wash will be required to assure a reproducible stationary phase is available for analytes.
The HILIC mode of separation is used intensively for separation of some biomolecules by polar differences, organic and some inorganic molecules (see: http://www.lcgcmag.com/lcgc/issue/issueDetail.jsp?id=4734). 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 (see: http://www.lcgcmag.com/lcgc/issue/issueDetail.jsp?id=4734) due to the ease of volatilization of the high organic content solvent.