HILIC is a novel type of chromatography based on a mixed mode of retention mechanisms. It does not suffer from the problems associated with traditional normal phase chromatography (NPC). Compatible with mass spectrometric detection, HILIC can be used with sample preparation procedures without drying down organic extracts. HILIC is especially suited for the analysis of small polar analytes, but, based on its unique selectivity, the technique may be tried for any compound where traditional approaches have failed.
Hydrophilic interaction chromatography (HILIC) is a novel mode of chromatography that is increasingly used in a variety of fields. As early as 1951, hydrophilic stationary phases were reported to contain a water-enriched layer on the surface. Although initially, in the 1970s, HILIC was used for the analysis of sugars, the more recent much wider application of the technique is evidenced by the explosion of publications that involve the use of this technique [Figure 1], especially as an alternative to separate polar and hydrophilic analytes. Several good reviews exist on this subject, and the reader is referred to these for a more in-depth and technical discussion [1-5]. Broadly, HILIC is novel type of chromatography that uses water as the stronger eluting solvent and is compatible with mass spectrometric detection. It is especially suited to separate small polar molecules where conventional reversed phase chromatography often fails, but may be worth trying in any situation where traditional approaches do not succeed.
There is occasionally some confusion about the definition of HILIC and the distinction from normal phase chromatography (NPC). The definition provided by Alpert  sums it up as follows: the term HILIC should be used if a) the strongly eluting solvent is water, and b) the retention mechanism is by partitioning. The latter is the main distinction from NPC. Whereas in both HILIC and NPC water is the stronger solvent, in NPC, the analyte is in equilibrium between a state of adsorption to the polar stationary phase and dissolution in the non-polar mobile phase, whereas in HILIC the analyte partitions between a water-enriched layer immobilised on the hydrophilic stationary phase and the less polar mobile phase. However, HILIC is not this easily characterised.
Chromatography based on adsorption can be described by the following formula:
Log(k’) = C + D • log(Nb), where k’ the capacity factor and Nb is the molar fraction of water and C and D are constants.
Chromatography based on partitioning can be described by the following formula:
Log(k’) = B + A • ϕ, where ϕ is the volume fraction of water and B and A are constants.
When plotting Log(k’) values of analytes versus either the aqueous fraction or the log-transformed aqueous fraction, the linearity of those relationships indicate the mechanism of retention. However, when this is done for analytes determined by HILIC, the retention mechanism may shift between adsorption and partitioning as the composition of the mobile phase is varied . This suggests that HILIC is based on a mixed-mode chromatography consisting of partitioning and occasionally adsorption. Furthermore, the stationary phase in HILIC is often charged (silica columns have negative silanolate moieties, amino columns have positively charged ammonium moieties), and this introduces a third retention mechanism, that of ion-exchange. In summary, HILIC is based mainly on partitioning between the mobile phase and an immobilised water-rich layer, but depending on the type of analyte, composition of the mobile phase and pH, adsorption and ion-exchange phenomena may contribute to retention and chromatographic behaviour, resulting in a mixed-mode chromatography. The result is sometimes unpredictable elution order, but also an opportunity to effect chromatographic resolution of hard-to separate analytes where other approaches have failed.
The most simple and traditional stationary phase is the bare silica column. In this case, the purity of the silica is of particular importance. Trace amounts of metal ions can influence the acidity of the silanol moieties, which in turn has an impact on the charge of the solid surface and consequently the chromatographic performance. A variety of columns exist where the silica backbone is chemically modified, e.g. with 3-aminopropyl, 3-cyanopropyl, 2,3-dihydroxypropyl, amide, carboxymethyl, poly(succinimide), etc. . Yet another variation involves the use of an organic polymer as the backbone instead of silica. This can have an impact on chromatography as well, because the silica backbone can still play a role in the retention of an analyte. Furthermore, in our experience, silica based columns may bleed silica, which can be found deposited on the cone of hyphenated mass spectrometric detectors , and can cause a high background noise when mass spectrometric detection is used .
The stability of HILIC columns is no less than that of other columns. The instability of silica columns used in NPC is due to the adsorption of polar components on the column, which reduce the adsorptive properties and thereby reduce retention. In contrast, the use of polar solvents, washing away polar components, and the importance of partitioning rather than adsorption in HILIC ensures adequate column stability. Obviously, higher pH values can still dissolve silica from silica-based columns, and this should be taken into consideration .
Two noteworthy peculiarities of amino columns is the occurrence of mutarotation of sugars, catalysed by the amino functionalities. In addition, the reactivity of the amino moieties needs to be considered. No aldehydes or ketones should be injected on amino columns, because a solvent such as acetone, or sugar analytes may form a Schiff’s base/azomethine with the amino groups.
Because of the reversal of the solvent strengths, the elution order of analytes in HILIC is usually the opposite of that observed in reversed phase chromatography. The most common solvent system is a mixture of water and acetonitrile. Instead of acetonitrile, other water-miscible organic solvents can be used, such as tetrahydrofuran, isopropanol, and methanol. Occasionally, a solvent system is used whereby there is no water at all, e.g. with methanol as the stronger solvent, but this does not seem to confer added selectivity . Because of the pH-dependency of the charge of the stationary phase it is especially important to fix the pH of the mobile phase with a buffer. The components of such buffers need to be soluble in all compositions of the mobile phase, and commonly consist of ammonium acetate or formate salts. The choice of buffering salt may be of importance for the selectivity of the separation, as shown for tetracyclines, which could be separated by a citrate buffer, but not by an acetate buffer .
The use of gradients in HILIC should take into consideration the long equilibration times required . As a result, the first injection of a run will have aberrant retention times. Especially when developing a new assay, multiple injections should be performed under the same conditions before any modifications are made to the gradient to change the elution. In addition, the use of different solvent systems can influence retention over hundreds of column volumes. Some columns used in our laboratory are delivered filled with hexane, which needs to be washed out with isopropanol. Using such a new column, we were able to resolve four analytes with an acetonitrile-water solvent mixture, but that resolution slowly decreased as we proceeded with the assay development. Eventually, by looking again at the column history, we could regain resolution by including a small percentage of
isopropanol in our gradients .
Compatibility with sample preparation methods and mass spectrometric detection
The fact that water is the stronger solvent in HILIC opens up new possibilities for the design of assays. The often higher organic content of the mobile phase results in a lower head-pressure, thereby allowing for higher flow rates . Furthermore, the higher organic content makes for a mobile phase more easily evaporated in the ion-source of a mass spectrometric detector. As a result, mass spectrometric response can be enhanced. Lastly, organic sample extracts derived directly from liquid-liquid extractions or from eluting analytes from a solid phase extraction cartridge can be directly injected, without the usual drying down and reconstitution, if sensitivity considerations permit. The high organic content (weaker solvent) of the sample extracts result in strong retention on the front of the column by the sample focusing effect. Subsequently, the analytes are eluted by the mobile phase, which has a higher aqueous content than the sample injected.
In conclusion, HILIC is a novel type of chromatography based on a mixed mode of retention mechanisms. It does not suffer from the problems associated with traditional NPC, is compatible with mass spectrometric detection, and with sample preparation procedures without drying down organic extracts. HILIC is especially suited for the analysis of small polar analytes, but, based on its unique selectivity, may be useful for any compound where traditional approaches have failed.
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Jan H. Beumer, Pharm.D., Ph.D.
Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA
and Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15213, USA
Jan H. Beumer, Pharm.D., Ph.D.
University of Pittsburgh Cancer Institute
Room G27D, Hillman Research Pavilion
5117 Centre Avenue
Pittsburgh, PA 15213-1863, USA