High performance liquid chromatography (HPLC) in various flavors (normal phase, reversed phase) has long been a commonplace tool used in chemistry and biochemistry labs in industry and academia alike.
However, this fundamental analytical technique continues to evolve and find additional applications. Hydrophilic interaction chromatography (HILIC) is a form of HPLC that relies on a hydrophilic stationary phase and a highly organic mobile phase to achieve separations of highly polar compounds. In that regard, HILIC is similar to normal phase chromatography - HILIC is even referred to in the literature as aqueous normal phase chromatography. The goals of the techniques are the same – retention of polar compounds. However, under HILIC conditions, the stationary phase becomes coated by a layer of water with which polar analytes interact. The mobile phase is typically a high percentage organic (> 80% acetonitrile), and the low percentage of water in the mobile phase leads to longer retention times for polar compounds. Both isocratic and gradient HILIC methods can be used. The elution order observed for HILIC is similar to that of normal phase separations, and opposite that of reversed phase HPLC . In addition to hydrophilic interactions, hydrogen bonding and ionic interactions may also contribute to retention of the analyte and affect the separation. While this mode of separation is not new, it has experienced a considerable increase in popularity in the last decade. The increase in popularity is likely driven in part by (1) the pharmaceutical industry’s need to develop novel classes of soluble polar compounds in the search for new drugs and (2) the applicability of the technique to peptides and proteins. A recent online search (www.PubMed.gov) for the term HILIC returned 325 hits, including 15 reviews. A similar search for the term “hydrophilic interaction chromatography” returned over 660 hits.
HILIC has now matured to the point where review articles focused on different aspects of HILIC separations have appeared in the last two years. For example, the behavior of different stationary phases and column designs (particles vs. monoliths) were reviewed by Ikegami, et al.1 They tested bare silica, amino and amide modified, cyano, diol and poly(succinimide) phases, among others . These authors make useful recommendations regarding column performance and th types of analytes best separated by each stationary phase. The use of alternative stationary phases, such as cyclodextrins and glycopeptides has also been reviewed.2 The effects of mobile phase composition and column temperature were studied by Hao et al.3 They concluded that the effects of increased column temperature or changes in mobile phase (methanol vs. acetonitrile, for example) were not predictable due to the different retention modes (hydrophilic interactions, hydrogen bonding and ionic interactions) that can occur during a HILIC separation.
A distinct advantage of HILIC separations that contributes to their growing popularity is their compatibility with mass spectrometric methods. The volatile nature of mobile phases comprised mostly of organic solvent aids the desolvation of ions and leads to stronger signals. A recent search of PubMed for the terms HILIC and MS returned > 150 hits, many of them describing applications with HILIC as the “LC “ component of “LCMS”. For example, while analysis of the many polar constituents of blood plasma by reversed phase HPLC can be challenging, Cai et al. demonstrate that HILIC methods can be applied to metabonomics studies with good results. Their HILIC study revealed potential new biomarkers not identified when reversed phase HPLC methods were used4.
In summary, the power of HILIC to separate a wide variety of polar compounds is now widely recognized and an array of stationary phases is available to choose from. The factors affecting HILIC separations are not completely understood, and further research is required. The technique is MS-friendly and robust enough to be used in metabonomics studies.
1Ikegami et al. J Chromatogr A 2008 Mar 14; 1184(1-2) 474-503.
2Wang et al. J Sep Sci 2008 Jun; 31(11) 1980-90.
3Hao et al. J Sep Sci 2008 May; 31(9) 1449-64.
4Cai et al. Analyt. Chimica Acta 650 (2009) 10-15.