Impact of column temperature and mobile phase components on selectivity of hydrophilic interaction chromatography (HILIC)

Abstract

The retention mechanism and chromatographic behavior for different polar analytes under hydrophilic interaction chromatography (HILIC) conditions have been studied by application of different mobile phases and stationary phases to various analytes at different temperatures. In addition to the commonly accepted mechanism of analyte liquid-liquid partitioning between mobile phase and water-enriched solvent layer which is partially immobilized onto the surface of the stationary phase, hydrogen-bonding, hydrophobic interaction, and ion-exchange interactions may also be involved. The predominant retention mechanism in HILIC separation is not always easily predictable. It can depend not only on the characteristics of the analytes but also on the selection of mobile and stationary phase compositions. The objective of this review is to evaluate the potential application of column temperature and mobile phase composition toward improving HILIC selectivity. The functional groups from analyte structures, stationary phase materials and organic mobile phase solvents will be highlighted.

Theory and Practice of UHPLC and UHPLC–MS

“…(2.9) to the experimental data is considered as proof of an enthalpy-controlled exothermic process within the experimental temperature range (Melander and Horvath, 1980). In addition to changing enthalpy, the retention can be connected with entropic changes, namely conversion between different sample configurations (Hao et al, 2008). For example, an enthalpy-controlled retention process (positive B i terms in Eq.…”

“…HILIC mobile phases usually contain a buffer, whose pH and ionic strength are usually selected to enhance the sample ionization, retention, and separation selectivity, i.e., pH > 7 for acids and pH < 7 for bases (Jandera, 2008;Hao et al, 2008). The retention in HILIC systems with uncharged stationary phases usually increases with increased salt (buffer) concentrations, probably because of enhanced hydrogen-bonding interactions between the analyte and the stationary phase.…”

“…An excellent overview on recent progress in the development of polar stationary phases until 2006 was written by Hemström and Irgum (2006); more recent advances are included in reviews by Jandera (2011) and by Buszewski and Noga (2012). Other reviews have focused on the effects of the operating conditions on HILIC separations (Hao et al, 2008), separation efficiency (Ikagami et al, 2008), HILIC method development (Dejaegher et al, 2008), coupling HILIC systems with MS and MS/MS (Nguyen and Schug, 2008;Hsieh, 2008), implementation of HILIC systems in two-dimensional (2D) separation modes (Jandera, 2008), and on HILIC applications in biological (Yoshida, 2004;Jian et al, 2010), pharmaceutical (Dejaegher and Vander Heyden, 2010), and metabolite (Iwasaki et al, 2007;Spagou et al, 2010) analysis. An excellent book on HILIC has appeared recently, covering various aspects of the technique: separation mechanism, stationary phases, method development, and applications (Olsen and Pack, 2013).…”

The acetonitrile shortage: Is reversed HILIC with water an alternative for the analysis of highly polar ionizable solutes?

“…In HILIC separation is primarily achieved by partitioning between a water-enriched layer on the surface of a polar stationary phase and a mobile phase that contains a high percentage of organic solvent [3]. Although several solvents have been evaluated, and in some cases successfully used [4,5], ACN is by far the most generic solvent in HILIC. More information about the features and separation mechanisms in HILIC can be found in two papers [6,7].…”

An introduction to liquid chromatography–mass spectrometry instrumentation applied in plant metabolomic analyses

“…The simplest forms of LC thus far discussed, paper chromatography and TLC, are categorised as partition chromatography methods, here a solvent is retained on or within the stationary phase, thus sample components are retained by equilibrium between the liquid stationary and liquid mobile phases (Hao et al, 2008). Hydrophilic interaction chromatography (HILIC) is a modern example of normal-phase partition chromatography applied in HPLC; here polar analytes diff use into a bonded polar stationary phase (being more polar than the mobile phase) the interaction between which depends upon an analyte polarity dictated by the various functional groups that the analyte possesses (Hao et al, 2008;Cubbon et al, 2009). By starting the gradient elution with a highly organic mobile phase solvent, the non-polar metabolites elute rapidly, whereas the retention of polar metabolites is maximised.…”