Text Box: HYDROPHILIC INTERACTION CHROMATOGRAPHY— AN EXCELLENT METHOD FOR SEPARATION OF POLAR SAMPLES PAVEL JANDERA DEPARTMENT OF ANALYTICAL CHEMISTRY, FACULTY OF CHEMICAL TECHNOLOGY, UNIVERSITY OF PARDUBICE, STUDENTSKÁ 573, CZ-53210 PARDUBICE, CZECH REPUBLIC Introduction In reversed-phase (RP) systems, most frequently used in contemporary HPLC practice, the stationary phase is non-polar, usually an alkyl-silica type bonded phase, while the mobile phase is a polar mixture of one or more organic solvents and water or a buffer. As a rule, the retention increases with more lipophilic stationary phases and with decreasing concentration of the organic solvent(s) in the mobile phase; polar solutes are less strongly retained than the non-polar ones. On the other hand, the stationary phase in normal-phase (NP) chromatography is more polar than the mobile phase, and opposite to RP HPLC, the retention increases with increasing polarity of the sample and of the stationary phase and in less polar mobile phases. In non-aqueous mobile phases traditionally used in NP chromatography, the retention mechanism is based on the competition between the sample and the mobile phase for localized polar adsorption centres on the adsorbent surface (Snyder et al., 2009; Snyder, 1968; Syder, 1974; Soczewinski, 1969; Nawrocki, 1997). Weakly or moderately polar compounds can be separated either in RPLC or in NPLC, with significant differences in the elution order and separation selectivity. Very hydrophilic samples such as carbohydrates or small strongly polar compounds are weakly retained in reversed-phase LC systems and often elute close to the column hold-up volume, so that their separation from one another and from polar matrix interferences may be difficult to accomplish, even in highly aqueous mobile phases (Pereira et al., 2009). Strongly polar compounds are often excessively retained in non-aqueous normal-phase systems or are poorly soluble in non-polar or in weakly polar organic solvents. However they can be separated on polar stationary phases with water added to the mobile phase (Huber et al., 1984). The aqueous normal-phase liquid chromatography (ANPLC) had been occasionally used long time before Alpert introduced the name “Hydrophilic Interaction Liquid Chromatography” (HILIC) for this separation mode (Alpert, 1983; Alpert, 1990).

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