Journal of Chromatography A, 1217 (2010) 683–688 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Efficiency of the same neat silica column in hydrophilic interaction chromatography and per aqueous liquid chromatography Fabrice Gritti a , Alberto dos Santos Pereira b , Pat Sandra b , Georges Guiochon a,∗ a Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA b Research Institute for Chromatography, Kennedypark 26, 8500 Kortrijk, Belgium article info Article history: Received 13 July 2009 Received in revised form 20 November 2009 Accepted 2 December 2009 Keywords: Hydrophilic interaction HILIC Hydrophobic interactions Per aqueous liquid chromatography PALC Mass transfer mechanism Halo HILIC Caffeine abstract The dependencies on the mobile phase flow velocity of the efficiency of a column packed with shell particles of neat porous silica (Halo) was measured under two different sets of experimental condi- tions. These conditions corresponded to the retention mechanisms of per aqueous liquid chromatography (PALC) at low acetonitrile concentrations and of hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. The results are compared. Small amounts of a diluted solution of caffeine were injected in order to record the chromatograms under strictly linear conditions. These efficiencies were measured in both water-rich (PALC retention mechanism) and acetonitrile-rich (HILIC mechanism) mobile phases for the same retention factors, between 0.25 and 2.5. The mobile phases were mixtures of acetonitrile and water containing neither supporting salt nor buffer component. At low retention factors, the efficiency of caffeine is better in the PALC than in the HILIC mode. For k ′ = 0.5, the minimum reduced height equivalent to a theoretical plate (HETP) is close to 2.5 in PALC while it exceeds 5 in HILIC. The converse is true for high retention factors. For k ′ > 2.5, the HETP is lower in HILIC than in PALC, because the major contribution to band broadening and peak tailing in this latter mode originates from the het- erogeneous thermodynamics of retention and eventually restricts column performance in PALC. Most interestingly, the reduced HETP measured in HILIC for caffeine never falls below 4. This suggests that the mass transfer of caffeine between the multilayer adsorbed phase (due to the interactions of the strong solvent and the silanol groups) and the acetonitrile-rich bulk eluent is slow. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Hydrophilic interactions chromatography (HILIC) is an effective alternative to reversed-phase liquid chromatography (RPLC) for the separation of samples containing very polar compounds [1]. HILIC is based on the distribution of the sample between a polar adsor- bent surface (silica, diol, aminopropyl, and zwitterionic phases) and a bulk aqueous phase, usually rich in organic solvent such as ace- tonitrile [2,3]. While the mechanism of retention in HILIC is still debated [4], the analysis of the elution times of water insoluble samples [5] and the minor plateau perturbation method [6] showed that a water-rich adsorbed multilayer (at least two layers) builds up on the surface of polar stationary phases when acetonitrile-rich (80–90%) bulk mobile phases are used. On the one hand, the more polar the compound, the higher its concentration in the adsorbed layers of water at equilibrium, suggesting that HILIC could have a partition mechanism. On the other hand, the retention of polar compounds increases with increasing water concentration in the ∗ Corresponding author. Tel.: +1 865 974 0733; fax: +1 865 974 2667. E-mail address: guiochon@utk.edu (G. Guiochon). mobile phase [7]. These observations demonstrate that partition alone does not govern the retention mechanism of polar samples in HILIC. Analytes compete with water for adsorption onto the polar sites at the stationary phase surface (silanols of silica, amino and zwiterrionic groups of silica-bonded phases). The current shortage of acetonitrile forces scientists in academic and industrial laboratories to consider replacing either acetonitrile as a component of mobile phases or acetonitrile-consuming sep- aration modes with other ones using different solvents. The first option would be to use ethanol or propanol instead of acetoni- trile. Ethanol has about the same elution strength as acetonitrile, performs reasonably well, and costs much less, so it is a suitable alternative solvent [8]. A second option is to take advantage of the hydrophobic character of siloxane groups at the surface of silica by using water-rich eluents in per aqueous liquid chromatogra- phy (PALC) [7]. Although good retention factors can be achieved in PALC, the adsorption mechanism is heterogeneous and involves active adsorption sites, which results in column overloading tak- ing place with rather small samples. In contrast, HILIC shows less peak tailing upon overloading the column. This was illustrated by the measurement of the adsorption isotherm of pyridine by frontal analysis and by the calculation of its adsorption energy 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.12.004