Abstract
Hydrophilic interaction liquid chromatography (HILIC) is able to separate from polar to highly polar solutes, using similar eluents to those in the reversed-phase mode (RPLC) and a polar stationary phase, where water is adsorbed onto its surface. It is widely accepted that multiple modes of interaction take place in the HILIC environment, which can be far more complex than the interactions in an RPLC column. The behaviour in HILIC should be adequately modelled to predict the retention with optimisation purposes and improve the understanding on retention mechanisms, as is the case for RPLC. In this work, the prediction performance of several retention models is studied for seven HILIC columns (underivatised silica, and silica containing diol, amino and sulfobetaine functional groups, together with three columns recently manufactured with neutral, anionic, and cationic character), using uracil and six polar nucleosides (adenosine, cytidine, guanosine, thymidine, uridine, and xanthosine) as probe compounds. The results in HILIC are compared with those that were offered by the elution of several polar sulphonamides and diuretics analysed with two C18 columns (Chromolith Speed ROD and Zorbax Eclipse XDB). It is shown that eight retention models, which only consider partitioning or both partitioning and adsorption, give similar good accuracy in predictions for both HILIC and RPLC columns. However, the study on the elution strength behaviour, at varying mobile phase composition, reveals similarities (or differences) between RPLC and HILIC columns of diverse nature. The particular behaviour for the HILIC and RPLC columns was also revealed when the retention, in both modes, was fitted to a model that describes the change in the elution strength with the modifier concentration.
Highlights
Since the introduction of bonded phases, reversed-phase liquid chromatography (RPLC) has become one of the most important analytical techniques, owing to its separation capability, versatility, and reliability
The retention times of several polar compounds separated with seven Hydrophilic interaction liquid chromatography (HILIC) columns and two RPLC columns were used to evaluate the performance of the retention models that are described in Section 2, and measure the elution strength behaviour in each column
In HILIC, the hold-up times were 1.38 ± 0.32, 1.61 ± 0.09, 1.22 ± 0.12, 1.40 ± 0.11, 3.12 ± 0.20, 2.7 ± 0.4, and 2.74 ± 0.14 min. for the diol, silica, amino, zwitterionic, HILIC A, HILIC-B, and HILIC-N columns, respectively, whereas the retention time ranges for uracil and the group of nucleosides were 2.04–4.61, 2.01–4.73, 1.60–6.52, 1.90–11.76, 4.00–6.94, 3.44–20.19, and 3.40–15.89 min., respectively
Summary
Since the introduction of bonded phases, reversed-phase liquid chromatography (RPLC) has become one of the most important analytical techniques, owing to its separation capability, versatility, and reliability. A solution to succeed in the separation of polar compounds is replacing conventional C18 bonded phases by more polar phases where water is adsorbed, in the so-called hydrophilic interaction liquid chromatography (HILIC) [3,4,5,6,7,8]. Conventional non-bonded phases in normal phase liquid chromatography (NPLC), such as pure silica, can be used in HILIC. In HILIC, the water-rich layer that covers the surface of the polar stationary phase creates a liquid/liquid extraction system, with a mobile phase containing a high concentration of organic solvent (usually acetonitrile). The complete retention mechanism is still not clarified, it is known that multiple interactions, including partitioning into the adsorbed water-rich layer, and polar and electrostatic (ion-exchange) effects are possible in a HILIC stationary phase
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