Abstract
The interactions and dynamic behavior of a select set of polar probe solutes have been investigated on three hydrophilic and polar commercial stationary phases using saturation transfer difference 1H nuclear magnetic resonance (STD-NMR) spectroscopy under magic angle spinning conditions. The stationary phases were equilibrated with a select set of polar solutes expected to show different interaction patterns in mixtures of deuterated acetonitrile and deuterium oxide, with ammonium acetate added to a total concentration that mimics typical eluent conditions for hydrophilic interaction chromatography (HILIC). The methylene groups of the stationary phases were selectively irradiated to saturate the ligand protons, at frequencies that minimized the overlaps with reporting protons in the test probes. During and after this radiation, the saturation rapidly spreads to all protons in the stationary phase by spin diffusion, and from those to probe protons in contact with the stationary phase. Probe protons that have been in close contact with the stationary phase and subsequently been released to the solution phase will have been more saturated due to a more efficient transfer of spin polarization by the nuclear Overhauser effect. They will therefore show a higher signal after processing of the data. Saturation transfers to protons in neutral and charged solutes could in some instances show clear orientation patterns of these solutes towards the stationary phases. The saturation profile of formamide and its N-methylated counterparts showed patterns that could be interpreted as oriented hydrogen bond interaction. From these studies, it is evident that the functional groups on the phase surface have a strong contribution to the selectivity in HILIC, and that the retention mechanism has a significant contribution from oriented interactions.
Highlights
Hydrophilic interaction chromatography (HILIC) [1,2] has in recent years become a widely used liquid chromatographic separation mode, mainly due to its unique capability of separating highly hydrophilic compounds that are poorly retained in reversed phase liquid chromatography (RPLC)
In a recent study it has been shown that toluene, a hydrophobic solute widely used as zero volume marker in HILIC, is capable of direct interaction with the ligands of three different polar stationary phases [15]
The saturation transfer difference Nuclear magnetic resonance (NMR) method used in this work has been described and validated in a recently published paper [15], in which we showed that toluene, which is frequently used as a void volume marker in HILIC, is capable of penetrating into the polar ligand space where the water-enriched layer is supposed to be located [11]
Summary
Hydrophilic interaction chromatography (HILIC) [1,2] has in recent years become a widely used liquid chromatographic separation mode, mainly due to its unique capability of separating highly hydrophilic compounds that are poorly retained in reversed phase liquid chromatography (RPLC). This advantage is gained by the use of highly polar stationary phases, which offer a substantially higher selectivity potential compared to RPLC. In addition there is evidence that dipole-dipole interactions, molecular shape selectivity, and even “hydrophobic interaction” play important roles in HILIC mode retention [16,17,18]
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