Focusing and non-focusing modulation strategies for the improvement of on-line two-dimensional hydrophilic interaction chromatography × reversed phase profiling of complex food samples

- Different focusing and non-focusing modulations studied to improve HILIC × RP.
- Phenyl-hexyl trapping columns provided a satisfactory focusing effect.
- Active modulation is demonstrated as the most suitable approach for licorice profiling.
- Method sensitivity is improved by using focusing modulation strategies.
- Up to 94 compounds can be separated in the metabolite profile of licorice.

Comprehensive two-dimensional liquid chromatography (LC × LC) is ever gaining interest in food analysis, as often, food-related samples are too complex to be analyzed through one-dimensional approaches. The use of hydrophilic interaction chromatography (HILIC) combined with reversed phase (RP) separations has already been demonstrated as a very orthogonal combination, which allows attaining increased resolving power. However, this coupling encompasses different analytical challenges, mainly related to the important solvent strength mismatch between the two dimensions, besides those common to every LC × LC method. In the present contribution, different strategies are proposed and compared to further increase HILIC × RP method performance for the analysis of complex food samples, using licorice as a model sample. The influence of different parameters in non-focusing modulation methods based on sampling loops, as well as under focusing modulation, through the use of trapping columns in the interface and through active modulation procedures are studied in order to produce resolving power and sensitivity gains. Although the use of a dilution strategy using sampling loops as well as the highest possible first dimension sampling rate allowed significant improvements on resolution, focusing modulation produced significant gains also in peak capacity and sensitivity. Overall, the obtained results demonstrate the great applicability and potential that active modulation may have for the analysis of complex food samples, such as licorice, by HILIC × RP.

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