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

Abstract

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.

Untargeted profiling of Glycyrrhiza glabra extract with comprehensive two‐dimensional liquid chromatography‐mass spectrometry using multi‐segmented shift gradients in the second dimension: Expanding the metabolic coverage

“…Due to the high complexity of the sample, a considerable number of compounds overlapped (Supporting Information Fig. 2), which is in agreement with earlier studies on licorice using a 1D‐LC method . Therefore, a 1D‐LC separation technique is likely insufficient for a comprehensive analysis of a complex plant extract, and the use of multiple dimensions (i.e.…”

“…The major classes of compounds included flavanones, flavones, chalconoids, and triterpenes. Table reports the tentative identification of the G. glabra constituents on the basis of their MS and diode array spectra data with reference to reported literature . The gain in compound coverage can be readily observed; 1D‐LC analysis (Supporting Information Fig.…”

“…Recent studies by Montero et al. demonstrated the applicability of a HILIC × RP combination for analysis of food samples, which illustrated promising “orthogonal” separation of the phenolic compounds and saponins . Notably in refs.…”

“…Notably in refs. selected licorice samples were analysed with the aim to provide a typical metabolite pattern according to their geographical location and further increase the HILIC×RP‐LC method performance .…”

Separation of di- and trisaccharide mixtures by comprehensive two-dimensional liquid chromatography. Application to prebiotic oligosaccharides

“…In fact, the fraction solvent was by far stronger for the 2 D than the initial gradient conditions. This problem has been widely reported in HILIC×RP couplings [20]. In other applications, an increase of the fraction volume was shown to be positive towards the elimination of this issue; the use of sampling loops with internal volume larger than strictly needed to accommodate the 1 D fraction was demonstrated to be useful to produce a dilution effect with 2 D compatible solvent [21].…”