Combination of off-line two-dimensional hydrophilic interaction liquid chromatography for polar fraction and two-dimensional hydrophilic interaction liquid chromatography × reversed-phase liquid chromatography for medium-polar fraction in a trad

Two-dimensional liquid chromatography (2-D LC) has been widely used for the analysis of complex samples owing to its great improvement in separation selectivity and peak capacity. However, one 2-D LC system may not be enough to meet the separation requirements due to the complexity of certain samples and respective limitations of two separation modes. In this work, water extract of Scutellaria barbata D. Don, a traditional Chinese medicine, was fractionated into polar fraction and medium-polar fraction by means of solid phase extraction (SPE). The fraction preparation made it easy to select the corresponding combination of 2-D LC method from hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC). An off-line 2-D HILIC × HILIC to analyze the polar fraction and an off-line 2-D HILIC × RP-LC to analyze the medium-polar fraction were developed, respectively. In total, 749 peaks were detected: 206 peaks from the polar fraction by the 2-D HILIC × HILIC and 543 from the medium-polar fraction by the 2-D HILIC × RP-LC. The practical peak capacities obtained in both systems were 2698 and 2879, and the orthogonality reached 63.18% and 90.62%, respectively. The results demonstrated that the two systems were both highly orthogonal, and the peak capacities greatly increased.