Pulsed elution modulation for on-line comprehensive two-dimensional liquid chromatography coupling reversed phase liquid chromatography and hydrophilic interaction chromatography.

In this work, a reversed phase liquid chromatography (RPLC) coupling to hydrophilic interaction chromatography (HILIC) system has been constructed, combining with pulsed elution reversed phase liquid chromatography (PE-RPLC) and HILIC to comprehensively analyze P. ginseng root extract, which is rich in saponins. By the application of pulsed elution (PE) modulation technique, the proposed RPLC × HILIC system allows the chromatographic separation to be optimized independently in both dimensions. In the first dimension (1D), PE modulation is achieved by the separation of a complex mixture, such as P. ginseng root extract, with a PE gradient. This PE gradient contains a set of pulses where the solvent strength increases gradually. Thus, the modulation of 1D eluent is realized by stepwise-pulse fractionation, rather than by a traditional two-dimensional interface. Furthermore, the number of fractions and the fractionated period can be regulated independently, which leads to independent adjustment of the separation cycle in the second dimension (2D) separation without the loss of D1 separation efficiency. To overcome the inherent solvent incompatibility of RPLC × HILIC, we introduced a newly developed trapping interface, equipped with bypass. The result indicates excellent separation of saponins in P. ginseng root extract. Compared with the traditional modulation method, the proposed RPLC × HILIC system has extreme flexibility, those modulation time could be regulated in a large range without re-optimizing the 1D PE gradient. Worthily mentioned, the proposed RPLC × HILIC system shows excellent orthogonality, and 20% more peaks could be obtained with current method compared to the traditional value based modulation method. Independent regulation of both dimensions could enable the proposed modulation method to be widely applied for complex samples analysis in ordinary laboratory.