Development of an On-Line Two-dimensional Normal Phase Liquid Chromatography System for Analysis of Weakly Polar Samples

In this study, a novel on-line two-dimensional (2D) normal phase × normal phase liquid chromatography (NPLC × NPLC) was developed for the separation of weakly polar samples. The 2D NPLC was integrated by a unique designed solvent evaporation (SE) interface, including a 6-port 2-position valve and a 10-port 2-position valve in conjunction with two silica gel-packed enrichment columns. The enrichment columns played a pivotal role in evaporating normal phase (NP) solvent from the first dimensional NPLC under vacuum and elevated temperature condition. The working parameters of the interface were evaluated comprehensively. To demonstrate the resolving powerful 2D system, we analyzed three natural-sourced weakly polar compounds from the extracts of toad venom, dammar resin, and propolis. To this end, we studied the effects of mobile phase combinations, the second dimensional column length for separation of the extracts from toad venom, and propolis, respectively. In general, we have found that excellent separation can be obtained with diversity of NP solvent combinations and longer NPLC column (150 mm long). Moreover, compared with the generally used 2D reversed-phase liquid chromatography (RPLC × RPLC), the NPLC × NPLC method exhibited better separation orthogonality. In conclusion, the new NPLC × NPLC separation method provides potential advantages for analysis of weakly polar samples.