Enhancing the compatibility of normal-phase chromatography x reversed−phase chromatography by combination of low−temperature sensitive aqueous-phase compatible normal- phase chromatography and at−column dilution modulation

Abstract

The nearly opposite retention mechanism in the two-dimensional liquid chromatography (2D−LC), which combines normal phase liquid chromatography (NPLC) and reversed phase liquid chromatography (RPLC), shows extremely high orthogonality and theoretical peak capacity. However, peak breakthrough and peak distortion caused by the highly incompatible 2D mobile phases counteracts the advantages offered by high orthogonality. To address this difficulty, this study proposes a comprehensive two-dimensional NPLC × RPLC integrating temperature-sensitive aqueous-phase compatible normal-phase chromatography (TSACNPLC) and at-column dilution modulation (ACDM). The proposed 2D−LC system uses an aqueous-miscible acetonitrile/methanol eluent in the 1st D NPLC, instead of an aqueous-phase immiscible eluent, such as n-hexane/methanol, to increase the miscibility with the RP mobile phase system. Additionally, the system exploits temperature-sensitive retention behavior to enhance the retention ability of aqueous-phase compatible NPLC. To verify the feasibility of the proposed 2D−LC, this study selected three multi-component samples with mid-to-low polarity, including ethoxylated (n ≈ 6) bisphenol A (BPA-6EO), ethoxylated (n ≈ 6) tristearylphenol (TSP-6EO), and safflower methanol extract. Next, the effectiveness of the constructed 2D−LC was systematically investigated, including low temperature-induced retention enhancement of NPLC, overcoming solvent incompatibility by ACDM, and optimization of 2 D separation conditions, was systematically investigated.