| An analytical and a preparative two-dimensional normal phase liquid chromatography x reversed phase liquid chromatography(NPLC x RPLC)system was constructed in the research.At first,a thermal evaporation assisted adsorption(TEAA)interface was designed.This interface could achieve the fast removal of NPLC solvent under increased temperature and transference of solutes from the first dimension to the second,which solved the solvent incompatibility problem between NP and RP systems.Secondly,a series of parameters of the system,including temperature and vacuum condition during evaporation,eluting time and regeneration time,was evaluated with test solution of toad skin.Thirdly,the performance of the NPLC x RPLC system was illustrated by the analysis of toad skin with a modulation time of 190 s,which showed a good resolving power.Better orthogonality(75.2%)was achieved when comparing with RPLC(C18)× RPLC and RPLC(CN)× RPLC.Then,the 2D system was expanded to the on-line preparative process of toad venom with an improved vacuum evaporation assisted adsorption(VEAA)interface and 19 compounds with high purity were isolated within a single run.At last,with the instruction of on-line NPLC x RPLC analysis,ethanol extract of toad skin was separated with silica column and preparative HPLC,resulting in 17 compounds.This is the first time to realize the on-line comprehensive analysis of a moderate polar natural product by coupling NPLC with RPLC.Owing to the natural high orthogonality between NPLC and RPLC,this 2D system shows high separation efficiency for the complex samples and can be applied to the routine analysis and separation of complex natural products. |
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