The Construction Of On-line NPLC×NPLC System And Its Application In The Study Of Chemical Constituents Of Inonotus Obliquus

A two-dimensional NPLC x NPLC system was constructed in the research because of the insufficiency for the separation of weakly polar natural products in 2D-LC,and explore the application in the separation and preparation of the weakly polar component of Inonotus obliquus.First,a new NPLC×NPLC two-dimensional interface was designed.This interface could achieve the fast removal of NPLC solvent under increased temperature vacuum condition and transference of solutes from the first dimension to the second.Secondly,a series of parameters of the 2D interface,including temperature and vacuum condition during evaporation,mobile phase type,regeneration time and influence of the restrictor,was evaluated with test solution of model compound.Thirdly,the performance of the NPLC x NPLC system was illustrated by the analysis of model compound with a modulation time of 610 s,which showed a good separating power.Then,the 2D system was expanded to the on-line preparative process of 1 g of Inonotus obliquus chloroform layer and 9 compounds were isolated.And The NPLCxRPLC system was used to separate the 1 g of Inonotus obliquus ethyl acetate layer and 8 compounds were isolated within a single run.In this paper,the traditional methods were also used to separate Inonotus obliquus.The 2D system exhibits the superiority in separation time and the preparation process.Finally,22 compounds were isolated from Inonotus obliquus,including 2 compounds not isolated using conventional methods.Among them,20 compounds were traditionally isolated and identified,which exceeded the online 2D-LC in quantity,and the amount of preparation was larger,but the separation period of the online 2D-LC was short,and 16 compounds were obtained and identified from only 2 g of Inonotus obliquus extract.This is the first time to realize the on-line 2D analysis and separation of a weakly polar natural product by coupling NPLC with NPLC,which shows more efficient separating power than RPLC,and provides a new technical means for the preparation of weakly polar natural products.