| Objective: Based on a new type of two-dimensional chromatographic method,we built the coupled Sephadex LH-20 × RPLC system and the rhubarbs were set as research object to study the application in Chinese native medicine analysis.Moreover,the online Sephadex LH-20 × RPLC was applied in the preparation of rhubarbs,and its application in the preparation of traditional Chinese medicine's compounds was discussed.At last,used the new compound prepared by Sephadex LH-20 × RPLC as a content determination indicators,to establish the new content determination method.Material and method:1.First,Sephadex LH-20 was selected as the first dimension of the role of molecular sieve,distribution and hydrogen bondingwe built the coupled analytical on-line Sephadex LH-20 ×RPLC system.Secondly,we investigate the separation effect on column length,velocity and the elution solvent on Sephadex LH-20 columns,to establish the first dimension separation of parameters.Thirdly,we investigate the interface technology of two-dimensional liquid chromatography,and the quantitative ring and the two common enrichment column interfaces were analyzed to select the appropriate interface.Fourthly,compared the separation effect of Sephadex LH-20 × RPLC combination mode with conventional RPLC × RPLC combination mode.Lastly,we applied the online analytical Sephadex LH-20 × RPLC system to analysis the chemical constituents of R.officinale Baill.and Rheum hotaoense C.Y.Cheng et C.T.Kao.2.We magnified the separation scale of online Sephadex LH-20 × RPLC system,and 20 mm inner diameter,170 cm length Sephadex LH-20 column was chose as the 1D separation column to separate gram-scale of R.officinale Baill.and Rheum hotaoense C.Y.Cheng et.C.T.Kao.Most purified fragments isolated by the LH-20 × RPLC system were also analyzed with HPLC.Identification of purified compounds was performed by MS and NMR.3.A method for the quantitative determination of 2-cinnamyl glucose ester tautomer was established by High Performance Liquid Chromatography?HPLC?.And the procedure was applied to determinate the quantity of tautomer from three different batches of R.officinale Baill..Results:1.The 1st D separation condition of LH-20 chromatography was determined: the column length was 100 cm and flow rate was 0.03 m L/min and enrichment column as the interface between two dimensions,diluted with gradient flow rate.Sephadex LH-20 × RPLC and RPLC× RPLC compared with the model,the former is better than the latter.The orthogonality of Sephadex LH-20 × RPLC online analysis of R.officinale Baill.was 90.6%,however,the orthogonality of on-line RPLC × RPLC was only 30.8%.2.Nine compounds were separated from R.officinale Baill.,include cinnamic acid?1?,pleuropyrone A?2?,Gallicacid?3?,2-cinnamoyl-glucose?4?,aloe-emodin-3-?hydroxymethyl?-O-?-D-glucopyranoside?5?,2,5-dimethyl-7-hydroxychromone?6?,aloe-emodin?7?,chrysop hanol?8?,emodin?9?.Twelve compounds were separated from Rheum hotaoense C.Y.Che ng et C.T.Kao,such as Chrysophanol-8-O-?-D-glucopyranoside?1?,Physcion-8-O-?-D-gl ucopyranoside?2?,emodin?3?,1,3-dihydroxy-6,7-dimethy-aetone-1-O-?-D-glucopyranoside?4?,Rhaponiticin?5?,Resveratrol?6?,aloe-emodin-8-O-?-D-glucopyranoside?7?,Piceatan nol-3-O-?-D-glucopyranoside?8?,chrysophanol-8-O-?-D-?6'-galloyl?-glucopyranoside?9?,Rhaponticin-1 "-O-gallate?10?,Rhapontigenin?11?,Rhaponticin-6"-O-gallate?12?.3.The 2-cinnamyl glucose ester tautomer determination method was established: extracted by ultrasound extraction with methanol,and the procedure has good stability in 10 hours?RSD=2.9%?,and finely repeatability?RSD=1.2%?.Recovery rate of samples was between95%-105.0%.The relative standard deviation?RSD?value also complied with relevant regulations.The tautomeric contents of the three batches of R.officinale Baill.were 0.3816%,0.3745% and 0.3862%,respectively.Conclusion:1.The coupled on-line two dimensional Sephadex LH-20 × RPLC system was conducted successfully and the orthogonality was higher than the RPLC × RPLC mode.2.The separation effect of complex samples with on-line Sephadex LH-20 × RPLC two dimensional system was good,which saving time,energy and solvent.3.In this study,it is the first time to establish the 2-cinnamyl glucose ester tautomer content determination method. |
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