Construction And Application Of A Comprehensive Preparative Two-dimensional HSCCC×HPLC Chromatography System

The article aimedat buildingtwo-dimensional highspeed countercurrent chromatography (HSCCC) coupling with the preparative liquid chromatography (prep-HPLC) system.Due to the incompatible problems betweencountercurrent chromatography and preparative liquid chromatography,the study was designed for connecting flow dilution-solid phase capture interface.The first dimensional countercurrent chromatography elution should be diluted, and then with the electronic control two ten-port valve, two enrichment column was used for alternating high-speed countercurrent chromatography dilution enrichment, and then elution was segmented into preparative HPLC.It is the first timefor using two-dimensional continuous HSCCC×HPLC chromatography.The interface between the enrichment column and diluted operating parameters was investigated and optimized, which finally determined the working parameters:sample volume100mg, the15mm×30mm id column as C18pre-enrichment column, four timeswater dilution and dynamic mixer.Furthermore, Toad methanol extract was used to test the separation capacity of HSCCC×HPLC system, the first dimension HSCCC using theflow control to match the second dimension HPLC separation of time. Application of the system within8hours, onealkaloid and11cardiac steroids were achieved from100mg of methanol extract Toad.Deacetylbufotalin(2,5.8mg,90.6%),Gamabufotalin(3,2.7mg,99.7%),de-O-acetylcinobufotalin(4,1.5mg,77.7%),Arenobufagin(5,2.8mg,99.9%),Telocinobufagin (6,1.5mg,99.9%),Deacetylcinobufagin (7,1.9mg,90.4%),Bufotalin(8,3.6mg,99.9%),Cinobufotalin(9,6.1mg,52.3%), Bufalin (10,4.8mg,99.9%),Cinobufagin(11,3.5mg,99.3%), Arenobufagin Resibufogenin (12,4.1mg,85.0%).Shows a good separation and peak capacity.The HSCCC×HPLC system was usedto praeruptorin extract isolates,too.11compounds were achieved from1000mg praeruptorin ethanol extract within three hours, xanthotoxin (1,3.7mg,90.2%)、Isopimpinellin (2,1.5mg,93.1%)、d-laserpitin (3,4.3mg,96.2%)、Pd-Ib (4,19.7mg,90.2%)、PraeruptorinA (5,167mg,90.2%)、Nodakenetin tiglate (6,3.1mg,90.2%)、corymbocoumarin (7,3.5mg,90.2%)、Decursinol angelate (8,4.1mg,90.2%)、Decursinol angelate (9,2.1mg,90.2%)、PraerupterinB (10,12.1mg,90.2%)、PraeruptorinE (11,30.3mg,90.2%)。In summary, the study designed a new comprehensive two-dimensional HSCCC x HPLC instrument system. the system was successfullyachieveda one-step separation and preparation from traditional Chinese medicine Toad and praeruptorin.The results showed that the system can take advantage of differences between CCC and HPLC separation mechanism and was capable of complex natural product for fast, efficientseparation and preparation, thus speeding up the process of structural studies of natural products.