| The dissertation was catagerized to the natural product analysis andchromatographic analysis of analytical chemistry. High speed countercurrentchromatography was employed as a focal point, to carry out a gradient countercurrentchromatography and pressurized liquid extraction–coupled countercurrentchromatography analyses.The newly established hyphenated instrumentation of UPLC/MS andUPLC/SPE/NMR techniques have been applied for a fast identification of the majorchemical components from Chinese medicinal herb Apocynum venetum.High–performance counter–current chromatography (HPCCC) and High performanceliquid chromatography coupled with mass spectrometry (HPLC–MS) was efficientlyutilized for the separation and identification of the chemical components with a widerange of polarity from the mixed extract of A. venetum. The HPCCC separation wasinitiated by filling the column with the lower phase of n-hexane–ethylacetate–acetonitrile–water at a volumn ratio of1.5:3.5:2:5as a stationary phasefollowed by elution with the upper phase of n-hexane–ethylacetate–acetonitrile–water (1.5:3.5:2:5) to separate the hydrophobic compounds (tailto head). Then the mobile phase was switched to the upper phase of ethylacetate–acetonitrile–water (5:3:7) to eluted the moderate hydrophobic compounds,then the mobile phase was switched to the upper phase of ethylacetate–methanol–water (5:2:5) to eluted the moderate hydrophilic compounds, andfinally the hydrophilic compounds still retained in the column was eluted by the upperphase of n-butanol–methanol–water (5:1:5). A total of sixteen compounds includingadhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, avicularin, acetylatedisoquercetin, acetylated hyperoside, astragalin, trifolin, isoquercetin, hyperoside,querciturone, rutin, chlorogenic acid andquercetin-3-O-β-D-glucosyl-β-D-glucopyranoside were successfully separated via thefour sets of solvent systems in one step operation for130min.Hypericum perforatum L. is a typical traditional Chinese medicine. Exposure of theextracts of H. perforatum to light may lead to the degradation of phloroglucinols,which are extremely sensitive to oxidation and unstable in solution on exposure to air,therefore phloroglucinols are difficult to separate and isolate by conventional method.In this case, a method combining an extraction system with an isolation system onlineto avoid the exposure of the extracts to air and light is urgently needed. Quinic acids have previously been shown to possess a multitude of pharmacological activities.However, the supplies of tricaffeoylquinic acids have been limited due to their verylow content in natural plants and difficulties in isolating their pure compounds fromnatural sources. More efficient extraction and separation methods to provide bioactivecomponents with high sample recovery are also needed. In this case, pressurizedliquid extraction (PLE) coupled with high-performance counter-currentchromatography (HPCCC) was successfully used for the extraction and onlineisolation of five chemical constituents from the plant Hypericum perforatum L. Theupper phase of the solvent system of ethyl acetate–methanol–water (5:2:5) was usedas both the ASE solvent and the HPCCC stationary phase. Two hydrophobiccompounds including hyperforin and adhyperforin were isolated. The lower phaseof ethyl acetate–methanol–n-butanol–water (5:2:2.5:12) was used as both the ASEsolvent and CCC stationary phase. Three hydrophilic compounds of5-O-tricaffeoylquinic acid,1,3,5-O-tricaffeoylquinic acid and3-O-caffeoylquinicacid were obtained in a one–step extraction–separation process with less than3hfrom raw material of H. perforatum.Panax notoginseng (Burk.) F. H. Chen is a well-known natural medicine ofPanax species, The extracts of P. notoginseng contain a broad range of commonsaponins, these common saponins could be hydrolyzed to the rare saponins at highertemperature. It is dificult to separate the saponins with a wide range of polarities inone operation by using ASE/HSCCC. Therefore, three stage ASE/HPCCC wassuccessfully used for the structural modification, extraction and online isolation of thesaponins with a wide range of polarity from Panax notoginseng by oneextraction–separation operation with three stages. At the first stage, the upper phase ofthe solvent system of ethyl acetate–n-butanol–water (1:1:2or1.2:1:2) or ethylacetate–n-butanol–methanol–water (3:5:1.5:6) was used as both the ASE solvent andHPCCC stationary phase (extracted at60oC), and the polar target compounds whichpumped in the CCC column were eluted with the corresponding lower phase of thesolvent system mentioned above. At the second stage, the upper phases of the solventsystem of ethyl acetate–n-butanol–methanol–water (6:3:2:6or7:3:2:7) was used asboth the ASE solvent and HPCCC stationary phase (extracted at115oC), and the targetmoderate polar compounds were eluted with the corresponding lower phase of thesolvent system. Finally, the upper phase of the solvent system ofn-hexane–n-butanol–methanol–water (8:2:2:8) or n-hexane–ethylacetate–n-butanol–methanol–Water (0.2:10:0.5:1.5:8) was used as both the ASE solvent and HPCCC stationary phase (extracted at135oC), and the target low polarcompounds were eluted with the corresponding lower phase of the solvent system.More than nine pure compounds including notoginsenosides R6, R1, Spt1andginsenosides Rb1, F4, Rh3, Rg3, Rs3and Rk1with a wide range of polarity weresuccessfully separated via the seven sets of solvent systems in oneextraction–separation operation within400min.Before HSCCC sepration, the HSCCC solvent systems are usually prepare4–10candidate solvent systems, and the optimal solvent system has to be selected bydetermining the K values of these systems. However, this screening method has agreat deal of randomness and blindness, and the solvent system so obtained may notbe the best solvent system. Therefore, it is necessary to apply a mathematical methodto calculate the relationship between the solvent composition of the two-phase solventsystem and the K value of the target compound by using the obtained K values; theoptimal solvent ratios of the two-phase solvent system can then be calculated, and thesolvent system obtained so can be identified as the optimum solvent system in ourwork. The use of organic solvents such as n-heptane, ethyl acetate, methanol, and inparticular chloroform and methylene chloride are highly toxic, and their excessiveexposure can lead to serious diseases. Therefore, it is very important to develop asuitable instrumental setup for industrial separation and to prevent their coming intocontact with human population. In this case, automation of pressurized liquidextraction (PLE) coupled with HSCCC based on mathematical calculation was firstlydeveloped and applied to the systematic extraction and online isolation of chemicalconstituents with a wide range of polarity from Panax ginseng and Panaxquinquefolium. The experiments were designed under the guidance of optimizedmathematical model, the K values of the target compounds and the peak resolutionswere employed as the research indicators to calculate and optimize the solventsystems and the flow rates of the mobile phases. First, P. ginseng was employed as atest medicinal plant, the solvent systems of HSCCC were prepared manually. At thefirst stage of PLE/HSCCC, the solvent system of ethyl acetate–n-butanol–water at avolume ratio of1.1:1.0:2.1, ethyl acetate–n-butanol–methanol–water (3.1:0.6:1.0:2.6)and n-hexane–n-butanol–methanol–water (4.2:1.0:1.2:4.5) were used in three stages,extracted at60,110oC and125oC respectively. The upper phase of the solvent systemwere used as both the PLE solvent and HSCCC stationary phase, and the targetcompounds were eluted with the corresponding lower phase of the solvent system toseparate the polar common compounds, moderate polar compounds and low polar compounds respectively. Nine target compounds with purities above93.35%and twonon-target compounds with purities above73.65%were successfully semi-preparativeseparated in460min. Second, P. quinquefolium was employed as a test medicinalplant, the solvent systems of HSCCC were prepared automatically by pumps. PLEcoupled with HSCCC via was firstly developed to extract and isolate ginsenosidesfrom Panax quinquefolium. In three stages of PLE/HSCCC, ethyl acetate, n-butanol,and water were simultaneously pumped into the solvent separator at the flow rates11.0,10.0, and23.0mL/min, respectively. The upper phase of the solvent system inthe solvent separator was used as both the PLE solvent and the HSCCC stationaryphase, followed by elution with the lower phase of the corresponding solvent systemto separate the common ginsenosides. In the second and third stages, rareginsenosides were first separated by elution with ethyl acetate, n-butanol, methanol,and water (flow rates:20.0,3.0,5.0, and11.0mL/min, respectively), then withn-heptane, n-butanol, methanol, and water (flow rates:17.5,6.0,5.0, and22.5mL/min, respectively). Nine target compounds, with purities exceeding95.0%, andthree non-target compounds, with purities above84.48%, were successfully separatedat the semipreparative scale in450min. The separation results prove that thePLE/HSCCC parameters calculated via mathematical model and formulas wereaccurately and scientifically. This research has opened up great prospects forindustrial automation application. |
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