| Traditional Chinese medicines(TCMs)have been used for centuries due to their therapeutic efficacy in the treatment of many diseases.They also attract worldwide attention as an important source of new bioactive compounds.It is generally believed that the active TCM components are complex.It's undoubtedly become an important research content for the analysis of its active ingredients.TCMs are usually administrated by its water extracts which is a complex mixture.They contain some hydrophilic polar substances such as nucleosides,nucleobases,flavonoids,saponins,amino acids,carbohydrates,protein,etc.Therefore,highly efficient analytical tools are essential for isolating active compounds from TCMs and controlling their quality.Until now,various separation techniques,including reversed-phase liquid chromatography(LC),capillary electrophoresis(CE),normal-phase liquid chromatography,hydrophilic interaction liquid chromatography,ion-exchange chromatography,gas chromatography and supercritical fluid chromatography(SFC),have been applied in TCM analysis.Among these separation tools,LC is considered as the most useful technique for TCM analysis owing to its wide choice of stationary phases,the availability of different modes,its high capacity,and good reproducibility,etc.SFC is a chromatographic technique for the development and improvement of the solvation capacity of the mobile phase with supercritical fluid as the mobile phase(mainly carbon dioxide)in the 1980s.which showed great potential for the separation and isolation of active constituents in TCMs and natural products.This could be attributed to several interesting features of SFC,such as high separation efficiency,high mobile phase velocities and thus reduced analysis times,and eco-friendly characteristics.Nowadays,SFC coupled with different detectors has showed promising analytical applications to a wide range of TCM components,from nonpolar compounds like carotenoids,fatty acids,and terpenes,to more polar analytes like flavonoids,alkaloids,and carbohydrates.SFC has shown a significant achievement in the analysis of low polarity compounds and chiral drugs due to the advantage of SF.However,it's quite less reported for the analysis of achiral compounds especially for the TCM.Basing on the LSER model,the separation mechanism of solute and stationary phase and mobile phase in SFC was investigated.Through this model,it will be helpful for the reference and theoretical basis for the application in TCM analysis.Afterwards,a variety of polar compounds from TCM extracts were analyzed for the method development.After optimizing a series of separation parameters and conditions,the SFC methods were applied to the TCM for the quality control.Compared with LC,the technique made it possible to have a certain complementarity strategy.Some case studies were investigated for SFC's applications.Very few reports of these active ingredients(flavonoids,saponins,nucleoside,nucleobases,carbohydrate,amino acids)were analyzed by SFC.The established SFC method and the traditional LC method were compared in terms of speed or selectivity,which provided a new perspective for the existing separation method.Finally,in view of the existence of expensive and complicated preparation of commercial packed column,this paper has also creatively prepared a hydrophilic monolithic column,and combined the column with SFC for the first time.It showed a great selectivity for the nucleobases,nucleosides and benzoic acids.The on-line combination gave more ideas for polar compounds analysis.The specific research contents are as follows:Chapter?:TCMs are gaining increasing popularity throughout the world due to their long historical clinical practices.However,the quality control of TCM is a bottleneck in its application and development process,thus restricting its modernization and globalization process.Therefore,highly efficient analytical separation tools are essential for investigating the mysterious properties of TCMs and their quality control.SFC showed a great potential in TCM analysis for both nonpolar and polar components.In this paper,an overview of the experimental conditions(i.e.detection mode,stationary phase,mobile phase composition,pressure and temperature)used in SFC for achiral separations of TCM components is presented and recent applications to the analysis of different classes of compounds extracted from TCMs,such as lipids,terpene and terpenoids,phenolic compounds,flavonoids,alkaloids,saponins and carbohydrates,will be briefly described.Chapter?:There are few reports on the mechanism of separation and retention in SFC.The LSER model was reported to describe the retention behavior of solute molecules and stationary phases and mobile phases.The coefficients in the model are obtained by multiple linear regression analysis,which is used to evaluate the relative strength of the forces thus explaining the retention mechanism.In order to clarify the relationship between them,the stationary phase of 12 different bonding functional groups and 122 probe solute molecules were selected for LSER analysis.System constants were determined by multilinear regression.The results indicated that several types of silica columns,including Diol,RX-SIL,HILIC and X Amide were shown some similarity in their retention behavior.Also,some dissimilar stationary phases were observed such as NH2,RX-SIL,PFP and Phenyl.The results of linear solvation energy relationship in view of the retention process could be interpreted.Finally,some applications(nucleoside and nucleobases,benzoic acids,amines,phenols,ketones)were chosen for the investigation of these stationary phases.The results are expected to provide a data reference for the active ingredients in TCM extracts.Chapter?:Flavonoids from plants always show a wide range of biological activities.In the present study,a rapid and highly efficient SFC method was developed for the separation of 12 flavonoids.After careful optimization,the 12flavonoids were baseline separated on a ZORBAX RX-SIL column using gradient elution.A 0.1%phosphoric acid solution in MeOH was found to be the most suitable polar mobile phase component for the separation of flavonoids.From the viewpoint of retention and resolution,a backpressure of 200 bar and a temperature of 40~°C were shown to give the best results.Compared with a previously developed reverse phase liquid chromatography method,the SFC method could provide flavonoid separations that were about three times faster,while maintaining good peak shape and comparable peak efficiency.This SFC method was validated and applied to the analysis of five flavonoids(kaempferol,luteolin,quercetin,luteoloside,buddleoside)present in Chrysanthemum morifolium Ramat.from different cultivars(Chuju,Gongju,Hangju,Boju).The results indicated a good repeatability and sensitivity for the quantification of the five analytes with RSDs for overall precision lower than 3%.The limits of detection ranged from 0.73 to 2.34?g/mL,while the limits of quantification were between 2.19 and 5.86?g/mL.The method showed that SFC could be employed as a useful tool for the quality assessment of TCMs containing flavonoids as active components.Chapter?:Triterpenoid saponins(TSs)are the most important components of some TCMs and have exhibited valuable pharmacological properties.Due to its instability,high polarity and lack of UV absorption and other characteristics of its analytical instruments to the high demand,there are few reports with SFC technology to study so far.In this study,a rapid and efficient method was developed for the separation of nine kudinosides,six stauntosides and 11 ginsenosides using SFC-MS.The separation conditions for the selected TSs were carefully optimized after the initial screening of eight stationary phases.The best compromise for all compounds in terms of chromatographic performance and MS sensitivity was obtained when water(5-10%)and FA(0.05%)were added to the supercritical carbon dioxide/MeOH mobile phase.Beside the composition of the mobile phase,the nature of the make-up solvent for interfacing SFC with MS was also evaluated.Compared to reversed phase liquid chromatography,the SFC approach showed higher resolution and shorter running time.The developed SFC-MS methods were successfully applied to the separation and identification of TSs present in Ilex latifolia Thunb.,Panax quinquefolius L.and Panax ginseng C.A.Meyer.These results suggest that this SFC-MS approach could be employed as a useful tool for the quality assessment of natural products containing TSs as active components.Chapter?:Ginsenosides are often used as indicators of ginseng.The efficacy of TCM is the result of multi-component synergies.Nucleobases and nucleosides are also reported to be a class of ginseng with important physiological activities.However,these content in the ginseng content is quite low and the polarity difference is large.Few analytical methods have been developed so far to simultaneously analyze these three classes of compounds with different polarities present in ginseng extracts.In the present study,a simple and efficient analytical method was successfully developed for the simultaneous separation of four nucleobases,six nucleosides and seven ginsenosides in ginseng extracts using SFC-MS.The effect of various experimental factors on the separation performance,such as the column type,temperature and backpressure,the type of modifier and additive,and the concentration of make-up solvent were systematically investigated.Under the selected conditions,the developed method was successfully applied to the quality evaluation of 14 batches of ginseng extracts from different origins.The results obtained for the different batches indicate that this method could be employed for the quality assessment of ginseng extracts.Chapter?:Saccharides are one of the important molecules in the world,which are responsible for various roles in all living things.It's a challenge to develop a method due to its high polarity and lack of UV absorption.Compared with the LC method,the analysis of saccharides need to derivatize.In the present work,the combined use of the state-of-the-art MS together with SFC was proved to be a useful tool for the separation of eight saccharides(ribose,xylose,fructose,mannose,glucose,sucrose,lactose,mycose).Investigation and optimization of the influencing factors were resulted in a baseline separation such as the column type,additive,temperature,make-up solvent.It's found that a higher temperature(60°C)making anomeric peaks collapse into a single one and successfully suppress the anomer separation of saccharides.In addition,a rapid,highly selective and sensitive SFC-MS method was developed and validated for the separation with a representative mixture of saccharides(fructose,glucose and sucrose)in the different honey samples.Compared to LC-MS,the two approaches obtained diverse retentions and selectivities providing a good complementarity of these analytical strategies.Method sensitivity was found1.5-2-fold higher in SFC-MS mode than LC-MS mode.The latter demonstrated a lower selectivity for the separation of mannose and glucose.Finally,the SFC-MS method for conventional saccharide analysis demonstrated the potential of SFC-MS for sensitive and robust determination of saccharides in honey and may promote new perspectives in the research of other commercially available sample containing these saccharides.Chapter?:Tea is a complex mixture containing a wide range of biological activities.It is classified as Pu-erh tea,green tea,black tea and oolong tea according to the degree of tea fermentation.Traditional analytical methods of derivatization have the drawbacks of longer analysis time and derivatized reagents that are easily contaminated with columns and systems.Amino acids have also been reported as a class of active ingredients in tea.In this study,a novel method for SFC-MS has been developed to simultaneously determine the contents of 11 free amino acids in different types of teas(pu-erh tea,green tea,black tea and oolong tea).The separation conditions for the selected amino acids were carefully optimized such as the column type,temperature and backpressure,the type of additive.The best compromise for tested analytes in terms of chromatographic performance was obtained when water(5%)and trifluoroacetate acid(TFA)(0.4%)were added to the supercritical carbon dioxide/MeOH mobile phase.Finally,the developed SFC-MS method was successfully applied to the analysis of the 11 amino acids present in the teas and fully validated as well.The results indicated a good linearity(r?0.995),precision(RSD?2.99%),stability(RSD?2.88%)and accuracy(91.95%~107.99%).The limits of detection ranged from 1.42 to 14.69 ng/mL,respectively,while the limits of quantification were between 4.53 and 47.0 ng/mL.The content of the amino acids in six different tea samples were also determined and presented some difference basing on the fermentation processes.The contents of amino acids were the highest in green teas,followed by that black tea,and much lower pu-erh tea and oolong tea.The proposed SFC-MS method showed a great potential in further investigations to differentiate tea varieties basing on the content of amino acids.Chapter?:A poly(SPE-co-EDMA)monolith was prepared by in a 100 mm×2.0mm i.d.stainless steel column and was investigated for SFC.The porous structure of monolith was optimized by changing the conditions of polymerization.Afterwards,the chemical group of the monolithic column was confirmed by a Fourier transform infrared spectroscopy(FT-IR)method and the morphology of column structure was characterized by scanning electron microscopy(SEM).The good repeatability and stability of the column were obtained by evaluating the run-to-run,day-to-day,column-to-column and batch-to-batch with relative standard deviations(RSDs)for adenine,uracil and cytosine,which were less than 2.0%(n=10),7.0%(n=3),6.0%(n=3)and 2.0%(n=3),respectively.The results indicated that prepared monolithic columns showed good reproducibility and stability.Compared with HPLC,the SFC method provided better kinetic performance and selectivity.Finally,a series of low-molecular-weight organic compounds were utilized to evaluate the retention behaviors of the monolithic column.The results demonstrated that the prepared column exhibited a good separation performance and showed a great potential in SFC.The conclusive evidence filled the vacancy of the monolith with SFC.Chapter?:The conclusions of the thesis were summarized and the applications for these techniques of future perspectives were also pictured. |
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