| Paclitaxel analogs include paclitaxel（PTX）,baccatin III（BCT）,10-deacetylbaccatin III（10-DAB）,10-deacetylpaclitaxel（10-DAT）,and cephalomannine（CEP）etc.,mainly from the bark and branches of Taxus chinensis,in which paclitaxel is an excellent diterpenoid anticancer drug.BCT and 10-DAB can also be converted to anticancer drugs by semi-synthetic method.Therefore,the separation of paclitaxel analogs from yew is of great significance and has always been a scientific problem.Rosin is a diterpenoid resin acid featuring double bonds,a carboxyl group,and a phenanthrene-like skeleton.Rosin derivatives can be obtained through the structural modification of the double bonds and carboxyl group of rosin.Based on the above analysis,modified rosin was bonded to the surface of alkylated silica,and a series of modified rosin-bonded silica stationary phases were prepared to separate paclitaxel analogs（BCT,10-DAB,10-DAT,CEP,and PTX）and their chromatographic properties,separation effects and separation mechanisms were studied in detail.The main contents are as follows:1.A core-shell rosin-based polymer bonded silica（RP@Si O2）stationary phase was prepared to separate PTX analogs via the radical polymerization of fumaropimaric acid tri（β-acryloxyl ethyl）ester onto alkylated silica.The proposed RP@Si O2 stationary phase was characterized by scanning electron microscopy,transmission electron microscope,laser diffraction granulometry,thermogravimetric analysis,elemental analysis,Fourier-transform infrared spectroscopy,and nitrogen gas adsorption.The results showed that the core-shell RP@Si O2 stationary phase with a particle size of 5.0μm,a specific surface area of 321.85 m~2/g,a pore volume of 0.71 cm~3/g,and an average pore size of 6.52 nm,and the thermal stability is excellent.A RP@Si O2 column was prepared by wet packing,and its structural properties and background pressure were tested.The RP@Si O2 column with a total porosity of 0.86,an impedance factor 884.74,and a column permeability of 0.28*10-9 cm~2,the column is well filled,and the column bed is stable.In addition,its chromatographic performance was evaluated.The results showed that the RP@Si O2column has good chromatographic stability and reversed-phase chromatographic behavior.Compared with the C18 column,the steric selectivity of the RP@Si O2column is higher than that of the C18 column due to the phenanthrene-like skeleton of the bonded monomer.A RP@Si O2 column was used to separate paclitaxel analogs,achieving resolutions exceeding 6.6 for consecutively eluted species.Furthermore,a RP@Si O2 column was employed to separate and purify paclitaxel from crude yew-bark extract,increasing paclitaxel purity from 6%to 81%.2.A maleic rosin（β-acryloyloxyethyl）ester（MRE）-bonded silica（MRE@Si O2）stationary phase was prepared by"thiol-ene"click chemistry.In MRE@Si O2,MRE molecules,which are functional ligands,are bonded to the surface of a silica gel matrix using a coupling agent,（3-mercaptopropyl）trimethoxysilane.The proposed MRE@Si O2 stationary phase was characterized by Fourier-transform infrared spectroscopy,elemental analysis,thermogravimetric analysis,scanning electron microscopy,laser diffraction granulometry,and nitrogen gas adsorption.The results showed that the MRE@Si O2 stationary phase with a particle size of 5.0μm,a specific surface area of 314.13 m~2/g,a pore volume of 0.67 cm~3/g,an average pore size of 6.39 nm,and good thermal stability.An MRE@Si O2 column was prepared by wet packing,and its structural properties and background pressure were tested.The MRE@Si O2 column with a total porosity of 0.83,an impedance factor 877.79,and a column permeability of 0.28*10-9 cm~2,the column is well filled,and the column bed is stable.Moreover,its chromatographic performance was evaluated.The results showed that the MRE@Si O2 column has good reproducibility and reversed-phase chromatographic behavior.Compared with the C18 column,because its bonding monomer has two carboxyl groups and a phenanthrene-like ring skeleton,its hydrophobicity is weaker,but its stereoselectivity is better.An MRE@Si O2 column was used to separate PTX analogs,achieving resolutions exceeding 4.9 for consecutively eluted species.In addition,an MRE@Si O2column was employed to separate and purify PTX from crude yew-bark extract,increasing PTX purity from 6%to 78%.3.An acrylopimaric acid（β-acryloyloxyethyl）ester-bonded silica（ARE@Si O2）stationary phase was prepared by"thiol-ene"click chemistry.The ARE@Si O2 stationary phase was characterized by different analytical techniques.The ARE@Si O2 stationary phase with a particle size of 5.0μm,a specific surface area of 262.77 m~2/g,a pore volume of 0.57 cm~3/g,an average pore size of 6.39 nm,and good thermal stability.An ARE@Si O2 column was prepared by wet packing,and its structural properties and background pressure were tested.The ARE@Si O2 column with a total porosity of 0.74,an impedance factor 827.32,and a column permeability of 0.30*10-9 cm~2,the column is well filled,and the column bed is stable.Besides,its chromatographic performance was evaluated.The results showed that the ARE@Si O2 column has good reproducibility and reversed-phase chromatographic behavior.Compared with the MRE@Si O2 column,it has better hydrophobicity and stereoselectivity,because its bonding monomer contains a carboxyl group and a phenanthrene-like skeleton.An ARE@Si O2column was used to separate PTX analogs,achieving resolutions exceeding5.6 for consecutively eluted species.Moreover,an ARE@Si O2 column was employed to separate and purify PTX from crude yew-bark extract,increasing PTX purity from 6%to 78%.4.A new stationary phase comprising hydrogenated rosin（β-acryloxyl ethyl）ester（HRE）–bonded silica（HRE@Si O2）was prepared by"thiol-ene"click chemistry.The proposed HRE@Si O2 stationary phase was characterized by different analytical techniques.The HRE@Si O2 stationary phase with a particle size of 5.0μm,a specific surface area of 228.56 m~2/g,a pore volume of 0.57 cm~3/g,an average pore size of 7.16 nm,and good thermal stability.An HRE@Si O2 column was prepared by wet packing,and its structural properties and background pressure were tested.The HRE@Si O2 column with a total porosity of 0.81,an impedance factor 945.37,and a column permeability of0.26*10-9 cm~2,the column is well filled,and the column bed is stable.In addition,its chromatographic performance was evaluated.The HRE@Si O2column exhibited satisfactory performance reproducibility and typical reversed-phase chromatographic behavior.Compared with RP@Si O2 column,MRE@Si O2 column and ARE@Si O2 column,the hydrophobicity of HRE@Si O2 column is better because the bonded monomer HRE does not contain carboxyl group.HRE@Si O2 column has higher shape selectivity than that of C18 column due to the phenanthrene-like skeleton of the bonded monomer.An HRE@Si O2 column was used to separate PTX and its analogs,achieving resolutions exceeding 7.4 for consecutively eluted species.Furthermore,an HRE@Si O2 column was employed to separate and purify PTX from crude yew-bark extract,increasing PTX purity from 6%to 82%.The findings of this study provide insights for developing rosin-based stationary phases for the separation of natural products.5.An HRE-CM@Si O2 stationary phase was prepared by click chemistry with hydrogenated rosin（β-acryloyloxyethyl）ester（HRE）and cardanol methacrylate（CM）as mixed functional ligands.As a comparison,a cardanol methacrylate-bonded silica（CM@Si O2）stationary phase was prepared by the same method.The proposed HRE-CM@Si O2 and CM@Si O2stationary phases were characterized by different analytical techniques.The HRE-CM@Si O2 stationary phase with a particle size of 5.0μm,a specific surface area of 270.75 m~2/g,a pore volume of 0.59 cm~3/g,and an average pore size of6.42 nm;The CM@Si O2 stationary phase with a particle size of 5.0μm,a specific surface area of 227.11 m~2/g,a pore volume of 0.54 cm~3/g,and an average pore size of 6.85 nm.The structural characteristics,background pressures of HRE-CM@Si O2 and CM@Si O2 were tested.The HRE-CM@Si O2 column with a total porosity of 0.90,an impedance factor 935.68,and a column permeability of 0.27*10-9 cm~2;the CM@Si O2 column with a total porosity of 0.82,an impedance factor 835.16,and a column permeability of 0.30*10-9 cm~2;the columns are well filled,and the column beds are stable.In addition,their chromatographic performance was evaluated.The HRE-CM@Si O2column and CM@Si O2column showed excellent reproducibility and typical reversed-phase chromatographic behaviors.The hydrophobicity of HRE-CM@Si O2 column was weaker than that of CM@Si O2 column,and its shape selectivity was weaker than that of HRE@Si O2 column.An HRE-CM@Si O2 column was used to separate PTX and its analogs,achieving resolutions exceeding 5.0 for consecutively eluted species,but the leading peak appeared in the PTX chromatographic peak.Furthermore,an HRE-CM@Si O2 column was employed to separate and purify PTX from crude yew-bark extract,increasing PTX purity from 6%to 64%.A CM@Si O2 column was used to separate PTX and its analogs,and the resolution between adjacent components exceeds 4.3.Moreover,the CM@Si O2 column was applied to the separate PTX from crude yew-bark extract,and the PTX purity in the crude extract was increased from 6%to 80%.The results showed that the purification effect of HRE-CM@Si O2 column on the PTX crude extract was lower than that of HRE@Si O2 column and CM@Si O2 column.6.Stoichiometric displacement theory for retention（SDT-R）,van’t Hoff plots,and van Deemter equation were used to analyze the separation mechanism and properties of PTX analogs on the proposed six chromatographic columns.The results indicated that SDT-R can be used to quantitatively assess RPLC mode interactions during the separation of PTX analogs on the proposed six chromatographic columns.The strong hydrophobic interaction between paclitaxel analogs and the modified rosin-bonded silica column,and good match between the polycyclic structure of the paclitaxel analogs and the phenanthrene-like skeleton of the modified rosin are more conducive to the retention of paclitaxel analogs on the chromatographic column.Thermodynamics analysis showed that the interaction between PTX analogs and the columns was related to the enthalpy contribution,and the separation of PTX analogs on the columns was an enthalpy-driven exothermic process.Chromatographic kinetics indicated that mass transfer resistance played a major role in chromatographic peak shape expansion during the separation of PTX analogs. |
