Preparation Of Modified Rosin Ester Bonded Silica Chromategraphic Column And Its Separation Mechanism Of Panax Notoginseng Saponions

Sanqi is a kind of Chinese medicinal material used for both medicine and food,the main components responsible for the drug actions of sanqi are notoginseng R1,ginsenoside Rg1,ginsenoside Re,ginsenoside Rb1,and ginsenoside Rd,which account for more than 80%of the saponin content in sanqi.It is widely used in the treatment of anemia,hyperlipidemia,coronary heart disease,angina pectoris,hypertension,stroke sequelae,and other diseases.The《Chinese Pharmacopoeia》clearly stipulates the content of Panax notoginseng saponins（R1,Rg1,Re,Rb1,Rd）and the content of single saponins in clinical medication,and the separation and purification of Panax notoginseng saponins is one of the research hotspots.Therefore,the study of the separation of Panax notoginseng saponins is of great significance.As a natural renewable resource,rosin is mainly composed of resinic acid,which is highly hydrophobic,and has a unique ternary phenylene ring skeleton to provide better rigidity.In this paper,two active reaction centers in the structure of the double bond and carboxyl group were modified,and several kinds of modified rosin esters were bonded to the surface of alkylated silica via"sulfhydryl-ene"click chemical reaction.Modified rosin esters bonded silica chromatographic columns were prepared for the separation of Panax notoginseng saponins and its separation mechanism was studied.The main research contents are as follows:1.The fumaropimaric acid（ethylene glycol）tris ester（FATEGE）was bonded to the surface of alkylated silica to obtain FATEGE@SiO₂stationary phase.It was characterized by Fourier transform infrared spectrometer,thermogravimetric analyzer,specific surface area and microporous physical adsorption analyzer and element analyzer,etc.The results showed that FATEGE@SiO₂the stationary phase had regular spherical shape with porous surface,the specific surface area was 281.94m²/g,the average pore diameter was 6.19 nm,and the particle size was concentrated around 4.61μm.The prepared stationary phase was packed into the column by wet method.The performance evaluation for the column showed that the FATEGE@SiO₂column had typical reversed-phase chromatographic behavior,hydrophobicity,hydrophobic selectivity and reproducibility.Tanaka test showed that FATEGE@SiO₂column had good stereoselectivity and hydrogen bond capacity.FATEGE@SiO₂was used for separation of Panax notoginseng saponins,the resolution（R_s）among the five saponins（R1,Rg1,Re,Rb1,Rd）were 2.53,2.47,15.51 and 7.94,respectively.The FATEGE@SiO₂column could completely separate Panax notoginseng saponins.Although the R_sof ginsenoside Rg1 and Re was better than that of C18 column,the R_sof Rg1 and Rle was only 2.47.2.The acrylopimaric acid（16-hydroxyethyl-34-hydroxyethyl acrylate）ester（AAE）was bonded to the surface of alkylated silica to obtain AAE@SiO₂stationary phase.It was characterized by Fourier transform infrared spectrometer,thermogravimetric analyzer,specific surface area and microporous physical adsorption analyzer and element analyzer,etc.The results showed that AAE@SiO₂had regular spherical shape with porous surface,the specific surface area was 290.05m²/g,the average pore diameter was 6.24 nm,and the particle size was concentrated around 4.53μm.The prepared stationary phase was packed into the column by wet method.The performance evaluation for the column showed that the AAE@SiO₂column had typical reversed-phase chromatographic behavior,hydrophobicity,hydrophobic selectivity and reproducibility.Tanaka test showed that AAE@SiO₂column had good stereoselectivity and hydrogen bond capacity.AAE@SiO₂was used for separation of Panax notoginseng saponins,the resolution（R_s）among the five saponins（R1,Rg1,Re,Rb1,Rd）were 2.40,3.10,12.52 and 8.90,respectively.Although the R_sof ginsenoside Rg1 and Re was much higher than that of C18column,the R_sof notoginsenoside R1 and ginsenoside Rg1 was only 2.40.3.Theβ-Pinene（β-P）and longifolene（LGF）were respectively bonded to the surface of alkylated silica to obtainβ-P@SiO₂and LGF@SiO₂stationary phase.They were characterized by Fourier transform infrared spectrometer,thermogravimetric analyzer,specific surface area and microporous physical adsorption analyzer and element analyzer,etc.The results showed that the stationary phases ofβ-P@SiO₂and LGF@SiO₂had regular spherical shape with porous surface,the specific surface area ofβ-P@SiO₂was 333.04 m²/g,the average pore diameter was 6.23 nm,and the particle size was concentrated around 4.45μm,the specific surface area of LGF@SiO₂was 330.56 m²/g,the average pore diameter was 6.18 nm,and the particle size was concentrated around 4.41μm.The prepared stationary phase was packed into the column by wet method.The performance evaluation for the column showed that theβ-P@SiO₂and LGF@SiO₂columns had typical reversed-phase chromatographic behavior,hydrophobicity,hydrophobic selectivity and reproducibility.Tanaka test showed thatβ-P@SiO₂and LGF@SiO₂columns had good stereoselectivity and hydrogen bond capacity.β-P@SiO₂and LGF@SiO₂columns were used for separation of Panax notoginseng saponins,the resolution（R_s）among the five saponins（R1,Rg1,Re,Rb1,Rd）onβ-P@SiO₂column were 2.69,2.22,34.38 and 8.60,respectively.The resolution（R_s）among the five saponins on LGF@SiO₂column were 2.91,2.35,26.88 and 8.95,respectively.However,compared with FATEGE@SiO₂and AAE@SiO₂columns,the R_sof ginsenoside Rg1and Re decreased,which were 2.22 and 2.35,respectively.4.The hydrogenated rosin hydroxyethyl acrylate（HRHA）was bonded to the surface of alkylated silica to obtain HRHA@SiO₂stationary phase.It was characterized by Fourier transform infrared spectrometer,thermogravimetric analyzer,specific surface area and microporous physical adsorption analyzer and element analyzer,etc.The results showed that HRHA@SiO₂the stationary phase had regular spherical shape with porous surface,the specific surface area was 308.55m²/g,the average pore diameter was 6.78 nm,and the particle size was concentrated around 4.55μm.The prepared stationary phase was packed into the column by wet method.The performance evaluation for the column showed that the HRHA@SiO₂column had typical reversed-phase chromatographic behavior,hydrophobicity,hydrophobic selectivity.HRHA@SiO₂was used for separation of Panax notoginseng saponins,the resolution（R_s）among the five saponins（R1,Rg1,Re,Rb1,Rd）were 3.33,3.54,20.17 and 9.72,respectively.The separation effect of HRHA@SiO₂was better than that of C18 column.Compared the work in the previous chapters,the HRHA@SiO₂column had the best separation effect on Panax notoginseng saponins,which indicated that the modification of rosin to improve its hydrophobicity on the basis of retaining the ternary phenanthrene ring skeleton was beneficial to the separation of Panax notoginseng saponins.5.N-butanol and lauryl alcohol were introduced to propylene pimaric acid to obtain propylene pimaric acid（16-hydroxyethyl acrylate-34-n-butyl）ester（BRB）and propylene pimaric acid（16-hydroxyethyl acrylate-34-dodecyl）ester（BRLA）,they were respectively bonded to the surface of alkylated silica to obtain BRB@SiO₂and BRLA@SiO₂stationary phase,which the objective was to further explore the effect of increasing the hydrophobicity of stationary phase on the separation of Panax notoginseng saponins.They were characterized by Fourier transform infrared spectrometer,thermogravimetric analyzer,specific surface area and microporous physical adsorption analyzer and water contact angle meter,etc.The results showed that the stationary phases of BRB@SiO₂and BRLA@SiO₂had regular spherical shape with porous surface,the water contact angle of BRLA@SiO₂was greater than that of BRB@SiO₂,the specific surface area of BRB@SiO₂was 280.37 m²/g,the average pore diameter was 5.85 nm,and the particle size was concentrated around4.66μm,the specific surface area of BRLA@SiO₂was 276.69 m²/g,the average pore diameter was 5.81 nm,and the particle size was concentrated around 4.87μm.The prepared stationary phase was packed into the column by wet method.The performance evaluation for the column showed that the BRB@SiO₂and BRLA@SiO₂columns had typical reversed-phase chromatographic behavior,hydrophobicity,hydrophobic selectivity and reproducibility.Tanaka test showed that BRB@SiO₂and BRLA@SiO₂columns had good stereoselectivity and hydrogen bond capacity.BRB@SiO₂and BRLA@SiO₂columns were used for separation of Panax notoginseng saponins.The resolution（R_s）among the five saponins（R1,Rg1,Re,Rb1,Rd）on BRB@SiO₂column were 3.51,3.26,17.79 and 10.44,respectively.The resolution（R_s）among the five saponins on BRLA@SiO₂column were 3.66,2.67,25.07 and 10.57,respectively.The separation effect of BRB@SiO₂and BRLA@SiO₂columns were better than that of C18 column.Moreover,with the increase in the hydrophobicity of the stationary phase,the overall R_sof Panax notoginseng saponins increased.6.Hydrogenated cardanol methacrylate（HCM）with C15 side chain and the mixture（HRHA-HCM）of hydrogenated rosin hydroxyethyl acrylate and HCM were respectively bonded to the surface of alkylated silica to obtain HCM@SiO₂and HRHA-HCM@SiO₂stationary phase.They were characterized by Fourier transform infrared spectrometer,thermogravimetric analyzer,specific surface area and microporous physical adsorption analyzer and element analyzer,etc.The results showed that the stationary phases of HCM@SiO₂and HRHA-HCM@SiO₂had regular spherical shape with porous surface.The specific surface area of HCM@SiO₂was 312.56 m²/g,the average pore diameter was 6.18 nm,and the particle size was concentrated around 4.44μm,the specific surface area of HRHA-HCM@SiO₂was 317.32 m²/g,the average pore diameter was 6.62 nm,and the particle size was concentrated around 4.55μm.The prepared stationary phase was packed into the column by wet method.The performance evaluation for the column showed that the HCM@SiO₂and HRHA-HCM@SiO₂columns had typical reversed-phase chromatographic behavior,hydrophobicity,hydrophobic selectivity and reproducibility.Tanaka test showed that HCM@SiO₂and HRHA-HCM@SiO₂columns had good stereoselectivity and hydrogen bond capacity.HCM@SiO₂and HRHA-HCM@SiO₂columns were used for separation of Panax notoginseng saponins.The resolution（R_s）among the five saponins（R1,Rg1,Re,Rb1,Rd）on HCM@SiO₂column were 4.02,2.81,29.03 and 9.91,respectively.The resolution（R_s）among the five saponins on HRHA-HCM@SiO₂column were 3.17,4.12,22.16and 9.39,respectively.The separation effect of HCM@SiO₂and HRHA-HCM@SiO₂columns were better than that of C18 column.In conclusion,the R_sof Panax notoginseng saponins did not always increase with the increase of the hydrophobicity of the stationary phase.The separation effect of the column prepared by the mixed monomer of HCM containing C15 side chain and HRHA containing the ternary phenicyl ring skeleton of rosin was the best,and there was a synergistic effect between the two monomers.7.In summary,nine kinds of modified rosin ester bonded silica columns were prepared and used for the separation of Panax notoginseng saponins.The separation mechanism of the columns was studied by hydrophobicity,hydrogen bond interaction,Knox equation and quantum chemical calculation,the results showed that the nine chromatographic columns all had a reverse phase chromatographic separation mechanism,the order of hydrophobicity from large to small was BRLA@SiO₂、HCM@SiO₂、BRB@SiO₂、HRHA-HCM@SiO₂、HRHA@SiO₂、LGF@SiO₂、β-P@SiO₂、FATEGE@SiO₂and AAE@SiO₂column,the improvement of the hydrophobicity of the column was beneficial to the separation of Panax notoginseng saponins,but it was not the only impacting factor.The rosin ternary phenanthrene ring skeleton was beneficial to the separation of Panax notoginseng saponins,especially for the separation of ginsenosides Rg1 and Re.The strong hydrogen bond interaction was beneficial to the separation of polar compounds.The Knox equation had a good fit on the column（R²＞0.9）,indicating that the mass transfer resistance played a major role in the separation of Panax notoginseng saponins.The quantum chemical calculation of HRHA-HCM@SiO₂column with the best separation effect of Panax notoginseng saponins showed that the force between the saponins and monomers was consistent with the retention time of the saponins on the liquid chromatography,since the longer the retention time,the greater the force.There were hydrophobicity and hydrogen bond interaction exist in the isosurface between HRHA-HCM and saponin molecules,which was consistent with the experimental results.