改性松香键合二氧化硅高效液相色谱固定相的制备及其对三七总皂苷的分离

doi:10.3724/SP.J.1123.2021.07008

色谱 ›› 2022, Vol. 40 ›› Issue (3): 234-241.DOI: 10.3724/SP.J.1123.2021.07008

改性松香键合二氧化硅高效液相色谱固定相的制备及其对三七总皂苷的分离

谢文博¹, 夏璐¹, 李浩¹, 李文¹, 曹宇², 黄云¹, 雷福厚¹^,^*()

1.广西民族大学化学化工学院, 林产化学与工程国家民委重点实验室, 广西林产化学与工程重点实验室/协同创新中心, 广西南宁 530006
2.湖北民族大学化学与环境工程学院, 湖北恩施 445000

收稿日期:2021-07-19 出版日期:2022-03-08 发布日期:2022-03-04
通讯作者: 雷福厚
基金资助:
国家自然科学基金(32060325);广西林产化学与工程重点实验室开放基金资助项目(GXFC18-03)

Preparation of modified rosin bonded silica high performance liquid chromatographic stationary phase and separation of Panax notoginseng saponins

XIE Wenbo¹, XIA Lu¹, LI Hao¹, LI Wen¹, CAO Yu², HUANG Yun¹, LEI Fuhou¹^,^*()

1. Key Laboratory of Chemistry and Engineering of Forest Products, State Ethnic Affairs Commission, Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, Guangxi Collaborative Innovation Center for Chemistry and Engineering of Forest Products, College of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Nanning 530006, China
2. School of Chemical and Environmental Engineering, Hubei Minzu University, Enshi 445000, China

Received:2021-07-19 Online:2022-03-08 Published:2022-03-04
Contact: LEI Fuhou
Supported by:
National Natural Science Foundation of China(32060325);Open Fund of Guangxi Key Laboratory of Chemistry and Engineering of Forest Products(GXFC18-03)

摘要/Abstract

摘要：

三七中发挥药效的主要成分为三七皂苷R1、人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1和人参皂苷Rd,用于贫血、冠心病、高血压、脑卒中后遗症等疾病的治疗,但其化学成分多且难分离。将氢化松香丙烯酸羟乙酯(HRHA)通过巯基-烯点击化学反应键合到烷基化硅胶表面,制备出一种新型的改性松香键合二氧化硅高效液相色谱固定相(SiO₂@HRHA),用于三七总皂苷的分离。对色谱固定相进行一系列表征,表明SiO₂@HRHA固定相具有球形度好和表面多孔的特点,比表面积为308.55 m²/g,平均孔径达到6.78 nm。将制备的固定相湿法装柱,色谱柱性能评价结果表明,SiO₂@HRHA柱具备反相色谱行为、较好的流通性及重复性。从色谱柱的综合评价Tanaka测试可知,SiO₂@HRHA柱具有较好的立体选择性及氢键容量。将其用于三七总皂苷的分离,SiO₂@HRHA柱在一定程度上优于C18柱,而SiO₂@HRHA柱的分离效果又优于实验室同期合成的色谱柱,对5种皂苷(R1、Rg1、Re、Rb1和Rd)的分离度依次为3.33、3.54、20.17和9.72,表明SiO₂@HRHA高效液相色谱柱对三七总皂苷具有优异的分离效果,为从实际样品中分离纯化三七总皂苷提供了新思路。

关键词: 松香键合固定相, 高效液相色谱, 三七总皂苷

Abstract:

The sanqi is the dried root of Panax notoginseng (Burk.) F. H. Chen. The main components responsible for the drug actions of sanqi are notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd, which account for about 80% of the saponin content in sanqi. It is widely used in the treatment of anemia, coronary heart disease, hypertension, stroke sequelae, and other diseases. However, sanqi has many chemical components with complex and similar structures, which are difficult to separate. In this study, alkylated silica gel bonded with hydrogenated rosin hydroxyethyl acrylate (HRHA) was prepared via mercapto-ene click chemistry. A new type of modified rosin-bonded silica stationary phase (SiO₂@HRHA) for high performance liquid chromatography was prepared for the separation of five saponins (R1, Rg1, Re, Rb1, and Rd). It was characterized by thermogravimetric analysis, Fourier-transform infrared spectroscopy, specific surface area and microporous physical adsorption and elemental analysis. The results showed that SiO₂@HRHA had a regular spherical shape with porous surfaces, along with a specific surface area of 308.55 m²/g and an average pore diameter of 6.78 nm. Performance evaluation of the column revealed that the SiO₂@HRHA column showed typical reversed-phase chromatographic behavior with better flowability and reproducibility. Results of the Tanaka test showed that SiO₂@HRHA column had good stereoselectivity and hydrogen bond capacity. Compared to other stationary phases, e. g. silica modified with acrylopimaric acid (16-hydroxyethyl-34-hydroxyethyl acrylate) ester (AAE) and dihydroterpineol (DTP), which were prepared in our laboratory at the same time, the SiO₂@HRHA column demonstrated better resolution (R_s) for the separation of the five saponins under optimal chromatographic conditions. The R_s values for R1, Rg1, Re, Rb1, and Rd were 3.33, 3.54, 20.17 and 9.72, respectively on the SiO₂@HRHA column. R_s between Rg1 and Re was also better than that obtained on a C18 column. Panax notoginseng saponins were separated on the SiO₂@HRHA column using acetonitrile and water as the mobile phases at the flow rate of 1.0 mL/min at 25 ℃. The optimal UV detection wavelength was 203 nm. It was found that the five saponins could be separated better using the SiO₂@HRHA column than the SiO₂@AAE and SiO₂@DTP columns. Because the ternary phenanthrene skeleton of the rosin group in SiO₂@HRHA had structural similarity and good stereoselectivity to the polycyclic compounds (Panax notoginseng saponins). In addition, according to the hydrophobicity evaluation, the SiO₂@HRHA column showed the best hydrophobicity among the three columns, which may be conducive to the separation of the five saponins. Thus, this study can provide a new avenue for the separation and purification of Panax notoginseng saponins from actual samples.

Key words: rosin bonded stationary phase, high performance liquid chromatography (HPLC), Panax notoginseng saponins

中图分类号:

O658

谢文博, 夏璐, 李浩, 李文, 曹宇, 黄云, 雷福厚. 改性松香键合二氧化硅高效液相色谱固定相的制备及其对三七总皂苷的分离[J]. 色谱, 2022, 40(3): 234-241.

XIE Wenbo, XIA Lu, LI Hao, LI Wen, CAO Yu, HUANG Yun, LEI Fuhou. Preparation of modified rosin bonded silica high performance liquid chromatographic stationary phase and separation of Panax notoginseng saponins[J]. Chinese Journal of Chromatography, 2022, 40(3): 234-241.

图/表 8

图1 SiO2@HRHA的合成路线

Fig. 1 Synthesis route of SiO2@HRHA HRHA: hydrogenated rosin hydroxyethyl acrylate; MAPS: (3-mercaptopropyl) trimethoxysilane; AIBN: azobisisobutyronitrile.

图2 SiO2、SiO2-MAPS、SiO2@HRHA、SiO2@AAE和SiO2@DTP的(a)热重分析、(b)红外光谱及(c)氮气吸附-脱附等温线图

Fig. 2 (a) Thermogravimetric analysis, (b) FT-IR spectra and (c) nitrogen adsorption-desorption isotherms graphs of SiO2, SiO2-MAPS, SiO2@HRHA, SiO2@AAE and SiO2@DTP AAE: acrylopimaric acid (16-hydroxyethyl-34-hydroxyethyl acrylate) ester; DTP: dihydroterpineol.

表1 SiO2、SiO2-MAPS、SiO2@HRHA、SiO2@AAE和SiO2@DTP的比表面积、孔容和孔径

Table 1 Specific surface areas, pore volumes and pore sizes of SiO2, SiO2-MAPS, SiO2@HRHA, SiO2@AAE and SiO2@DTP

Sample	Specific surface area/(m²/g)	Pore volume/ (cm³/g)	Average pore size/nm
SiO₂	370.88	0.85	6.93
SiO₂-MAPS	349.78	0.71	6.87
SiO₂@HRHA	308.55	0.65	6.78
SiO₂@AAE	290.05	0.64	6.24
SiO₂@DTP	319.65	0.67	6.27

表2 SiO2、SiO2-MAPS、SiO2@HRHA、SiO2@AAE和SiO2@DTP的元素分析结果

Table 2 Elemental analysis results of SiO2, SiO2-MAPS, SiO2@HRHA, SiO2@AAE and SiO2@DTP

Sample	C/%	H/%	S/%
SiO₂	1.77	0.48	0
SiO₂-MAPS	3.61	1.15	2.13
SiO₂@HRHA	6.08	2.30	1.22
SiO₂@AAE	5.12	2.13	0.94
SiO₂@DTP	5.43	1.93	1.26

图3 SiO2@HRHA、SiO2@AAE和SiO2@DTP柱的流速与柱压关系曲线

Fig. 3 Relationships between the flow rate and the column pressure of SiO2@HRHA, SiO2@AAE and SiO2@DTP columns

图4 色谱柱反相色谱性能及疏水性能评价

Fig. 4 Evaluation of reversed-phase chromatography performance and hydrophobic performance of columns

图5 SiO2@HRHA、SiO2@AAE及SiO2@DTP柱的Tanaka测试参数图

Fig. 5 Tanaka test parameter diagrams of SiO2@HRHA, SiO2@AAE and SiO2@DTP columns. kPB: hydrophobicity;$α_{CH_{2}}$: hydrophobic selectivity; αT/O: stereoselectivity; αC/P: hydrogen bond capacity; αB/P 7.60: ion exchange capacity of pH>7; αB/P 2.70: ion exchange capacity of pH<3.

图6 SiO2@HRHA、SiO2@AAE、SiO2@DTP和C18柱分离三七总皂苷

Fig. 6 Separation of Panax notoginseng saponins by SiO2@HRHA, SiO2@AAE, SiO2@DTP and C18 columns For SiO2@HRHA, mobile phase: (A) acetonitrile-(B) water; gradient elution program: 0-20 min, 18%A; 20-45 min, 18%A-46%A; 45-55 min, 46%A-55%A; 55-60 min, 55%A; flow rate: 1.0 mL/min; temperature: 25 ℃; wavelength: 203 nm. For SiO2@AAE column, mobile phase: (A) acetonitrile-(B) water; gradient elution program: 0-20.00 min, 15%A; 20.00-27.23 min, 15%A-30%A; 27.23-60.00 min, 30%A; flow rate: 0.5 mL/min; temperature: 30 ℃; wavelength: 203 nm. For SiO2@DTP, mobile phase: (A) acetonitrile-(B) water (15:85, v/v), isocratic elution; flow rate: 1.5 mL/min; temperature: 25 ℃; wavelength: 203 nm. For C18, mobile phase: (A) acetonitrile-(B) water; gradient elution program: 0-20 min, 20%A; 20-45 min, 20%A-46%A; 45-55 min, 46%A-55%A; 55-60 min, 55% A; flow rate: 1.5 mL/min; temperature: 25 ℃; wavelength: 203 nm.

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改性松香键合二氧化硅高效液相色谱固定相的制备及其对三七总皂苷的分离

Preparation of modified rosin bonded silica high performance liquid chromatographic stationary phase and separation of Panax notoginseng saponins

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