腰果酚键合硅胶固定相的制备及其色谱性能

doi:10.3724/SP.J.1123.2021.12023

色谱 ›› 2022, Vol. 40 ›› Issue (6): 547-555.DOI: 10.3724/SP.J.1123.2021.12023

腰果酚键合硅胶固定相的制备及其色谱性能

曾磊¹, 姜利娟¹, 姚兴东¹, 王婷¹, 史伯安¹^,², 雷福厚¹^,^*()

1.广西民族大学化学化工学院, 广西林产化学与工程重点实验室, 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:2021-12-22 出版日期:2022-06-08 发布日期:2022-05-26
通讯作者: 雷福厚
基金资助:
国家自然科学基金项目(32060325);广西自然科学基金项目(2019GXNSFBA245084);广西林产化学与工程重点实验室开放项目(GXFK2004)

Preparation and chromatographic performance of cardanol-bonded silica stationary phase

ZENG Lei¹, JIANG Lijuan¹, YAO Xingdong¹, WANG Ting¹, SHI Bo’an¹^,², LEI Fuhou¹^,^*()

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

Received:2021-12-22 Online:2022-06-08 Published:2022-05-26
Contact: LEI Fuhou
Supported by:
National Natural Science Foundation of China(32060325);Natural Science Foundation of Guangxi Province, China(2019GXNSFBA245084);Open Fund of Guangxi Key Laboratory of Chemistry and Engineering of Forest Products(GXFK2004)

摘要/Abstract

摘要：

天然产物作为一种绿色低毒、来源广泛、功能位点丰富的单体,已被广泛应用于色谱固定相的研制与开发。该文以天然可再生资源腰果酚为配体,通过一步法开环反应将其接枝到由γ-缩水甘油醚氧丙基三甲氧基硅烷(KH-560)修饰的硅胶上,制备得到腰果酚键合硅胶固定相。利用傅里叶红外光谱、元素分析、热失重分析和N₂吸附脱附实验对固定相进行表征,结果表明成功制备了腰果酚键合硅胶色谱固定相。采用Tanaka实验试剂、烷基苯、多环芳香烃、苯酚类化合物和芳香族位置异构体为探针评价其分离性能和保留机制,并与C₁₈柱进行对比。研究发现,腰果酚键合固定相除疏水作用外,还具有π-π和氢键作用。基于上述保留作用,腰果酚键合硅胶固定相对测试探针表现出良好的分离性能。重复进样10次,各探针保留时间的RSD为0.052%~0.079%,峰面积的RSD为0.104%~0.847%,峰高的RSD为0.081%~0.272%,表明该色谱柱具有良好的重复性和稳定性。此外,腰果酚键合硅胶色谱柱对中药喜树果和吴茱萸果的粗提物具有良好的分离性能,验证了其在实际样品分析中的巨大潜力。将天然产物腰果酚用于色谱固定相的制备,为分离纯化喜树碱和吴茱萸提供了新的方法,同时拓展了腰果酚在色谱分离材料方面的应用。

关键词: 腰果酚, 键合固定相, 色谱性能, 中药分离

Abstract:

As green, less toxic, widely available, and site-rich functional ligands, natural products are widely used for the development of chromatographic stationary phases. In this work, a novel stationary phase, cardanol-bonded on silica (CBS) was prepared using γ-glycidoxypropyltrimethoxysilane (KH-560) as the coupling reagent and cardanol as the functional ligand. The synthesized stationary phase was characterized by Fourier transform-infrared spectra (FT-IR), thermogravimetric analysis (TGA), elemental analysis (EA), and N₂ adsorption-desorption analysis. The results revealed that cardanol was successfully immobilized on the surface of spherical silica by the ring-opening reaction of the epoxy groups in phenolic hydroxyl. The retention mechanism and chromatographic performance of the CBS column were further evaluated and compared with those of a commercial C₁₈ column using different classes of analytes, e. g., Tanaka standard test mixtures, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), phenols, and aromatic positional isomers. The retention of alkylbenzenes under different chromatographic conditions revealed that the CBS column was a typical reversed-phase liquid chromatographic column, similar to the commercial C₁₈ column. From the results of the Tanaka test, it was concluded that CBS could provide various interactions for different solutes e. g., hydrogen bonding and π-π interactions, along with hydrophobic interactions. The synergistic effects resulting from the aromatic rings, the hydroxyl and alkyl linkers in the new stationary phase improved the separation selectivity via multiple retention mechanisms. Based on these interactions, different solute probes such as hydrophobic alkylbenzenes, PAHs, and phenols were successfully separated in the reversed-phase liquid chromatography (RPLC) mode. For example, the aromatic positional isomers o-terthenyl, m-terphenyl, and triphenylene were used to investigate the chromatographic performance of the CBS column. These PHAs were baseline separated with good peak shapes. The resolution of m-terphenyl and triphenylene was as high as 6.81, while the two isomers could not be separated on the C₁₈ column under the same chromatographic conditions. The repeatability and column stability of the CBS column was evaluated, and excellent repeatability and column stability were observed. The relative standard deviations (RSDs) of the retention time, peak area, and peak height for alkylbenzenes with 10 replicate injections were 0.052%-0.079%, 0.104%-0.847%, and 0.081%-0.272%, respectively. Traditional Chinese medicines have contributed notably to the Chinese civilization and human health. However, the complicated chemical compositions, unclear medicinal action mechanisms, and low purification efficiency for the traditional Chinese medicines have limited further development. Therefore it is necessary to establish an efficient, simple and feasible method for the separation and purification of herbal medicines. HPLC has been widely used in traditional Chinese medicines for the separation and detection of various components. In order to explore the CBS column for analysis of the traditional Chinese medicines, the ethanol extracts of fruits of Evodiae fructus and Camptotheca acuminata were used to test the separation performance of this column. The resolution of camptothecin from the preceding and following impurity peaks was 4.23 and 2.71. The resolution between evodiamine and rutaecarpin was 5.43, while the resolution from the adjacencies of impurity peaks was 2.20 and 1.69, respectively. The above mentioned results indicated that the CBS column shows good separation performance for the main active ingredients in the ethanolic extracts of these drugs, this validating its great potential for the analysis of real samples. Overall, the present study not only provides a new approach for the preparation of chromatographic stationary phases but also opens a new possibility for the separation and purification of camptothecin and evodiamine in real samples. This is an extension of the application of cardanol to chromatographic separation materials.

Key words: cardanol, bonded stationary phase, chromatographic properties, separation of traditional Chinese medicines

中图分类号:

O658

曾磊, 姜利娟, 姚兴东, 王婷, 史伯安, 雷福厚. 腰果酚键合硅胶固定相的制备及其色谱性能[J]. 色谱, 2022, 40(6): 547-555.

ZENG Lei, JIANG Lijuan, YAO Xingdong, WANG Ting, SHI Bo’an, LEI Fuhou. Preparation and chromatographic performance of cardanol-bonded silica stationary phase[J]. Chinese Journal of Chromatography, 2022, 40(6): 547-555.

图/表 15

图1 腰果酚键合硅胶色谱固定相的制备

Fig. 1 Synthesis of cardanol-bonded silica stationary phase

表1 硅胶、环氧硅胶和固定相的元素分析结果

Table 1 Elemental analysis results for BS, KBS, and CBS

Analyte	Elemental contents/%
Analyte	C	H
BS	0.735	1.121
KBS	8.465	1.564
CBS	15.130	2.181

表2 BET比表面积和孔径数据

Table 2 BET surface area, pore volume, and pore size data

Analyte	BET surface area/ (m²/g)	Pore volume/ (cm³/g)	Pore size/ nm
BS	354.82	0.847	7.65
KBS	293.27	0.632	6.30
CBS	212.16	0.458	6.28

图2 (a)硅胶、(b)环氧硅胶和(c)腰果酚键合硅胶固定相的红外光谱图

Fig. 2 FT-IR spectra of (a) BS, (b) KBS, and (c) CBS

图3 硅胶、环氧硅胶和腰果酚键合硅胶固定相的热失重曲线图

Fig. 3 Thermogravimetric analysis curves of BS, KBS, and CBS

图4 CBS柱上烷基苯保留因子的对数(log k)与甲醇体积分数的关系

Fig. 4 Relationship between log k of alkylbenzenes and methanol volume fraction on CBS column Mobile phase: methanol/water; flow rate: 1.0 mL/min.

图5 CBS柱和C18柱上烷基苯侧链长度与log k关系

Fig. 5 Relationship between log k and alkyl chain length of alkylbenzenes on CBS and C18 columns Mobile phase: methanol/water (80∶20, v/v); flow rate: 1.0 mL/min.

图6 CBS柱与C18柱的Tanaka参数雷达图

Fig. 6 Radar plots of the Tanaka parameters of CBS and C18 columns kPB: retention factor of n-pentylbenzene, kn-pentylbenzene, used to evaluate the carbon content or density of ligands in the stationary phases; α C H 2: kn-pentylbenzene, used to evaluate the hydrophobicity of the stationary phase; αT/O : ktriphenylene/ko-terphenyl, used to evaluate the shape selectivity of the stationary phase; αC/P: kcaffeine/kphenol, used to evaluate the hydrogen-bonding interaction of the stationary phase; αB/P: kbenzenamine/kphenol, used to evaluate the apparent silanol activity of the stationary phase; αPB/O: kn-pentylbenzene/ko-terphenyl, used to describes the aromatic selectivity of the stationary phase.

图7 CBS柱与C18柱的Tanaka色谱图

Fig. 7 Chromatograms of Tanaka standard test mixture on CBS and C18 columns Mobile phase: methanol/water (75∶25, v/v); flow rate: 1.0 mL/min. Tanaka standard test mixture: 1. uracil; 2. benzenamine; 3. phenol; 4. caffeine; 5. n-butylbenzene; 6. n-pentylbenzene; 7. o-terphenyl; 8. triphenylene.

图8 多环芳烃在CBS和C18柱的分离色谱图

Fig. 8 Chromatograms of PAHs on CBS and C18 columns Mobile phase: methanol/water (80∶20, v/v); flow rate: 1.0 mL/min. Peaks: 1. benzene; 2. naphthalene; 3. biphenyl; 4. fluorene; 5. anthracene; 6. pyrene; 7. benz(a)anthracene.

图9 溶质在CBS和C18柱上log k与log P之间的关系

Fig. 9 Relationships between log k and log P of solutes on CBS column and C18 columns Mobile phase and flow rate are the same as those in Fig. 8. Solutes: 1. benzene; 2. toluene; 3. ethylbenzene; 4. n-propylbenzene; 5. n-butylbenzene; 6. n-pentylbenzene; 7. naphthalene; 8. fluorine; 9. anthracene; 10. benz(a)anthracene.

图10 多环芳烃异构体在CBS柱和C18柱上的色谱图

Fig. 10 Chromatograms of aromatic positional isomers on CBS and C18 columns Mobile phase and flow rate are the same as those in Fig. 8.

图11 苯酚类化合物在CBS柱和C18柱上的色谱图

Fig. 11 Chromatograms of phenols on CBS and C18 columns Mobile phase: methanol/water (60∶40, v/v); flow rate: 0.8 mL/min. Peaks: 1. phloroglucinol; 2. resorcinol; 3. phenol; 4. 2-cresol; 5. 3-nitrophenol; 6. 2-naphthol.

图12 CBS柱的分离重复性

Fig. 12 Separation repeatability of the CBS column Mobile phase: methanol/water (80∶20, v/v); flow rate: 0.8 mL/min.

图13 (a)喜树果粗提物和(b)吴茱萸粗提物在CBS柱上的色谱图

Fig. 13 Chromatograms of (a) Camptotheca acuminate extract and (b) Fructus evodiae extract on the CBS column

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腰果酚键合硅胶固定相的制备及其色谱性能

Preparation and chromatographic performance of cardanol-bonded silica stationary phase

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