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.
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。
Keywords: bonded stationary phase; cardanol; chromatographic properties; separation of traditional Chinese medicines.