【作者】 陈会明；

【导师】 李来生；

【作者基本信息】 南昌大学 ， 分析化学， 2010， 硕士

【摘要】 我国蕴藏着丰富的药用植物资源,在防病治病中发挥着重要的作用。本研究就地取材利用药用天然化学成分做色谱配体,设计开发新型色谱固定相,并进行相关的基础应用研究。直接利用这些天然配体不仅能降低制备成本,还可避免繁杂的合成与纯化过程,对深层次开发利用我国丰富的中草药资源具有重要意义。本论文以天然产物大黄的药用成分大黄素为配体,将大黄素稳定地键合到硅胶表面,制备了一种具有抗菌和多作用位点的新型色谱固定相-大黄素配体键合硅胶固定相(EDSP),采用红外光谱、元素分析和热重分析进行结构表征,并评价了新固定相基本的色谱性能,探讨了该固定相的分离机制,为将来的开发利用提供理论依据。主要研究内容如下：1、从色谱固定相的基质选择、配体键合方法、色谱保留机理及应用研究的角度,较全面地总结了近年来国内外新型高效液相色谱键合硅胶固定相的研究进展,其中对传统固定相和新型固定相的研究开发进行了较详细的评述,以此作为本论文工作的理论依据。2、采用有机溶剂提取法,从药用大黄中提取并纯化得到大黄素。采用固液表面连续反应法,以γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)为偶联剂,首次将大黄素配体键合到球形硅胶表面,制备得到一种新的大黄素键合硅胶固定相。对配体的选择、制备路线的设计和反应条件的优化进行了探讨。并采用红外光谱、元素分析和热重分析表征了新固定相的结构。结果表明,通过蒽醌环上的配羟基与环氧基的开环反应,大黄素配体被成功地键合到球形硅胶表面,测得大黄素配体的键合量是0.180 mmol/g,不同批次的产品有良好的重现性,该制备方法简便,所制备的固定相色谱性能稳定。3、采用高压泵将固定相的匀浆直接填入不锈钢柱中(4.6 i.d.×150mm),以联苯为溶质探针,甲醇-水为流动相(50:50, v/v,0.8 mLmin-1,室温),测得EDSP的柱效为8227块/米。采用纯有机溶剂或酸性流动相长时间冲洗,发现该固定相对溶质的保留能力和柱效变化较小。采用Kimata等建立的一种国际上公认的C18反相键合硅胶固定相的色谱性能综合评价方法,评价了大黄素键合固定相的基本色谱性能。其中包括疏水性能、氢键能力、立体选择性和离子交换能力等。然后以中性的多环芳烃、酸性的酚类化合物和碱性硝基苯胺异构体等为溶质探针,初步研究EDSP对芳香性溶质的分离能力,并在相同条件下与传统的反相色谱固定相ODS进行比较,从不同的角度较系统地表征出EDSP对不同类型化合物的色谱分离性能和特点,结合色谱数据来探讨其分离机理,为新固定相实际应用提供理论依据。上述结果表明,所制备的大黄素键合硅胶固定相具有良好的反相色谱性能,除与溶质间的疏水作用外,还能提供氢键、n-π或π-π电荷转移作用以及偶极-偶极作用,这与大黄素配体的多羟基蒽醌环大π平面性结构有关,多位点的协同作用使得大黄素键合固定相不仅拥有类似ODS的反相色谱优良的色谱选择性和广泛的分离对象,同时在极性化合物的分离方面显示出较强的分离能力和快速分离等特点。4、进一步评价EDSP对极性芳香性化合物的分离能力,溶质包括9种嘧啶、4种嘌呤、3种核苷、4种蝶呤和5种农药等药物或生物活性密切相关的极性化合物,分别研究了它们在大黄素键合硅胶色谱固定相(EDSP)上的色谱行为,考察了流动相中常见的溶剂甲醇含量和pH对分离选择性的影响,并以ODS做对照,探讨了EDSP对这类含氮的碱性极性化合物的色谱分离机理。结果表明,采用通用和价廉的甲醇-水体系流动相,大黄素键合硅胶固定相能在较短的时间内分别实现上述物质的基线分离。在相同的流动相中,5-硝基尿嘧啶、异黄蝶呤、敌稗在EDSP上的洗脱顺序与ODS不同,说明EDSP和ODS对上述溶质的保留机理存在差异。明显地,由于EDSP中大黄素配体中含有蒽醌大π共轭体系,丰富的π电子与溶质间能发生ππ-π作用,羟基和羰基上的孤电子与溶质之间发生氢键作用和偶极-偶极作用,多种作用力的分离机制使得EDSP能够克服ODS单一疏水性作用的缺陷,在对极性化合物中显示其特有的优越性能。5、为考察新制备的EDSP固定相对复杂样品的分离能力,选用常见的中草药如虎杖、葛根、半枝莲和半边莲提取液做实际分离样品,采用液相色谱质谱电喷雾离子化联用技术,在分离的同时,对相关色谱峰进行了初步的质谱定性分析。结果表明,在未经前处理的情况下,EDSP对上述植物中复杂的提取液有较好的分离能力,明显地,这与多作用位点有关。此外,因地取材得到的天然大黄素配体的药物功能和分离功能相结合,更能满足工业色谱的需要。更多还原

【Abstract】 It is well known that there is abundant medicinal plants in our country, which play an important role in disease prevention. In this study, we make use of narural active components in Traditional Chinese Medicine as chromatographic ligands to design, prepare and apply new-style chromatographic separation materials. Direct exploitation of the natural ligands can not only reduce production cost, but also to avoid the complicate synthesis and purification process, and it also has an importantance in the development and utilization of Chinese herbal medicine deeply. In this thesis, a new emodin-bonded silica gel stationary phase (EDSP) with the ligand from Rheum officinale Baill for HPLC was prepared for the first time. Its structure were characterized by Fourier transform infrared spectrometry, elemental analysis and thermal analysis. The chromatographic performance of EDSP were evaluated. Related separation mechanism was systematically studied. The new stationary phase was used for the separations of complicate samples. The major contents are as follows.1. The recent advance in the proceeding from the matrix, preparation, chromatographic retention mechanism and their application of novel bonded silica gel stationary phases for HPLC at home and abroad, were reviewed. The study of bonded silica gel stationary phases for HPLC was reviewed from two aspects, including the improvement of traditional bonded-silica gel stationary phases and the design of new bonded phases. Above all will contribute to the research basis and start point of this thesis.2. Emodin was extracted and purfied from Rheum officinale Baill by organic solvent extraction method. And emodin was chemically immobilized to the surface of silica gel as stationary phase by the solid-liquid phase reaction with y-glycidoxy-propyltrimethoxy-silane (KH-560) coupling reagent for high performance liquid chromatography(HPLC). The new emodin-bonded stationary phase (EDSP) was brown-red monodispersal particles. According to. the preparative scheme, the reaction conditions were also partially optimized via investigation under different reaction ratios. The structure of new emodin-bonded stationary phase (EDSP) was characterized by Fourier transform infrared spectrometry, elemental analysis and thermal analysis. The results showed that emodin was successfully immobilized onto the surface of spherical silica gel with a 0.18 mmolg-1 of bonded amount and highly physical and chemical stability. In addition, the bonded amounts of the packings in different batches were similar to each other, which indicated that the above preparation procedure was practical and reliable.3. The slurry of EDSP was packed into a stainless steel column by using common LC pressure pump. The column efficiency was 8827 plates/m by using biphenyl as probe in methanol-water (50/50, v/v) mobile phase at room temperature. The chromatographic stability of EDSP in strong organic solvent and acidic mobile phase were investigated, respectively. That an authorized column validation method proposed by Kimata’s group was used to systematically evaluate the basic chromatographic properties of EDSP, including hydrophobicity, charge transfer ability, hydrogen-bonding, steric selectivity and ion exchange interactions. The chromatographic performance of new EDSP was studied in detail by using different structural solutes as probes and ODS as reference. These chromatographic data can be used as the theoretical basis for its applications in future. The tested solutes included neutral aromatics, acid phenols and basic aromatic amines etc. The results indicated that EDSP performed as a versatile reversed-phase material with a relatively weak hydrophobicity, especially, various sites located in the emodin ligand, such as hydrophobic, hydrogen bonding,π-πor n-πcharge transfer and dipole-dipole interactions. EDSP has excellent abilities for the fast separations of neutral, acid and basic compounds. It is related with the existence of largeπsystem and planar rigid structure of EDSP. Hence, EDSP was endowed advantage over only hydrophobic ODS in rapid separations of polar compounds.4. In order to further evaluate chromatographic performance in the separation of polar compounds, the behavior of nine pyrimidines, four purines, three nucleosides, four pterins and five pesticides on EDSP were studied by HPLC. Meanwhile, the comparative study in the retention behavior of the above solutes on both EDSP and ODS were also done under the same conditions. The influence of mobile phase variables, such as methanol content and pH value on the retention behavior of the polar compounds were investigated. The results showed that baseline separation on EDSP were easily achieved by using common and cheap methanol-water mobile phases. The elution orders of 5-nitrouracil, isoxanthopterin and propanil were very different on EDSP and ODS under the same condition, which showed that EDSP and ODS had different retention mechanism. Obviously, the EDSP exhibited advantage over only hydrophobic ODS in seperations of the above polar compounds. The results can be explained as follows:theπ-πinteraction, hydrogen bonding, n-πinteraction existed between phenolic hydroxyl or carbonyl groups of emodin anthraquinone ligands containingπ-conjugated system and the solutes.5. To investigate the chromatographic performance in the separation of complicate compounds, EDSP was also employed for the separation of complex samples, which were extracted from common herbs, including polygonum cuspidatum S. et Z., Radices puerarire, Scutellaria barbatat and lobelia. Some major components were identified by LC-ESI-MS in the absence of standard substances. Good separations of the above samples were achieved without pre-treatment, which was because the carbonyl and hydroxyl on emodin anthraquinone ligands could provide various sites. In addition, new separation material with natural emodin ligand will exhibit a bright future for industrial chromatography separation applications because it has both pharmaceutical and chromatographic functions.更多还原