【作者】 许丽丽；

【导师】 李来生；

【作者基本信息】 南昌大学 ， 分析化学， 2007， 硕士

【摘要】 利用我国丰富的植物药用资源，将植物药用成分均匀地化学修饰到硅胶表面，能制备得到一种具有色谱分离和药用价值的双功能新材料，其色谱分离功能的研究及其应用是色谱学与相关应用科学交叉的前沿研究课题，具有重要的学术和应用价值。本学位论文工作首次利用药用姜黄素作配体，成功地制备了一种新型的姜黄素键合硅胶固定相，旨在先期研究其色谱功能和初步的分析应用。除采用常规的红外光谱、热重分析和元素分析进行结构表征外，还首次展示了电喷雾离子化质谱表征硅胶表面姜黄素配体结构的新用途。选用多种结构特征的溶质为探针，从新固定相基本色谱性能的评价研究入手，由浅入深，分别较系统地考察新固定相对各种溶质探针、含系列极性化合物的简单样品到复杂中草药提取液样品等的综合色谱分离能力，并探讨了姜黄素键合固定相的色谱保留机理。研究表明，由于姜黄素配体含易极化的较大芳环电子系统，与溶质间存在多种协同作用，在含氮的碱性化合物和极性化合物的分离方面显现出优势，有较好的色谱应用开发前景。此外，这种表面含天然药用成分的材料在抗菌医药等领域将有应用前景，这为中草药有效成分深加工利用提供了一条新途径。包括如下研究内容：1．从色谱固定相制备、色谱保留机理和分析应用的角度，较全面地综述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。同时展望了植物活性成分作为固定相键合配体在色谱分离科学中的应用前景，并以此作为本论文的选题、研究内容和技术路线设计的依据。2．以γ-[（2，3）-环氧丙氧]丙基三甲氧基硅烷（KH-560）为偶联剂，采用固液表面连续反应法，在温和的反应条件下，将植物药用成分姜黄素键合到硅胶上，首次制备了姜黄素液相色谱键合固定相（CCSP）。采用红外光谱、热重分析、元素分析和质谱对其结构进行表征，并对键合反应的制备路线和反应条件进行了一定的优化。结果表明，配体姜黄素被成功地键合到球形硅胶表面，测得配体键合量为0.129mmolg^-1，该固定相有良好的物理和化学稳定性。不同批次的键合相具有相近的键合量，表明该键合反应路线简便易行，制备方法重现性较好。3．以联苯为溶质探针，甲醇-水（55∶45，v／v）为流动相，测得CCSP的柱效为8221块／米。采用纯有机溶剂或酸性流动相长时间冲洗，发现该固定相仍保持稳定的色谱性能。首先采用公认的Kimata小组发展的方法，对新固定相的基本色谱性能进行了综合评价，这其中包括疏水性能、氢键能力、立体选择性和离子交换能力等。然后根据新固定相的特点，侧重细评其疏水性、电荷转移能力和对芳香烃位置异构体的分离能力。通过对中性、碱性和酸性化合物分离的考察，展示新固定相在复杂样品体系分离分析中的优势，溶质包括：非极性难电离的芳烃、极性大易电离的芳香酸和芳香胺、水溶性极性强且有生化分析意义的维生素和核苷碱基等特征结构的探针群体。并引用传统的商品化反相填料ODS柱作参比相。力求从多个方面较全面地把握CCSP对不同类型化合物的色谱分离性能，结合机理研究，为其未来的实际应用提供理论依据。研究结果表明：CCSP是一种疏水性较弱的反相色谱填料，能与溶质发生疏水、氢键、偶极-偶极以及电荷转移等作用，协同作用的结果使得CCSP能够克服ODS单一疏水性作用的缺陷，在对极性化合物的快速分离中占优势。4．为进一步评价新的硅胶固定相（CCSP）对碱性溶质的分离能力，实验选用11种含氮的碱性化合物为溶质探针，考察了流动相中甲醇含量、pH值对分离的影响，研究了该固定相的亲硅醇基活性和相关色谱保留机理。结果发现，在不含任何添加剂的甲醇水简单流动相中，未经封尾处理的姜黄素键合硅胶固定相能分别实现三组碱性化合物的基线分离。在相同色谱条件下，CCSP对碱性物质的选择性比ODS高。且N，N-二甲基苯胺和β-萘胺、N-甲苯胺和间硝基苯甲醛在新固定相的洗脱顺序与ODS不同，说明二者的保留机理存在差异。明显地，新固定相在反相色谱条件下对碱性化合物的分离除与疏水作用有关外，还与氢键作用、偶极-偶极作用、n-π和π-π电荷转移等作用有关。新固定在分离碱性化合物方面体现出优势：一是由于其较弱的疏水性和多种作用位点，更易接近碱性化合物，进行丰富的相互识别作用，从而在短时间内能够实现良好的分离；二是偶联剂间隔臂的屏蔽作用和配体的立体保护效应能有效地抑制硅胶表面残存的硅醇基效应，克服了碱性溶质的拖尾现象，在CCSP上的色谱峰对称性明显优于ODS，且碱性化合物的容量因子κ’均随甲醇含量的增大而减小，这个变化过程中始终未观察到不理想的“U”形曲线。5．以易电离的13种极性取代芳香酸为探针，考察不同流动相和pH值的条件下，CCSP对取代芳香酸色谱行为的影响，探讨新固定相对芳香酸类化合物的色谱保留机理。结果表明，在不同含量的甲醇-0.02molL^-1NaH₂PO₄（pH=2.5）流动相体系中，三组芳香酸溶质在CCSP上实现基线分离。在相同条件下，CCSP对13种取代芳香酸类化合物的洗脱顺序与ODS具有较大的不同，其中5种溶质在CCSP上比在ODS上的保留更强，与两固定相的疏水性和键合量不符，这归因于CCSP的协同作用。例如，姜黄素配体的酚羟基和羰基能与酸性化合物的极性基团发生氢键作用、偶极．偶极作用，与此同时，n-π和π-π等电荷转移作用也普遍存在于姜黄素配体的芳环与溶质的苯环之间。在分离芳香酸时，含姜黄素配体的CCSP有多种作用机制，这使得它对极性的和离子化的溶质如：水杨酸、对硝基苯甲酸、3，4，5-三羟基苯甲酸、邻苯二甲酸和邻溴苯甲酸等具有较强的保留，同时由于本身较弱的疏水性，苯甲酸和邻氯苯甲酸在CCSP上的保留不会像在ODS上那么强，这样分离时间缩短，分离度也有明显的改善。6．分别研究了5种水溶性维生素和5种核苷在CCSP上的色谱行为，考察了甲醇含量、流动相pH值和离子强度对CCSP分离这类极性化合物的影响，探讨了色谱保留机理。结果表明，CCSP能较好地分离5种水溶性维生素，而核苷的分离是仅用水为流动相的条件下实现的。与ODS柱相比，在相同条件下，CCSP对5种水溶性维生素具有较高的选择性，洗脱顺序与ODS具有较大的差异。相同色谱条件下，CCSP对核苷的保留比ODS弱，胸苷与黄苷的洗脱顺序也与ODS不同，仅用水做流动相就可快速分离核苷，节省了分析时间和费用。在相对短时间内分离选择性却有提高，这归因于极性溶质与姜黄素配体的极性基团间的各种非疏水性相互作用，这些作用使新固定相更适用于极性化合物的分离分析。7．将CCSP分别用于四种较复杂的实际样品的色谱分析，样品包括：复方水杨酸搽剂、四川大蒜粉、常见中草药五味子和白术的提取液。以ODS为参比相，初步地考察了新固定相对复杂样品的分离能力，探讨了相关色谱保留机理。结果表明，该固定相在对复杂样品的分离中以疏水作用为主，同时存在氢键、偶极-偶极以及n-π和π-π电荷转移等作用，这有利于改善分离选择性，协同作用使CCSP能够在较简单的条件下实现复杂样品的有效快速分离。更多还原

【Abstract】 It is well known that there is abundant herb plant resource in our country. Traditional Chinese Medicine is very valuable treasure derived from nature. In order to make full and deeply use of them, this thesis focused on the exploration of their new chromatographic separation usage based on the natural chemical structural ligand. The scheme dealt with the preparation of separation materials with both chromatographic and pharmaceutical functions by chemical modification of the medicinal components onto the surface of silica gel for high performance liquid chromatography （HPLC）. It is an attractive subject with great significance for the researches of theory and practice in the overlapping area among modern chromatography and related applied sciences to study the chromatographic retention mechanism and application of such novel separation materials.In this thesis, a new curcumin-bonded silica gel stationary phase （CCSP） was prepared so as to study its chromatographic functions and exploit its application for the first time. Its structure was characterized by electrospray mass spectrometry creatively, besides ordinary Fourier transform infrared spectrometry, elemental analysis and thermal analysis. Different structural solutes were used as probes to study the chromatographic property of CCSP systematically, including the basic chromatographic performance and the characteristic chromatographic separation evaluations. The probes covered a wide structural range from common probes, simple polar compound samples to complicate Chinese herbs extracts. Meanwhile, the retention mechanisms were also discussed.The results showed that CCSP exhibited excellent separation abilities for many polar compounds due to its large aromatic rings electronic system and various action sites. It has a promising prospect in the separations of basic compounds and many polar compounds for chromatography. Additionally, materials modifed with natural medical ingredients have potential application in the field of antibacterial medicines, which provides a new way pointing to the modern deep-processing of active components of TCM in the future. The major contents are as follows: 1. The recent advances of the study in the preparation, chromatographic retention mechanisms and their application of novel bonded silica gel stationary phases for HPLC at home and abroad were reviewed systematically. The application prospect of the active components from plants as the bonded ligands for HPLC and related separation technologies in the future was also discussed. Above all will contribute to the research foundation and start point to explore new natural medicinal ligand bonded stationary phases.2. An herb medicinal active component curcumin was chemically immobilized to the surface of silica gel as stationary phase by the solid-liquid phase reaction method under mild conditions by usingγ-glycidoxy-propyltrimethoxy-silane （KH-560） as coupling reagent for high performance liquid chromatography. The structure of new curcumin-bonded stationary phase （CCSP） was characterized by Fourier transform infrared spectrometry, elemental analysis, thermal analysis and mass spectrometry. The preparative scheme and reaction conditions were also partially optimized via investigation under different reaction conditions. The result showed that the curcumin ligand was successfully immobilized on the surface of spherical silica gel with a 0.129 mmolg^-1 of bonded amount, and CCSP had high 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. Biphenyl was used as probe to determine the column efficiency （8221 plates per meter） in methanol-water （55:45, v/v） mobile phase. The stability of CCSP in strong organic solvent and acidic mobile phase were investigated, respectively. The basic chromatographic properties of CCSP, including hydrophobicity, charge transfer capacity, hydrogen bonding capacity, steric selectivity and ion exchange capacity were systematically evaluated, according to an authorized column validation method proposed by Kimata’s group. Subsequently, various structural probes and conventional ODS were employed so as to profound study of new CCSP for a wide apllications in the future. The intensive chromatographic properties lie on the hydrophobicity, charge transfer and recognization abilities for aromatics and their positional isomers. The advantages of CCSP were showed in the separation procecesses by using probes, including many neutral, basic and acidic compounds. For example, nonploar aromatic carbonhydrons, polar and ionized aromatic acids and amines, water-soluble vitamins and nucleosides etc. Some comparative study was also carried out by adopting as reference under the same conditions. According to the chromatographic behavior of probes on CCSP, some retention mechanisms of new packing were also proposed, which will partially be used as the theoretic foundation for its practical application in the future. The results indicated that CCSP performed as a versatile reversed-phase material with a relatively weak hydrophobicity, especially, various action sites located in the curcumin ligand, such as hydrophobic, hydrogen bonding, dipoledipole, charge transfer action sites for different analytes. Hence, CCSP was endowed advantage over only hydrophobic ODS in rapid separations of polar compounds.4. Eleven basic compounds containing nitrogen-atom were employed as solute probes so as to further study the separation ability of new curcumin bonded stationary phase for basic compounds, which is important to the bonded silica gel stationary without end-capped process. The effects of mobile phase variables, such as methanol content and pH in mobile phases on the chromatographic behaviors of the basic compounds, as well as the silanophilic activity of CCSP were investigated in detail via the comparative study with ODS. The behavior change was used to clarify the related retention mechanism of the analytes on CCSP. The results indicated that baseline separation of three series of basic compounds on CCSP （no end-capped） could be achieved only with methanol-water as binary mobile phases without addition of buffer solution, respectively. CCSP exhibited better separation selectivity for the above basic compounds than that of ODS. It could also be noticed that the elution order of N,N-dimethylaniline andβ-naphthylamine, N-methylaniline and m-nitrobenzaldehyde were very different under the same reversed-phase chromatographic conditions. Obviously, CCSP had different retention mechanism to the basic compounds as compared with conventional ODS. According to the chromatographic data, some conclusion could be made that the versatile separation ability of CCSP derived from the synergetic interactions between the curcumin and analytes, including hydrophobic, hydrogen-bonding, dipole-dipole, n-πandπ-πcharge transfer interactions. CCSP had two advantages in the separation for basic compounds. On one hand, the rapid separation of basic compounds can easily be realized because of weak hydrophobicity and multi-sites of CCSP. On the other hand, the side effect of residual silanols on the surface of silica gel was despressed by the polar linkage spacer. Thus, symmetrical peak shapes can be observed by using only methanolwater as mobile phase. Moreover, the unfavorable "U" shape of retention factor （k’） of the basic solutes never appeared with increasing of methanol content.5. Subsequently, thirteen aromatic carboxylic acids were used as acidic probes to characterize new CCSP. In the same way, the comparative study with ODS was also done under the same reversed-phase chromatographic conditions. After some investigation of influence factors, it could be found that baseline separation of three series of aromatic carboxylic acids on CCSP were easily achieved, instead of ODS. CCSP behaved as a reversed-phase packing and its separation selectivity for aromatic carboxylic acids was better than that of ODS. The different elution orders of some acidic compounds were also observed on both CCSP and ODS. Among them, five of solutes exhibited stronger affinities to CCSP, which was inconsistent with the hydrophobicities and bonded amount of both packings. It was obvious that CCSP exhibited better separations whether acidic or basic compounds, which was mainly independent of the ionization property of solutes and dependent on the synergetic interactions providing by CCSP. For example, hydrogen bonding, dipole-dipole interactions existed between the phenolic hydroxyl or carbonyl groups of the curcumin and polar groups of acidic compounds. The CCSP containing curcumin ligand had two advantages over ODS. On one hand, the polar and ionized analytes, such as salicylic acid, p-nitrobenzoic acid, 3,4,5-trihydroxybenzoic acid, phthalic acid and o-bromobenzoic acid showed stronger affinities to CCSP because of various synergistic mechanisms. On the other hand, the retention of benzoic acid and o-chlorobenzoic acid can be successfully separated on CCSP within a short time because of its weak hydrophobicity.6. Five water-soluble vitamins and five nucleosides were chromatographied for the further study. Their chromatographic behaviors were studied on both CCSP and ODS, respectively. Some influence of mobile phase variables such as methanol content, pH and ionic strength on the retention behavior of analytes was investigated so as to clarify the retention mechanism. The results showed that a better separation of five water-soluble vitamins on CCSP was achieved by using methanol-0.01molL^-1 NaH₂PO₄ （40:60, v/v, pH3.5） as mobile phase, and the separation of nucleosides was carried out with only water as mobile phases. CCSP had better separation ability for vitamins and provided the different retention order for the analytes as compared With ODS. CCSP could also achieved effective separation of nucleosides and with different selectivity for thymidine and xanthosine from ODS within a relatively short analytical time. The various interactions and weak hydrophobicity of CCSP contributed to its advantages over only hydrophobic ODS in the separations of the above polar compounds.7. CCSP was employed for the preliminary separations of four complicate samples, including salicylic acid liniment, Sichuan garlic Allium Sativum L powder extracts, Sichsandra sphenanthera Rehd. et wils. seed extract and Atractylodes macrocephala Koidz. extract. The separation ability for complicate samples and the chromatographic retention mechanisms on CCSP were studied as compared with ODS. It was found that besides hydrophobic interaction other interactions such as hydrogen bonding interaction, dipole-dipole interaction, n-πandπ-πcharge transfer interaction could also tailor the chromatographic retention behaviors of analytes and facilitate to the separations. Rapid separations of the above samples can be achieved on CCSP under simple chromatographic conditions with the help of the synergistic interactions.更多还原