【作者】 周尚；

【导师】 杨季冬；

【作者基本信息】 西南大学 ， 分析化学， 2008， 硕士

【摘要】 当物质分子受到光（电磁波）的照射时，会发生光的吸收、反射或透射以及光的发射、散射等现象。分子光谱即是将这些光现象进行分光所得到的光谱，它包含着物质的组成、结构、理化特性、反应历程等丰富的信息。分子光谱法则是以此为基础对物质进行测量和表征的分析方法，分子光谱分析是定性、定量分析研究物质的常用分析技术，是测定和鉴别分子结构的重要实验手段，是化学反应理论发展和实验验证的基础。分子光谱根据吸收电磁波的范围不同，可分为紫外、可见光谱和近红外光谱、红外光谱等；根据电磁波的发射方式不同又可分为分子荧光、磷光和散射光谱等。本文采用分子光谱法研究染料及生物大分子与某些药物的相互作用及其分析应用：1、纳它霉素对牛血清白蛋白的荧光猝灭作用，计算了不同温度下结合位点数n，结合常数K₄，以及对应的热力学参数△G，△H和△S；2、硫酸软骨素与牛血清白蛋白的相互作用，研究体系的RRS和SOS光谱特征及其影响因素和分析应用；3、研究赤藓红与扑尔敏的相互作用及其分析应用；4、研究卤代荧光素与西布曲明的相互作用及其分析应用；5、研究阿霉素与金属离子和赤藓红三元离子缔合物体系的光谱特征及其分析应用。1牛血清白蛋白-纳它霉素体系在中性介质中，采用荧光光谱和同步荧光光谱，研究了纳它霉素与血清白蛋白间的相互作用。依据F（o|¨）rster非辐射能量转移机理探讨了纳它霉素与血清白蛋白相互结合时其给体、受体间的距离（r=1．08 nm）和能量转移效率（E=22．99％），并用同步荧光技术考察了纳它霉素对血清白蛋白构象的影响。并根据热力学常数确定了该药物与血清白蛋白之间的作用力类型主要为氢键和范德华力。此外，还研究了在中性介质中，纳它霉素与血清蛋白反应形成结合产物，此时将导致蛋白质内源荧光(λ_ex/λ_em=285 nm／347 nm)猝灭，其猝灭值（△F）与纳它霉素浓度成正比，据此可建立以血清蛋白为探针的荧光猝灭法测定酸奶中纳它霉素的残留量的新方法。2硫酸软骨素-牛血清白蛋白体系在pH 3．5的B-R缓冲溶液中，带正电荷的牛血清白蛋白（BSA）与带负电荷的硫酸软骨素（CS）在水溶液中能通过静电相互作用形成BSA-CS聚合物体系。实验以△λ=0的方式和荧光扫描的方式扫描分别得到体系的RRS光谱和SOS光谱，研究了牛血清白蛋白与硫酸软骨素聚合物体系的共振瑞利散射（RRS）光谱和非线性二级散射光谱（SOS）光谱特征，实验还发现当牛血清白蛋白与硫酸软骨素作用时会增强其RRS光谱和SOS光谱，且体系的RRS光谱增强程度与其中任一组分浓度增加成正比，基于此可建立起灵敏度高的测定CS和BSA的RRS光谱分析的新方法，此外还探讨了RRS增强的机理。3．扑尔敏-赤藓红体系3．1赤藓红褪色分光光度法测定扑尔敏在弱酸性介质中，马来酸氯苯那敏（CPM）与赤藓红（ET）或曙红Y（EY）阴离子借静电引力和疏水作用力而形成离子缔合复合物。溶液的吸收光谱发生变化，赤藓红体系发生明显的褪色作用，最大褪色波长位于525 nm，CPM浓度在0．1～4．0μg·mL-1范围内遵从比尔定律，ε为3．5×10⁴ L·mol（-1）·cm^-1。方法灵敏度较高，选择性好，操作简便快速，用于片剂及尿样中马来酸氯苯那敏的测定，结果满意。3．2扑尔敏与赤藓红的荧光猝灭和共振散射光谱及其分析应用在pH 4．4 B-R缓冲介质中，扑尔敏（CPM）与赤藓红（ET）相互作用形成离子缔合物，导致荧光和同步荧光猝灭及共振瑞利散射（RRS）的显著增强并产生新的RRS光谱，最大RRS峰位于578 nm附近。其荧光猝灭程度及散射增强程度与扑尔敏浓度成线性关系，线性范围分别为：0．24～8．μg·mL^-1、0．08～3．6μg·mL^-1。将方法用于尿样中扑尔敏含量的快速测定，结果满意。此外还讨论了共振散射增强机理及荧光猝灭的原因。4西布曲明-卤代荧光素体系4．1卤代荧光素与西布曲明的荧光猝灭和褪色光谱及其分析应用在弱酸性缓冲溶液中，乙基曙红（EE）、赤藓红（ET）和荧光桃红（PX）三种卤代荧光素与盐酸西布曲明（SH）形成离子缔合物，导致吸收光谱发生变化和荧光猝灭。文中研究了反应产物的吸收和荧光光谱特征，适宜的反应条件，据此发展了以卤代荧光素为光谱探针的灵敏、简便、快速测定SH的新方法。其中ET体系褪色反应灵敏度最高，对SH的线性范围为0．1～4．0μg·mL^-1，检出限为0．06μg·mL（-1）；PX-SH体系的荧光猝灭法对SH的线性范围是0．2～4．6μg·mL^-1（λ_ex/λ_em=540 nm／560 nm），检出限为0．09μg·mL^-1。讨论了离子缔合反应对荧光及吸收光谱的影响及卤代荧光素荧光猝灭原因。4．2卤代荧光素与西布曲明相互作用的共振散射光谱及其分析应用用共振瑞利散射（RRS）法研究了，研究了乙基曙红（EE）、赤藓红（ET）和荧光桃红（PX）三种卤代荧光素与盐酸西布曲明（SH）的相互作用，发现乙基曙红、赤藓红和荧光桃红三种卤代荧光素与盐酸西布曲明（SH）形成了离子缔合物导致两体系产生强烈的RRS，它们的最大散射峰分别位于316 nm、345 nm和370 nm。考察了适宜的反应条件及共存物质的影响，在一定范围内SH的浓度与RRS强度成正比，其检出限分别为32 ng·mL^-1（EE-SH体系）、28ng．mL^-1（ET-SH体系）和21 ng·mL^-1（PX-SH体系）。以ET-SH体系为例，由光偏振实验，测得体系573 nm处的共振散射光谱偏振度P为0．95，表明体系的共振散射光谱主要由散射光构成，基本不含共振荧光成分。本实验还用讨论了体系的共振散射增强的原因。5阿霉素-Me（Ⅱ）-卤代荧光光素体系在弱酸性NaAc-HAc介质中，研究了阿霉素与金属离子Cu（Ⅱ）和Pd（Ⅱ）形成的阳离子配合物和阴离子卤代荧光素染料结合生成三元离子缔合物的反应，结果表明，三元离子缔合物的形成使体系的共振瑞利散射强度（RRS）显著增强，两个金属离子所形成的结合产物的RRS光谱特征相似，最大RRS峰均位于570 nm附近。但引起的RRS增强的程度Pd（Ⅱ）>Cu（Ⅱ），RRS的增量与阿霉素的浓度在一定范围内线性相关，据此建立了一种测定阿霉素的高灵敏度的分析方法。该方法的检出限（3σ）为8．2ng·mL^-1，为阿霉素的测定提供了一种新的方法。更多还原

【Abstract】 When the material molecule receives the light （electromagnetic wave）, the absorption, reflection or transmission and emission, scattering will take place. The molecule spectrum is obtained from dividing these light phenomena, it includes abundant information such as the composition, structure, physical and chemical characteristics, reaction mechanism and so on. Molecular Spectrometry is the analytical method of measuring and characterizing, molecule spectral analysis is commonly used analysis technology of the qualitative and quantitative analysis, it is the important experimental method of determining and indentifing molecular structure, it is the development foundation of chemical reaction theory and experimental verification. According to difference ranges of absorbing the electromagnetic wave, it can be divided into ultraviolet-visible spectrum, the near-infrared spectrum and infrared spectrum etc; according to the difference electromagnetic wave emission method, it can be divided into the molecular fluorescence, the phosphorescence and the scattering spectrum and so on. This article studies on the interaction of some pharmaceuticals with biological macromolecule and dyes by Molecular Spectrometry and their analytical applications: 1. Fluorescence Quenching of Bovine Serum Albumin by Natamycin, The binding site number n and the binding constant K_A , corresponding thermodynamic parametersΔG,ΔH andΔS at different temperatures were calculated; 2. The interaction of chondroitin sulfate with bovine serum albumin, we studied the RRS and SOS spectra characheristics of the system; 3. Study on the interaction of Chlorphenamine with erythrosine and its analytical applications; 4. Study on the interaction of Sibutramine with halofluorescein and their analytical applications; 5. Study on the spectra characheristics of ternary ion-association complexes of Doxorubicin-Me（II）-Erythrosine system and thair analytical applications.1 Natamycin - bovine serum albumin systemThe interaction of natamycin and bovine serum albumin （BSA） was studied with fluorescence spectroscopy and synchronous fluorescence. The binding distance（r=1.08 nm） and transfer eficiency （E=22.99%） between natamycin and BSA were obtained according to the theory of F（o|¨）rster non-radiation energy transfer. Furthermore, the effect of natamycin on the conformation of BSA was also analyzed using synchronous fluorescence spectrometry. Finally, the thermodynamic data show that the interaction forces of BSA with natamycin is the hydrogen bond and van der waals interaction. In addition, in neutral medium, natamycin could bind with BSA to form complexes, which would result in the quenching of the intrinsic fluorescence (λ_ex/λ_em=285 nm/347 nm) of the BSA. The quenching intensity （ΔF） was directly proportional to the concentration of the natamycin. Therefore, a new analytical method was established to determine trace ntamycin in yoghurt products.2 Chondroitin sulfate- bovine serum albumin systemIn pH 3.5 B-R buffer solution, BSA with positive charge and chondroitin sulfate （CS） with negative charge formed self-assembled polymer via electrostatic interaction in aqueous solution. We studied the resonance rayleigh scattering （RRS） and second-order scattering （SOS） spectra of CS-BSA system, RRS and SOS spectra were found by synchronous scanningΔλ=0 and scanning the fluorescence spectrum mode, respectively; we researched the RRS and SOS spectra characheristics of CS-BSA and BSA-CS system, it is found that RRS intensity can be enhanced after the interaction of CS and BSA, and there was a linear relationship between the RRS intensity and CS or BSA concentration. So a new method for the sensitive and rapid determination of CS and BSA can be developed. In addition, the RRS enhanced mechanism has been discussed.3 Chlorphenamine maleate - erythrosine system3.1 Fading spectrophotometric determination of chlorphenamine maleate with erythrosineIn weak acidic medium, Chlorphenamine Maleat （CPM） react with halogenated fluoresce dyes such as erythrosine （ET） and eosinY （EY） to form the ion-association complex through electrostatic and hydrophobic interaction, the color of solutions changes obviously, erythrosine system has a remarkable color fading reaction and the fading wavelengths is 525 nm, the concentration of CPM obeys Beer’s law in the range of 0.1-4.0μg·mL^-1, the molar absorptivity （ε） is 3.5×10⁴ L·mol^-1·cm^-1. The method is sensitive, simple and rapid, it has been applied to the determination of CPM in tablets and urine with satisfactory results.3.2 The fluorescence quenching and resonance Rayleigh scattering enhancement of ET-CPM system and its analytical applicationIn pH 4.4 BR buffer medium, erythrosine （ET） and Chlorphenamine Maleate （CPM） could form ion-association complex, which leads to the quenching of fluorescence and synchronized fluorescence, and the significant enhancement of resonance Rayleigh scattering （RRS） of erythrosine. The fluorescence and RRS intensities have linear relationship with the CPM concentration in the range of 0.24-8.0μg·mL^-1, and 0.08-3.6μg·mL^-1, respectively. The method could be applied to the determination of CPM in urine samples with satisfactory results. The mechanisms of the RRS enhancement and fluorescence quenching were discussed as well.4 Sibutramine - halofluorescein systems4.1 Study on determination of sibutramine by fluorescence quenching and fading spectrophotometryIn weak acid buffer solutions, 3 halofluorescein dyes, such as, ethyl eosin （EE）, erythrosine（ET） and phloxine（PX）, can react with Sibutramine（SH） to form ion-association complexes, which not only results in the quenching of fluorcence, but also results in the great decrease of absorbance. In this work, the spectral characteristics of fluorcence and absorption spectra, the optimum conditions of the reaction were investigated. A new method for the determination of Sibutramine by using halofluorescein as a probe has been developed. ET-SH system has the highest sensitivity for the fading reaction. There is a linear relationship between the absorption intensities and SH concentration in the range of 0.1-4.0μg·mL^-1, The detection limit for SH is 0.06μg·mL^-1; PX-SH system has the highest sensitivity for the fluorcence quenching reaction, the linear range is 0.2-4.6μg·mL^-1(λ_ex/λ_em=540 nm/560 nm). The detection limit for SH is 0.09μg·mL^-1. In addition, the reasons for the the quenching of fluorcence and the fading of absorption spectra were discussed.4.2 Studies on the interaction of Sibutramine with halofluorescein and their analytical application by resonance Rayleigh scattering spectraThe interaction of halofluorescein, such as, ethyl eosin （EE）, erythrosine （ET） and phloxine（PX） with Sibutramine（SH） have been investigated by resonance rayleigh scattering spectra（RRS）. SH can react with halofluorescein to form ion-association complexes, which can result in the significant enhancement of RRS intensity. The maximum scattering peaks esist at nm for EE-SH, nm for ET-SH and nm for PX-SH. The RRS spectral characteristics, the the optimum conditions and influencing factors have been studied for EE-SH, ET-SH and PX-SH reaction systems. The intensity of RRS is directly proportional to the concentration of SH in a certain concentration range, the detection limit for SH was 32 ng·mL^-1 （EE-SH system）, 28 ng·mL^-1（ET-SH system） and 21 ng·mL^-1（PX-SH system）, respectively. The polarization of light was measured, the polarization value P of the RRS at 573 nm is measured to be 0.95, the experimental facts revealed that the scattering peak of ET-halofluorescein mainly consists of resonance scattering and few resonance fluorescence. In addition, the reasons for intensity enhancement of RRS have been discussed. 5 Doxorubicin-Me （II）-erythrosine systemsIn NaAc-HAc buffer solution, the Doxorubicin reacted with Cu（II） and Pd（II） ion to form cation chelates which further reacted with anion halofluorescein dyes, such as ethyl eosin（EE）, erythrosine（ET） and phloxine（PX） to form ternary association-complexes. As a result, the significant enhancements of RRS intensities were observed. Their spectral characteristics of RRS were similar, and the maximum RRS wavelengths were at 570 nm. The increments of RRS intensity were different in the series of Pd （II）>Cu （II）. The enhanced RRS intensities were proportional to the concentration of DOX, the detection limit for DOX is 8.2 ng·mL^-1; a kind of new experimental method of measuring DOX was provided更多还原