【作者】 罗红群；

【导师】 刘绍璞；

【作者基本信息】 西南师范大学 ， 分析化学， 2002， 博士

【摘要】 共振瑞利散射(RRS)和倍频散射、二级散射等共振非线性散射(RNLS)是二十世纪90年代发展起来的一种新兴的分析技术，因其高灵敏度、简易性和较好的选择性而引起了人们的广泛兴趣和关注。近年来研究和应用日益增多。特别是它对于生物大分子的一些非键合作用极为敏锐，已成为研究生物大分子的一种十分有用的手段，目前主要集中于核酸和蛋白质的研究，但另一类最重要的生物分子——糖类，至今尚很少涉及，此外，利用这种方法对一些物理-化学参数的测定，也尚未见报道。因此，本文在国家自然科学基金的资助下以糖胺聚糖的肝素为例，研究RRS和RNLS法在多糖分析中的应用，并以β-环糊精的包结常数和表面活性剂的临界胶束浓度(CMC)值为例，研究这类方法在测定物理-化学参数方面的应用。 本文的主要研究内容及成果如下： 1．肝素-碱性二苯基萘基甲烷染料及肝素-碱性吩噻嗪类染料体系的共振瑞利散射光谱及其分析应用 以肝素为分析对象，研究了碱性二苯基萘基甲烷染料和碱性吩噻嗪类染料等两类碱性染料与肝素相互作用的共振瑞利散射光谱特征及其分析应用，结果表明，在弱酸性至弱碱性溶液中，维多利亚蓝4R(VB4R)、维多利亚蓝B(VBB)和夜蓝(NB)三种碱性二苯基萘基甲烷染料及亚甲蓝(MB)、甲苯胺蓝(TB)、天青Ⅰ(Azure Ⅰ)和天青Ⅱ(Azure Ⅱ)四种吩噻嗪类碱性染料与肝素结合后，能导致共振瑞利散射光谱强度急剧增强并产生新的RRS光谱，它们分别在523nm(维多利亚蓝4R体系)、534nm(维多利亚蓝B体系)、540nm(夜蓝体系)、346nm(亚甲蓝体系)、329nm(甲苯胺蓝体系)、337nm(天青Ⅰ体系)和338nm(天青Ⅱ体系)处出现最大RRS峰。考察了RRS光谱特征，影响因素和适宜的反应条件，结合紫外-可见分光光度法和量子化学计算探讨了反应机理。指出这些碱性染料与肝素的结合物的RRS光谱特征与吸收光谱密切相关，两者之间具有对应关系。研究了共振瑞利散射强度与肝素浓度之间的关系，建立了用共振瑞利散射技术测定痕量肝素的新方法，方法灵敏度高， 西南师范大学博士学位论文 摘 要七种反应体系的检出限在 3．35－35．93 ＂g／mL之间。其中维多利亚蓝 4R体系的灵敏度最高，用于肝素钠注射液的效价测定获得满意结果。说明这一技术用于糖类的研究和测定也有很好的前景，这是共振瑞利散射在生物大分子分析应用中的一个新的发展。2．肝素－亚甲蓝体系的二级散射和倍频散射光谱研究 用倍频散射和二级散射技术研究了吩嚏嚎类碱性染料亚甲蓝（MB）与肝素的结合反应，结果表明，亚甲蓝与肝素的结合也能导致倍频散射（FD）和二级散射（SO）强度急剧增强，它们分别在350 run 倍频散射）和700 urnc级散射）处出现最大散射峰。考察了适宜的反应条件，影响因素及两种散射强度与肝素浓度之间的关系，提出了用倍频散射和二级散射技术测定痕量肝素的方法。方法灵敏度高，倍频散射和二级散射法的检出限分别为 4．36 ＂g／ffiL和 3．55 gb。探讨了倍频散射和二级散射的光谱特征与共振瑞利散射的内在联系。3．肝素－结晶紫体系的共振瑞利散射、二级散射和倍频散射光谱及其分析应用研究 用共振瑞利散射、倍频散射和二级散射技术研究了碱性三苯甲烷类染料结晶紫（CV）在 pH 6刀司二的 Britton－Robinson缓冲溶液中，与肝素的结合反应，结果表明，该反应能导致共振瑞利散射及倍频散射和二级散射强度急剧增强，它们分别在AeX／lem 为 492 Inn／92 urn（共振瑞利散射），984 nln／92 urn（倍频散射）和 492Inn／84 urn二级散射）处出现最大散射峰。研究结果表明肝素的浓度在 0＋0 pg／25mL范围内与散射强度有良好的线性关系，提出了用共振瑞利散射、倍频散射和二级散射技术测定痕量肝素的方法。方法灵敏度高，共振瑞利散射、倍频散射和二级散射法的检出限分别为：2．92 ＂g／ffiL，3．46 ＂g／inL和 3二8 ＂g／ttiL。并探讨了共振瑞利散射与倍频散射和二级散射之间的关系，由此验证了倍频散射和二级散射是由共振瑞利散射引起的共振非线性散射的观点。4．阳离子表面活性剂与肝素反应的共振瑞利散射光谱特性及其分析应用 研究了氯化十六烷基二甲基芋基按（CDBAC）、氯化十四烷基二甲基芋基铰记ePh＼澳化十六烷基毗陡（CPB＊滨化十四烷基毗陡（TPB）和澳化十六烷基三甲铰（CTAB）等5种阳离子表面活性剂与肝素的相互作用，结果表明，它们均能产生强的RRS，且反应条件接近，RRS光谱特征相似，但灵敏度差异较大，其中含芳基且分子量大的氯化十六烷基芋按（CDBAC）最灵敏，而不含芳基分子量又较小的澳化十六烷基三甲镀（CTAB）灵敏度最低，前者对于肝素的检出限为 11＂g／thL，后者则为 33 ＂g／ffiL。方法对于肝素有较好的选择性，可用于痕量肝素的测 且二 西南师范大学博士学位论文 摘 要定。研究还发现RRS强度与阳离子表面活性剂的结构和分子量有关。5．共振瑞利散射和共?更多还原

【Abstract】 Resonance Rayleigh scattering (RRS) and resonance nonlinear scattering (RNLS) such as double frequency scattering and second order scattering etc. is a new technology developed in 1990s. For its remarkable characteristics of high sensitivity, simple operation and good selectivity, this method brings to more attention and interesting and has been studied and applied more and more in recent years. Particularly, it is very sensitive to the nonbonding action of biological macromolecules, therefore, has been used as a useful means to study biological macromolecules. At present, this technology is mainly used to the study of nucleic acids and proteins. However, up to now it is seldom applied to the study of another most important biological molecule, saccharide. Moreover, There is no report about this technology used to the determination of some physicochemical parameters. Therefore, supported by the National Natural Science Foundation of China and using heparin, a glycosaminoglycan, as an example, this thesis has studied the application of the RRS and RNLS methods. Furthermore, using the inclusion constant of p-cyclodextrin and the critical micelle concentration (CMC) of some surfactants as examples, the methods have been applied to the determination of the physicochemical parameters.The main contents and some conclusions of the thesis are as follows:1. Resonance Rayleigh Scattering Study of Interaction of Heparin with Some Basic Diphenyl Naphthylmethane Dyes and Some Basic Phenothiazine Dyes and Their Analytical ApplicationUsing heparin as an analyte, the RRS spectral characteristics of the interaction of heparin with some basic diphenyl naphthylmethane dyes and some basic phenothiazine dyes and their analytical application have been studied. The results show that in weak acidic and weak basic medium, binding of heparin with some basic diphenylnaphthylmethane dyes such as victoria blue 4R (VB4R), victoria blue B (VBB) or night blue (NB) and some basic phenothiazine dyes such as methylene blue (MB), Azure A Azure B or toluidine blue (TB) can result in a significant enhancement of resonance Rayleigh scattering (RRS) and appearance of a new RRS spectrum. Their maximum scattering wavelengths max appear at 523 run, 534 nm, 540 nm, 346 nm, 337 nm, 338 run and 329 nm for VB4R, VBB, NB, MB, Azure A, Azure B and TB, respectively. The RRS spectral characteristics, the influencing factors and the optimum conditions of these reactions have been investigated. By combining ultra violet visible spectrophotometry and the quantum chemistry calculation, the reaction mechanism has been discussed. The results show that the RRS spectral characteristics of the combination products of heparin with these basic dyes are closely related to their absorption spectra. There is a corresponding relationship between them. And the relationship between the RRS intensity and the concentration of heparin has been investigated. A new method for the determination of trace amounts of heparin based on the RRS method has been developed. The methods exhibit high sensitivities and the detection limits for heparin of the seven systems are between 3.35 ng/mL and 35.93 ng/mL. Among them the VB4R system is the most sensitive and was applied to the determination of heparin in heparin sodium injection samples with satisfactory results. It also illustrates that this technology has a good application foreground to the study and determination of saccharide. This is new development of the RRS technology used in the analytical application of the biological macromolecules.2. Study on Frequency Doubling Scattering and Second-Order Scattering Spectra of Heparin-Methylene Blue SystemThe combination of heparin with methylene blue, a basic phenothiazine dye, has been studied by means of the frequency doubling scattering and second-order scattering technology. The results show that the binding of heparin with methylene blue can result in a significant enhancement of frequency doubling scattering (FDS) and second-order scattering (SOS). Their maximum scatte更多还原