光散射光谱法测定蛋白质和表面活性剂的研究
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摘要
本论文主要应用光散射光谱法和紫外可见光谱法对蛋白质及阳离子表面活性剂进行了定量分析,并系统研究了蛋白质与盐析物质、蛋白质与阴离子表面活性剂以及氯金酸与阳离子表面活性剂之间的相互关系。
利用共振光散射技术对蛋白质进行了定量分析。着重研究了以盐析类物质及阴离子表面活性剂为探针的共振光散射技术对蛋白质的测定,提出了两种测定蛋白质的共振光散射方法:(1)以盐析类物质硫酸铵为共振光散射探针,用于牛血清白蛋白、人血清白蛋白及卵清蛋白等三种蛋白质的测定;(2)以阴离子表面活性剂SDBS为共振光散射探针,并分别以β巯基乙醇(βME)和二硫苏糖醇为强还原剂,测定了牛血清白蛋白。详细研究了各探针物质与蛋白质之间的相互作用,解释了β巯基乙醇(βME)和二硫苏糖醇作为强还原剂的作用,揭示了共振光散射强度增强的原因。
同时利用氯金酸与阳离子表面活性剂之间的离子缔合作用,提出了一种新的测定阳离子表面活性剂的光散射方法。详细探讨了离子缔合作用发生的可能机理,揭示了体系光散射强度增加的原因。
考察了上述三种光散射体系的各种实验条件对反应的影响,并分别将方法用于实际样品的测量,所得结果证实了方法的可靠性和实用性。
In this thesis, light scattering(LS), especially resonance light scattering(RLS),was reviewed and two simple, rapid and sensitive RLS methods were developed todetermine proteins and a sensitive and accurate LS method was built to detect CS.The possible mechanisms of the interaction between probes and target analytes havebeen disccussed and the applications of the methods were used to the sampleanalysis.
Firstly, (NH4)2SO4 protein systems were investigated by RLS spectrometry.Protein solution has a colloidal propety and its molecular diameter is the same as thatof colloidal particles. When the concentration of a neutral salt[such as (NH4)2SO4] isin a definite range, the activity of aqueous molecules decreased greatly, which canresult in the precipitation of proteins. Ammonium sulphate, (NH4)2SO4, is one of themost important salts used for precipitation of proteins because of its high solubility,low density, good stabilization and low price. Based on the precipitation happenedbetween (NH4)2SO4 and proteins which can lead to the deposition and collection ofproteins and can induce the enhancement of RLS intensity, (NH4)2SO4 was used as anew RLS probe to determine bovine serum albumin(BSA), human serumalbumin(HSA) and ovum albumin(OVA). The effects of pH value, (NH4)2SO4saturation and coexistence substances were examined. Under the opitical conditions,the calibration curves were constructed, and the detection limits of BSA, HSA andOVA can all reach to the level of 10 9 mol/L. The synthetic and real samples were also analyzed by this assay and the results are in agree with the results obtained byCoomassie Brilliant Blue(CBB) method. It can be concluded that the proposedmethod is suitable for the determination of proteins.
Secondly, the RLS spectra of proteins reduction agents anionic surfactantssystems were investigated. The reason for the use of anionic surfactant(AS) todetermine biomacromolecules by RLS is that when pH Finally, chloroauric acid anion(HAuCl4) cationic surfactant system wasinvestigated by light scattering(LS) spectrometry. When pH value is about 2.0, themain formation of cationic surfactant(CS) is CS+. If the concentration of cationicsurfactant is much lower than critical micelle concentration(cmc), the main form of cationic surfactant(CS) is CS+. The main formation of HAuCl4 is AuCl4 when pHvalue is ca. 2.0. The reaction between AuCl4 and CS+ is due to the electrostaticattraction and the stable ion association complex is formed. Because of the formingof complex of [AuCl4 ][CS+], the size of molecule becomes larger which canenhance the light scattering intensity. Based on this mechanism, HAuCl4 was used aslight scattering(LS) probe to determine three kinds of cationic surfactants,cetyltrimethyl ammonium bromide(CTMAB), cetyltrimethyl ammoniumchloride(CTMAC) and cetylpyridinium bromide(CPB). The effects of pH value,HAuCl4 concentration, reaction time, reaction temperature and coexistencesubstances were discussed. Under the opitical conditions, the dynamic range and thedetection limit of the method were obtained. The detection limits of CTMAB,CTMAC and CPB can all reach to the level of 10 8 mol/L. Some real samples wereanalyzed by the proposed method and the results obtained were satisfactory.
利用共振光散射技术对蛋白质进行了定量分析。着重研究了以盐析类物质及阴离子表面活性剂为探针的共振光散射技术对蛋白质的测定,提出了两种测定蛋白质的共振光散射方法:(1)以盐析类物质硫酸铵为共振光散射探针,用于牛血清白蛋白、人血清白蛋白及卵清蛋白等三种蛋白质的测定;(2)以阴离子表面活性剂SDBS为共振光散射探针,并分别以β巯基乙醇(βME)和二硫苏糖醇为强还原剂,测定了牛血清白蛋白。详细研究了各探针物质与蛋白质之间的相互作用,解释了β巯基乙醇(βME)和二硫苏糖醇作为强还原剂的作用,揭示了共振光散射强度增强的原因。
同时利用氯金酸与阳离子表面活性剂之间的离子缔合作用,提出了一种新的测定阳离子表面活性剂的光散射方法。详细探讨了离子缔合作用发生的可能机理,揭示了体系光散射强度增加的原因。
考察了上述三种光散射体系的各种实验条件对反应的影响,并分别将方法用于实际样品的测量,所得结果证实了方法的可靠性和实用性。
In this thesis, light scattering(LS), especially resonance light scattering(RLS),was reviewed and two simple, rapid and sensitive RLS methods were developed todetermine proteins and a sensitive and accurate LS method was built to detect CS.The possible mechanisms of the interaction between probes and target analytes havebeen disccussed and the applications of the methods were used to the sampleanalysis.
Firstly, (NH4)2SO4 protein systems were investigated by RLS spectrometry.Protein solution has a colloidal propety and its molecular diameter is the same as thatof colloidal particles. When the concentration of a neutral salt[such as (NH4)2SO4] isin a definite range, the activity of aqueous molecules decreased greatly, which canresult in the precipitation of proteins. Ammonium sulphate, (NH4)2SO4, is one of themost important salts used for precipitation of proteins because of its high solubility,low density, good stabilization and low price. Based on the precipitation happenedbetween (NH4)2SO4 and proteins which can lead to the deposition and collection ofproteins and can induce the enhancement of RLS intensity, (NH4)2SO4 was used as anew RLS probe to determine bovine serum albumin(BSA), human serumalbumin(HSA) and ovum albumin(OVA). The effects of pH value, (NH4)2SO4saturation and coexistence substances were examined. Under the opitical conditions,the calibration curves were constructed, and the detection limits of BSA, HSA andOVA can all reach to the level of 10 9 mol/L. The synthetic and real samples were also analyzed by this assay and the results are in agree with the results obtained byCoomassie Brilliant Blue(CBB) method. It can be concluded that the proposedmethod is suitable for the determination of proteins.
Secondly, the RLS spectra of proteins reduction agents anionic surfactantssystems were investigated. The reason for the use of anionic surfactant(AS) todetermine biomacromolecules by RLS is that when pH
引文
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