Study On The Interaction And Application In Analytical Chemistry Between Serum Albumins Or Chitosan And Dyestuff By Spectroscopy

Fluorescence spectrophotometry is a common qualitative and quantitative analytical method with high sensitivity and good selectivity, and using little specimen and operation rapid and simple. We study spectral character of dyes band to serum Albumins by Fluorescence quenching method. We explained the type of fluorescence quenching and the type of effection of protein and dyes by experimental data. We calculated the features parameter of the reaction system such as binding constants, sites and distance. We found the intensity fluorescence quenching is linearly proportional to the concentration of dyes after interaction between the serum albumins and dyes, then a new method for determination of dye could be established. However the fluorescence intensity of dye can be enhanced because of the protein biding to dyes in different condition, and the enhanced intensity is linearly proportional to the concentration of BSA. Thus a new method for the analysis of BSA was developed.Resonance Rayleigh Scattering(RRS) is a recent developed molecular spectral analytical method with high sensitivity. At present RRS technique has been extensively and successfully applied in various research fields such as biomacromolecular analysis, organic substance and medicine analysis, determined inorganic ion and characterized nanometer ion. In the thesis we study assay of carbohydrate substances with this technique, a new RRS method of determination of chitosan was established. At the same time, we study the experimental conditions and influenced factors. The method was applied to determinate real samples of chitosan with satisfied results. It has higher sensitivity and good electivity than other determinated methods.The research contents as follows: 1. Serum albumin-phenazine dye systemBovine serum albumin(BSA) or human serum albumin(HSA) bind to phenazine dye such as phenosafranine, neutral red and Safranine T in the Tris-HCl buffer solution at pH 7.4.The recation lead to fluorescence quenching of serum albumin. The relative values of fluorescence quenching is linearly proportional to the concentration of dyes. It can be used for determination of dyes with the protein for reaction probe. The fluorescence quenching data at different temperature were analyzed according to Stern-Volmer equation and Lineweaver-Burk double reciprocal equation. The dynamic quenching constants, the binding constants of static quenching of the reaction, the binding sites number and the basic thermodynamic parameters of the binding process were obtained.The binding distance between dye and Serum Albumins at 20℃was obtained according to the theory of Foster s non radiation energy transfer mechanism. However we study the fluorescence enhancement of phenosafranine in Tris-HCl buffer solution at pH 9.5. The relative fluorescence intensity of phenosafranine is linearly proportional to the concentration of Bovine Serum Albumins. It could be used for determination of Bovine Serum Albumins. We study the three-dimensional fluorescence spectra of neutal red bind to serum albumin.It display there are energy transfer in system. bovine serum albumin transfer partis energy to neutral red. This caused fluorescence quenching of bovine serum albumin.2. Serum albumin-bisphenylnaphthylmethane dyes systemThe binding feature of bisphenylnaphthylmethane dyes to bovine serum albumins or Human serum albumins was studied by fluorescence quenching spectra, the synchronous fluorescence spectrum and ultraviolet spectrum. The reaction of bisphenylnaphthylmethane dye and serum albumins causes fluorescence quenching of serum albumins. The relative fluorescence quenching is linearly proportional to the concentration of VB,VBB,VB4R. The fluorescence quenching data were analyzed according to Stern-Volmer equation and Lineweaver-Burk double reciprocal equation. The dynamic quenching constants, the binding constants of static quenching of the reaction, the binding sites number and the basic thermodynamic parameters of the binding process were obtained. The binding distance between dye and Serum Albumins was obtained according to the theory of Foster s non radiation energy transfer mechanism.3. Chitosan-metal ion-serum albumin systemThe ternary complex system was produced from the three composes of human serum albumin, copper ion and chitosan reaction in Britton-Robinson (BR) buffer solution at pH 6.5.It results in a significant enhancement of resonance Rayleigh scattering (RRS) intensity and the maximum RRS peak is at 470 nm.But the resonance Rayleigh scattering (RRS) intensity at 305-340 nm are stable. The enhanced RRS intensity (â–³I_RLS) at 318 nm is proportional to the concentration of chitosan in the range of 0.01～4.0μg/mL. The method exhibits high sensitivities and the detection limit is 3.7 ng/mL for chitosan. The method are applied to the determination of practical samples with satisfactory results.4. Chitosan-Tetraiodofluorescein systemTetraiodofluorescein and chitosan form compound in Britton-Robinson (BR) buffer solution at pH 4.0.It results in a great enhancement of resonance Rayleigh scattering intensity. The maximum RRS peak is at 575 nm. Optimum conditions of the reaction and the influence of foreign substances have been investigated. It was found that the RRS intensities were in proportion to the concentrations of chitosan at peak 576 nm in the range of 0.1².0μg/mL. The detection limits was 17 ng/mL.A rapid, simple and sensitive RRS method for the analysis of chitosan was developed.This method was applied to the determination of chitosan in capsule with satisfactory results. This method and above method in thrid system are more sensitive than other method of fluorescence quenching and ultraviolet light spectroscopy. The reaction systems are more stable too.