Study On The New Methods Of Fluorescence Quenching And Its Analytical Application

With the rapid development of science and the pharmaceutical industry, a lot of new drugs have been found. In order to strictly monitor the quality of drugs, we must adopt good analysis methods for the determination of them. Some of the original analysis methods are gradually replaced because of low sensitivity,bad selectivity, complex operation and other reasons. With the new kinds of instruments have been published and a variety of detection methods have been improved, the establishment of a analysis method of higher sensitivity, better selectivity, less dosage, simple operation has become a research hotspot for analysis workers. Fluorescence analysis method has been widely used in pharmaceutical analysis, environmental analysis and biochemical analysis because of its advantages of high sensitivity, high selectivity and low detection limit. However,most of the drugs are not fluorescent or weak fluorescence signals, unable to make quantitative analysis by direct fluorescence method. Through the experimental study, we want to establish some indirect fluorescence analytical method for the determination of these drugs, and study on the quenching mechanism between drug and system. The study can not only provide the possibility for fluorescence determination of some drugs, but also can expand the application range of the fluorescence analysis method.Firstly, this paper gave an overview of the fluorescence analysis method, and then some drugs without fluorescent properties or endogenous fluorescence weak were studied by fluorescence quenching method. Using bovine serum albumin, 5- bromine salicyl fluorine, eosin Y and C-Dots as fluorescence probes, the fluorescence spectra of the interaction between sulfasalazine, cefixime, chlorpromazine hydrochloride, Cefetamet Pivoxil hydrochloride, Cr(VI) and them were studied. New fluorescence quenching methods for the determination of these drugs were established, which were applied to analyses real samples. In addition, the quenching mechanism between drug and system were discussed. The main contents are as follows:1. The interaction between sulfasalazine(SASP) and bovine serum albumin(BSA) was studied using fluorescence quenching technique in this paper. The experimental results showed that SASP could strongly quench the intrinsic fluorescence of BSA in p H=7.40 borate buffer solution, the relative fluorescence intensity(ΔF=F0-F) showed a good linear relationship with the concentration of SASP in the certain range. The linear regression equation of the method was ΔF=3.03×109c(mol/L)+20.20(r = 0.9980), the detection limit and the relative standard deviation were 7.2×10-10 mol/L and 0.43%(n=5, c=4.0×10-8 mol/L), respectively. The method has been used for the determination of SASP in tablets, the recovery rate was 96.3-104%. Additionally the quenching mechanism between SASP and BSA was investigated by fluorescence lifetime, effect of different temperature on the quenching constants and UV absorption spectrometry. The results showed that the fluorescence quenching between SASP and BSA was a static quenching process. The binding distance between SASP and BSA was calculated according to the F?rster’s theory. In addition, the thermodynamic parameters were obtained according to the Van’t Hoff equation. This indicated that the interaction between SASP and BSA was a spontaneous process and the main interaction forces were van der Waal’s force and hydrogen bonds. The synchronous fluorescence spectroscopy was used to further investigate the effect of SASP on BSA molecule.2. The interaction between bovine serum albumin(BSA) and cefixime(CEF) was studied by fluorescence quenching in p H=3.80 sodium acetate buffer solution. The results showed that CEF made the fluorescence intensity of BSA decreased significantly, and the concentration of CEF in the range of 8 ×10-9~1.2×10-7 mol/L, there was a good linear relationship between the fluorescence quenching degree ΔF of the system at λem=342nm and the concentration of CEF. According to this, a novel method for the determination of cefixime was established. The linear regression equation of the method was ΔF=1.9×109c(mol/L) +5.65(r=0.9994), the detection limit of the method was 2.0 ×10-9 mol/L, the relative standard deviation was 0.86%(n=5, c=8.0×10-8 mol/L). The method has been used for the determination of SASP in tablets with satisfactory results, the recovery rate was 94.5~97.3%. The influence of temperature on the quenching constants, fluorescence lifetime and UV absorption spectrum all explained that the quenching process between BSA and CEF was a static quenching procedure. Through the calculation of the thermodynamic parameters, which showed that the interaction between BSA and CEF was a spontaneous process, and the forces were mainly van der Waal’s force and hydrogen bonds. According to the F?rster’s theory, the binding distance between BSA and CEF was 2.14 nm. In addition, the synchronous fluorescence method showed that the cefixime caused changes in the conformation of bovine serum albumin.3. In p H =7.40 phosphate buffer solution(PBS), a novel fluorescence quenching method for the determination of cefetamet pivoxil hydrochloride(CPH) with 5-bromo-salicyl-fluorone(5-Br SAF) as a fluorescent reagent was established. The linear regression equation of the method was ΔF =12.14×109c(mol/L) +25.06(r= 0.9944), the linear range was 8.0×10-10~1.8×10-8 mol/L, the detection limit was 3.8×10-10 mol/L and the relative standard deviation was 1.8%(n=5, c=8.0×10-9 mol/L). The method has been used for the determination of CPH in tablets, the recovery rate was 98.0-104%. At the same time,the interaction mechanism of CPH and 5-Br SAF was investigated by fluorescence lifetime, the different temperature effect on the quenching constants and UV absorption spectrometry, the results showed that the interaction mechanism between CPH and 5-Br SAF was a static quenching process.4. In p H=2.80 Britton-Robinson(B-R) buffer solution, a novel fluorescence quenching method for the determination of chlorpromazine hydrochloride(CPZ) with eosin Y as a fluorescent reagent was established. Under the selected experimental conditions, the linear range of the method for the determination of CPZ was 8×10-8 ~8.2×10-6 mol/L, the detection limit was 1.6×10-9 mol/L and RSD is 1.3%(n=5, c=1.2×10-6 mol/L). The method was used to determine the CPZ tables, the recovery was 98.6-105%. In addition, the influence of temperature on the quenching constants, fluorescence lifetime and UV absorption spectrum all explained that the quenching process between EY and CPZ was a static quenching procedure.5. The C-Dots was prepared using bovine serum albumin as the carbon source, by one-pot method direct acidification in aqueous medium. And it was characterized by fluorescence spectroscopy and UV absorption spectra. In p H=7.40 three hydroxymethyl aminomethane-HCl(Tris-HCl) buffer solution, synchronous fluorescence spectrometry was used to study on the interaction of C-Dots and Cr(Ⅵ). The results showed that the synchronous fluorescence intensity of C-Dots was decreased by Cr(Ⅵ). Moreover, the synchronous fluorescence quenching value(ΔF) of the system further increased when added sixteen alkyl three methyl ammonium bromide(CTMAB). If the concentration of Cr(Ⅵ) in the range of 2.4×10-6~ 8.0×10-5 mol/L, ΔF value has a good linear relationship with the concentration of Cr(Ⅵ). The linear regression equation was ΔF =4.4×106c(mol/L) +21.58(r=0.9953), the detection limit was 8.4×10-7 mol/L and RSD was 0.58%(n=5,c=4.0×10-5 mol/L). This method was used to determine Cr(Ⅵ) from simulated waste water and the water of Yan He River with satisfactory results. In addition, the effect of temperature on the quenching constants and UV absorption spectrum were used to study on the mechanism of interaction between C-Dots and Cr(Ⅵ).