Study On The Determination Of Cu²⁺ Concentration In Water Based On Fluorescence Quenching Of Calcein

The situation of copper pollution is serious while the measurement methods of coppercontent is high cost,complicated sample pretreatment and time-consuming.So the determination method of copper???ion concentration in water based onfluorescence quenching of calcein was studied in this paper.Exitation-Emission matrix?EEM?was used in this paper to study the properties of calcein and calcein-copper???ion system.Three-dimensional fluorescence quenching method for the determination of copper???ion concentration in water was established,and it was successfully applied in actual water samples.Meanwhile,Fourier Transform Infrared Spectroscopy?FT-IR?was used to research the complexing mechanismbetween calcein and copper ions,and the fluorescence quenching mechanism of calcein and copper ion system was discussed based on the complex mechanism and fluore-scence quenching theory.There are 5 fluorescence peaks of calcein,and the fluorescence peak Ex/Em is 490⁴95/515 nm,365³70/515 nm,320³25/515 nm,285²90/515 nm and 235²40/515 nm.Among them,the fluorescence peak Ex/Em 365³70/515 nm had a better linear relationship?R²=0.9942?with the concentration of calcein,and this fluorescence peak was selected for the experiment.The study found that under the conditions of within 40 min reaction time and the temperature between 20?to 50?,the fluorescence intensity of calcein-copper ion system was stable.Although at the pHvalue between 2⁵ and the pH value between 8¹2 the fluorescence intensity of calcein-copper ion system changed obviously,at the pHvalue between 5⁸ it was basically stable.So the pH value between5⁸ was restricted strictly in the whole experimental process.Under the measuring conditions?pH=5⁸,25??,it was found that the relative calcein intensities??F/F₀<0.9?increased nearly linearly with increasing the concentration of Copper???ion in the range of0.00023⁰.063mg/L.And?F/F₀-C(Cu²⁺)afforded a good linear relationship with plotting equation:?F/F₀=0.6987C(Cu²⁺)+0.1336 and a high linear coefficient?R²=0.9933?.The limit of detection?LOD?for Cu²⁺was 5.66×10^-5mg/L.The variations of fluorescence quench were not evident when calcein was mixed with the resolution of Li⁺,Na⁺,K⁺,Ca²⁺,Mg²⁺,Ba²⁺,Al³⁺,Pb²⁺,Zn²⁺,Fe³⁺and Hg²⁺,respectively.Among them,Li⁺,Na⁺,K⁺,Ca²⁺,Mg²⁺,Ba²⁺had hardly influence on the accuracy of the Cu²⁺test results with the error less than 2%.The accuracy of Cu²⁺determination results was not affected when the ratios of Al³⁺,Pb²⁺,Zn²⁺concentrations and Cu²⁺concentration was less than 100 times.When the ratios of Fe³⁺and Hg²⁺concentrations and Cu²⁺concentration was 10 times,the Cu²⁺test resultswere affected a little with the error of only 2.69%and 8.76%,respectively.The precision and accuracy of the method were studied by using the actual water sample.The relative standard deviation,recovery rate of the water samples in the three gorges reservoir during dry season were 0.00⁰.91%,86.13%¹11.96%,respectively.The relative standard deviation,recovery rate of industrial wastewater were 0⁰.27%,108.4%¹10.8%,respectively.Applying this method to determine Yangtze three gorges reservoir area plentiful water samples,Cu²⁺concentration was between 0.07-0.176?mol/L.According to the variations of width and intensity of FT-IR peak and the fluorescent quenching of calcein with the ratio of 1:2 between calcein concentration and copper???ion concentration,the complexing mechanism was that a calcein molecule providingtwo carboxyl and another calcein molecule providing two carboxyls together formed linear complexation with the complexationratio of 1:2.The exciplexwhich was formed by calcein and copper???ion was on the one handmore efficient with the process of channeling non-radiative peaks decaying to the ground state.On the other hand the ground state complexes generated competing with fluorescent material of ground state molecules absorbing light excitation?Filter effect?caused the decrease of fluorescence intensity of fluorescence material.Therefore,the fluorescence quenching mechanism of calcein and copper???ion was static quenching.