Experimental determination of the quantum yield of dichlorofluorescein

Summary

This experiment is from the official website of Wuhan University School of Pharmacy

Operation method

Experimental determination of the quantum yield of dichlorofluorescein

Principle

Fluorescence analysis has been widely used in the fields of organic electroluminescence, biomedicine and clinical diagnosis. The preparation of high-performance fluorescent materials has become the research hotspot and frontier in these fields, and the fluorescence quantum yield of these fluorescent materials directly affects their performance advantages and disadvantages. The fluorescence quantum yield (YF) is the ratio of the number of photons of fluorescence emitted by a fluorescent material after absorbing light to the number of photons of the absorbed excitation light. Its value is usually less than 1. The larger the YF value, the more fluorescent the compound is, while the fluorescence quantum yield of a non-fluorescent substance is equal to or very close to zero. The fluorescence quantum yield is generally determined by the reference method. That is, under the same excitation conditions, were determined to be tested fluorescent specimens and known quantum yield of the reference fluorescent standard material two dilute solutions of the integral fluorescence intensity (i.e., corrected fluorescence spectrum of the area included), and the same excitation wavelength of the incident light (UV-visible) of the absorbance, and then substituting these values into the specific formula for the calculation, you can get to the fluorescent specimen to be tested quantum yield: Yu = YundefinedFu/Fs *As/AuYu, Ys - the fluorescence quantum yield of the substance to be tested and the reference standard; Fu, Fs - the integrated fluorescence intensity of the substance to be tested and the reference substance; Au, As - the absorbance of the incident light of the substance to be tested and the reference substance at the excitation wavelength (A = εbc). When using this formula, the absorbance As, Au is generally required to be lower than 0.05. It is best to choose a reference standard sample with a similar excitation wavelength value of the fluorescent substance. Fluorescent compounds with analytical application value of Yu is generally often between 0.1-1.

Move

1. Prepare dichlorofluorescein (0.25 μg-mL-1) solution of the sample to be tested (containing 1.0 mol-L-1 NaOH aqueous solution), rhodamine B (0.25 μg-mL-1) reference standard solution (solvent is anhydrous ethanol);

2. Turn on the molecular fluorescence spectrometer and ultraviolet-visible spectrophotometer;

3. Pipette the required concentration of dichlorofluorescein and rhodamine B solution, dilute to 10.0 mL with the corresponding solvent (A505 nm < 0.05), and measure its absorption spectrum curve on the UV-visible spectrophotometer; determine their absorbance at 505 nm, respectively.

4. Pipette the same solution as above in a fluorescence cuvette, and scan its fluorescence excitation and emission spectra on a fluorimeter; determine their fluorescence emission spectra at 505 nm as the excitation wavelength, respectively.

Common Problems

data processing

1. Calculate the relative integral area of the fluorescence spectra of dichlorofluorescein and the standard substance rhodamine B.

2. Check the quantum yield of the reference standard Rhodamine B in ethanol solvent from relevant sources.

3. Calculate the quantum yield of dichlorofluorescein solution by substituting the obtained data into the fluorescence quantum yield formula.

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