I want to determine the quantum yeild of FITC in PBS buffer, I used Rhodamine 6G in ethanol as standard (quantum yield 0.95) and i excited at 480 nm and keeping the absorbance at 0.072. I use the same instrumental condition for the sample but the calculated quantum yeild was greater than 1 (2.19). I try various wavelenght and obtained the same result. How can i accurately determine the quantum yeild of FITC, what standrad is most appropriate
Fluorescein seems like the obvious choice, since FITC is fluorescein isothiocyanate, an amine-reactive derivative of fluorescein. The quantum yield of fluorescein in basic pH is 0.95, according to this source:
http://www.iss.com/resources/reference/data_tables/FL_QuantumYieldStandards.html
Here is a paper that should interest you:
"Fluorescence quantum yields and their relation to lifetimes of rhodamine 6G and fluorescein in nine solvents: improved absolute standards for quantum yields"
https://www.ncbi.nlm.nih.gov/pubmed/12003120
Regarding QY exceeding 1:
Did you keep the absorbance same for sample and standard at the excitation wavelength (480 nm)? Since your sample is highly emissive, please keep absorbance in the range of 0.03-0.05. To avoid error use same cuvette for sample and standard (as different cuvette have different transmittance). If you are not too familiar with QY measurement then please go through this paper
http://pubs.acs.org/doi/abs/10.1021/ed076p1260
Thanks Tuhin Khan.
I kept the absorbance of the sample (FITC dye) and standrad (R6G) at the same concentration (0.702) . I use lower absorbance value to prevent inner filter effect and excited at both at 480 nm. for my calculation i considered the area under the graph as suggested in the article you sent to me. But my quantum yeild grater than 1
If the problem persist even after using fluorescein as a standard then the problem might be in absorption measurement. Since in QY measurement we use very low absorbance (0.03-0.05) a wrong baseline will significantly affect your results. A simple way of checking baseline is to see whether the absorbance is zero (within error limit of 0.0002) at the wavelength where your sample does not absorb. In other word, if your sample absorption starts at 500 nm then the absorbance should be within +-0.0002 in the range of 500-800 nm.
Another point, when you measure QY after excitation at different wavelength does the the QY value change? If yes, then the problem is in the absorption measurement.
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