I performed QY measurements for two of my synthesized, novel compounds (L1 and L2) using tryptophan (solvent: water, 250C, QY literature value: 13%) as the reference sample. Test samples were dissolved in methanol. First, I recorded the UV absorbance values (between 0.01-0.1) at the excitation wavelengths to be used. For tryptophan it was at 280 nm while for the test samples it was 260 nm.
The absorbance values for,
Tryptophan: 0.015, 0.031, 0.045, 0.055, 0.064, 0.068, 0.086, 0.099
L1: 0.031, 0.044, 0.056, 0.069
L2: 0.032, 0.064, 0.067, 0.097
Then, fluorescence spectra were recorded (excitation wavelengths were 280nm and 260nm for the standard and the two test samples respectively) for each in the same solutions used to take absorbance. Then the integrated fluorescence intensity was calculated from the spectra (for tryptophan - from 300nm to 450nm, for L1 and L2 - from 280 nm to 400 nm) after subtracting the effect from the solvent background . Finally the graph of integrated fluorescence intensity vs absorbance was plotted and the QY values were calculated (L1 = 1.4% , L2 = 2%). The comparative method was used to calculate the QY values.
This is my first attempt in QY measurements for the novel compounds and I would be much grateful if I could get feedback on this and suggestions to improve are highly appreciated.
The graphs are attached.
Thanks in advance!
1. Both tryptophan and test sample should be excited at same wavelength. So I suggest you excite tryptophan and test at 280 nm. Additionally the absorbance value at the excitation wavelength should be similar and around 0.05 (to minimize inner filter effect).
2. Fluorescence is not always proportional to absorbance. So ideally integrated fluorescence intensity should be divided by (1-10^-Absorbance) not just (Absorbance). At low absorbance limit (1-10^-Absorbance)~=(Absorbance). Though I do not think it is critical for your case.
3. Since the solvent for tryptophan and test samples are different a refractive index correction should be done.
You can have look in this paper. It gives a quite detailed description of QY determination https://www.nature.com/articles/nprot.2013.087
Thank you for the suggestions!
The refractive index correction was done.
- Badges
- Science topic