In general we see that the highest absorption wavelength of a solution is the probable excitation wavelength in fluorescence. But I am seeing quenching behavior at lower excitation wavelength for my sample. What could be the possible reason? Any literature suggestion would be highly appreciated. Thank you!
Any excitation wavelength in the absorption band will excite the fluorophore to an excited level. So even though an excitation wavelength lower than the absorption maximum will not excite as many fluorophores as the absorption maximum it will still create a population of excited fluorophores that will be quenched as usual.
Perhaps you should read Chapter 7 in my book which discusses quenching.
The principle of fluorescence excitation is based on the Jablonski diagram, where the highest absorption wavelength corresponds to probable excitation. Fluorescence emission usually occurs at higher energy (shorter wavelength) due to relaxation to the lowest vibrational level of the first excited singlet state (Stokes shift). However, quenching can occur at lower excitation wavelengths due to the inner filter effect, scattering, ground-state complex formation, photobleaching, non-radiative decay pathways, solvent effects, and aggregation.
https://www.spectroscopyonline.com/view/key-errors-avoid-consideration-fluorescence-quenching-data
David M Jameson Thank you for your answer. But, the excitation wavelength where I am getting a good quenching result, there was no absorption peak and in the absorption wavelength region, the quenching is not that good. I was wondering what could be the possible reason for that. Could you give me some insights on this?
Well I guess another possibility is that the compound is not 100% pure. Let me ask this question. If you compare the emission spectra for excitation at the 2 wavelengths (at the peak and at the lower excitation wavelength) do you get identical spectrum (in terms of shape - of course the intensities will differ)? If you don't then there is more than one fluorophore and quenching results are much more complicated.
Hi again Samia,
Could I ask exactly what your probe is and what quencher you are using. It may be easier to understand your system if we know the details.
Thanks,
Dave
Hi David M Jameson ,
Yes I am getting the same shape of emission wavelengths at two different excitation, and the quenching process I can see after adding different lower milimolar concentration of acetone. I hope this might help you to understand. Thanks!
Acetone? Acetone is not usually a quencher. How is the quantum yield of your dye affected by solvent polarity? It will help if you can specify the actual molecule.
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