I am performing experiments on a suspension of fluorescent material that is quenched in the presence of a target analyte. I excite at 279nm and see a peak around 560nm. When I add analyte solution (a very small volume relative to suspension volume) this 560nm peak responds very well to its presence illustrating a "quenching" effect. Is this second order scattering? I would not expect such a phenomena to respond to my analyte solution. For the record, I am adding 15uL to 2mL in the cuvette. A first order peak is observed and is very small compared to the peak at 560nm, which is sharp.
You mean that the peak at 560 is the 2nd harmonic of the 279 nm excitation? If this is being quenched by the analyte, it could be due to the inner filter effect, whereby the absorbance of the analyte reduces the amount of light reaching the fluorescent material.
Adam B Shapiro, thank you for your response, that is what I mean.
I have run UV-Vis for one of my analytes and it appears to have substantial absorbance between 200-300nm, but not at all outside of this range.
If I understand the scattering correctly, it is happening at the diffraction grating. This would mean for the "quenching" observed to be due to the inner filter effect the analyte would need to absorb around 560nm. Is this the case?
I would like to add the peak at 560nm is ~20x more intense then that of the peak at 279nm.
I'm not sure what you mean by "the peak at 560nm is ~20x more intense then that of the peak at 279nm." Aren't you exciting at 279 nm and measuring emission at 560 nm? How do you have a peak at 279 nm? That would mean you were setting excitation and emission monochromators to the same wavelength, which is not a good practice.
In any case, if the emission measured at 560 nm is not fluorescence but is only stray 2nd harmonic light from the monochromator, and if the analyte does not absorb at 560 nm, then my earlier suggestion must be incorrect. Are you sure the signal is not fluorescence? Have you taken an emission spectrum of your fluorophore and a background spectrum of the solvent?
Adam B Shapiro If that is a bad practice I will discontinue it.
With some additional tests I have found that the peak at 560nm must be due to 2nd harmonic light from the monochromator.
That being said, I have done UV-Vis and emission on both my solvent and analyte- neither absorb in that range or appear to fluoresce. I am still observing a linear relationship between this peak and my analyte but it is not so important as there is another emission peak I can consider.
I suspect it has something to do with my suspension, though I have no idea how.
Thank you again for your help in this problem of mine.
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