I have recorded emission spectrum for my drug solution in water and methanol. The FI values were very high. The emission lambada max was in the range of 380- 400 nm. what type of reference standard can i use to calculate Relative fluorescence intensity of my drug. I recorded emission spectrum over a concentration range of 0.5-5 ppm. I have attached a word document of my FI values in water and methanol?
Anthracene and 1-napthol may be considered as reference. Please see this link
https://pure.uva.nl/ws/files/1189483/104485_354696.pdf
Utilize this software for absorption and emission spectra of known molecules.
http://omlc.org/spectra/PhotochemCAD/
When working with fluorescence please deal with absorbance not concentration. The intensity that you have listed is quite high (107) you need to dilute it to make sure no non-linearity takes place. Go through the instrument manual to see the accepted intensity range (generally it is 106). If you cannot dilute the sample decrease the path length (smaller pathlength cuvette) to decrease the absorbance and thereby fluorescence intensity.
You do not need a standard for relative fluorescence intensity, because it is relative. It depends on many factors, some if which are specific to the instrument used.The important parameters are the lambda max and the signal-to-background ratio.
If you want to measure the quantum yield (number of photons emitted/number of photons absorbed), that requires a standard. Methods for this determination may be found by searching on-line.
As long as the compound concentration is not too high and the detector is not saturated, the dependence of the fluorescence intensity on the compound concentration should be linear. This does not seem to be the case for your measurements, so something has gone wrong.
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