i want to record the emission spectra of unknown compound. To do this i have record the absorbance of the compound, used the lamda max as the excitation wavelength and recorded the emission spectra. then i fixed the emission maxima obtained from the previous spectra and record the exitation spectra. Now i am using the exitation max obtained from the previous spectra to get the new emission spectra of my compound. Let us say new excitation wavelength is 229 nm and new emission wavelength is 356nm. Now, my compound is dissolve in the water, hence i want to check whether the emission i got is due to my compound or something water additive. Hence i further check the emission spectra of water only by using the same excitation wavelength that is 229nm. And i got he emission maxima at 346nm wavelength. here i need the clarification that is whether the emission spectra i got of my compound is due to my compound only or due to water. The emission spectral intensity of water alone (e.g. 400) is half of that of compound in the water (e.g.800)
Is the emmision spectrum from the water the same shape as the emission spectrum of the water plus your compound? This might indicate carryover.
Are you sure the water is pure?
Are you seeing a stray light effect from your spectrofuorimeter? What if you use a different (non-fluorescent) solvent?
These are the questions I would be asking myself in this situation.
Hope this helps to focus your thoughts.
The apparent fluorescence spectrum of water is caused by Raman scattering. The emission maximum will change as you change the excitation wavelength. This will not happen for your fluorescent compound.
As Adam said, you might be looking at the raman emission. He is also correct in that you can easily check if it is Raman by irradiating your sample at a different excitation wavelenght. The relationship between the raman peak and that wl is known (see attachment). Is your compound fluorescent at all? if you're only seeing Raman after irradiating at the maximum absorption wl, then it doesn't really look like it is.
You can also have some contamination in the water (as Alan said).
Adam/eugenio
looks like the excitation and detection lambda are far apart: can this be raman scattering? also: the emission is not at the double frequency which could have been detection of the overtone stray light
according to the formula, for 229 exc the raman would be at about 248 nm, which does not match the 356 emission. ¿Probably the solvent is contaminated?
Good point Harry and Eugenio. Another possibility is that the cuvette is dirty. Try cleaning it with chromic acid or some other suitable cleaning agent.
I agree with the very good answers of the aswerers. BUT you must have a PURE and DRY compound for your fluorescence spectra investigations.
Have you got a good elemental analysis, MS, NMR, TLC ...of your unknow compound.
Traces of impurities (e.g. impurities with own fluorescence) will give false results.
Please give infos about your compound and your purification methods and the qualities of solvents (e.g. spectranal quality /supplier) and what a kind of cuvettes are you using (quartz a.s.o...) and what a kind of spectrometer (Supplier).
The hints of Eugenio and Adam (see above) are very important too!!
JRG
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