I am trying to compare compound fluorescent activity in different solvent. first i observed Absorption spectra of compound, then found max absorption wavelength/ excitation wavelength. on this excitation wavelength excited to observe emission of the compound in different solvent. and also run the blank (solvents) on same excitation wavelength. now blank showing the peak (solvents) on this excitation wavelength. now i want to know that, should i subtract the solvents emission spectra from compound emission spectra. And solvents emit spectra or do not?. or only compound can emit, solvent not.
please, please suggest me, my work is stopped at this stage.
The apparent fluorescence you observe for the solvent is probably Raman scatter, or a contaminant. Subtract the solvent spectrum from the compound-in-solvent spectrum to obtain the compound spectrum in that solvent. If the compound spectrum is very weak compared to the solvent spectrum, this subtraction may be inaccurate. To improve the result, use a high concentration of the compound and minimize the dimensions of the cuvette. By minimizing the dimensions of the cuvette, you reduce the amount of solvent through which the light passes, thus reducing the solvent background. The compound concentration can be increased up to a point, but once the concentration gets too high, the absorption of light by the compound will begin to affect the spectrum (inner filter effect). Measure the compound spectrum at a range of compound concentrations. You should see that the fluorescence intensity increases in direct proportion to the compound concentration until the inner filter effect starts to become significant, at which point the increase will be sublinear.
Mt. Kumar,
Could you provide more detail about the emission band position?
According the very valuable answer posted from Adam, please give us infos about :
- Purity of your solvents (spectranal quality or not)
- for UV-Vis and fluorescence spectra, you must purify your solvents and , important, you must dry them.
- Have you used new cuvettes (what a kind ? Quartz...?)
- Please note that Ethanol , Methanol, DMSO for spectroscopy contain water to a specified extent. (see suppliers infos).
Thank you in advance and good luck
JRG
That is nothing but the ramman peak due to the scattering, please substract them. By the way what is the dye (probe) you are using?
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