My Copper complexes shows fluorescence.. and I have got fluorescence intensity (a.u.) vs wavelength(nm) plot and I have also checked solvent (acetonitrile) purity (no emission). But I have seen some journal, the authors have written Normalized Intensity vs Wavelength or Integrated Intensity vs Wavelength. How will I get the normalized or Integrated intensity? Another point is that some authors have written corrected emission spectrum? How will I correct emission spectrum?
When you want to draw a graph independent of a variable (e.g. concentration), you can use normalized spectra: Just divide the fluorescence intensity values with the corresponding values of the variable and redraw the spectra using these new values.
Integrated fluorescence intensity is the area under a fluorescence spectrum. You can use the software of the fluorometer to calculate this value.
Mr. Maji,
Those different representations of Fs spectra are associated with the fact that experimentally there is obtained a technical Fs spectrum. The reason for this is that Fs as physical phenomenon permits lost of Fs intensity due to set of mechanisms, on the one hand. Here can be added also cases when intensity of a Fs band becomes independent from the number of fluorophores in GS. Because of, intensity of Fs spectrum (IFs) according Einstein's theory about transition probability between two e- states is given by: IFs ca. dNi-j. The intensity should be proportional to number of fluorophore molecules first excited to Ej state and then turned back to Ei state. In other words, for ideal case IFs should increases in linearly with increasing in Ni. And this is true, when the solutions are very diluted. However at high concentrations, it is occurred independence of IFs from Ni. Or there is deviation from the linear relationship Ifs=f(Ni), due to an effect of density of the medium.
On the other hand because of you do not know wavelength-dependent effects of the detector, you cannot "claim" that all emitted irradiation by sample is detected. Or when, there is taken into consideration wavelength-dependent response of the detector, the corresponding Fs spectra are called "corrected". This correction can be obtained by direct measurements showing however a inaccuracy about 10 %. The common way is to correct your technical spectrum using a Fs of a standard for which you know the wavelength-dependent efficiency of the detector. There is possible to obtain corrected spectrum using for example measurements of detector response as a function of a standard source of light as well. But generally use of Fs spectrum of standard is a common practice.
Note, you should distinguish between "corrected" Fs spectrum and "baseline correction" applied to Fs spectra.
Integrated spectrum means that integrated Fs bands are divided by corresponding widths to each of the bands.
Normalized Fs spectrum is this, where are taken into consideration all processes associated with absorption or scattering. Because of you can have lost of Fs emission due to re-absorption, for example.
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