Re: Fluorescent Spectrum: Is this an artifact?

Friedrich Menges wrote:
philip wrote:

Marvin wrote:

philip wrote:

Hi
I'm performing emission scans on some unknown organic mixtures at an excitation wavelength of 248nm. In some of the spectra, there appears to be a peak at 620nm. Since peaks beyond the 2nd order raman are seldom reported, I wonder if this signal originates from organics or could it be artifacts?

I posted the spectra at:
http://img82.imageshack.us/img82/5264/image1lp2.jpg

Spectrum 'B' is what I get most of the time. Spectrum 'A' at wavelengths > 575nm is what I am not sure about.

thanks
p



Are you sure about the emissions below 400nm that are not in both spectra. If so, what are they? Do the short wavelength emissions always appear with the peak about 620 nm. Unlike the strong peaks, both of the extra sets have broad half-widths and structure that may or may not be noise. Are their shapes repeatable?



The emissions below 400nm are likely proteins. 'A' apparently is more concentrated. For samples similar to 'A', the short and long wavelength emissions appear together most of the time and are repeatable. The signal at 620 nm can be stronger or weaker than that at 320nm. If the noise are smoothed out, the the 'peak' at 620nm would still appear as a bump unlike that in 'B'. The broad shapes are mainly because its a mixture of compounds.

Posing the question in another way, is it reasonable to see Stokes shift as much as 372nm?


It is possible, but most unusual. To find out about the nature of your 620nm band, it would be easiest to vary the excitation wavelength. If the band stays in place, it is caused by molecular fluroescence, otherwise it can be a solvent Raman band or an artifact caused by your spectrometer. The spectra seem to be heavily smoothed, this not always helps to better interpretation.
Which sort of instrumentation do you use, what about more detailed information about your mixture?!

Regards,
Friedrich


I did that too. Here's a nicer picture of sample 'A' with the Rayleight and Raman peaks removed for clarity:
http://img82.imageshack.us/img82/5236/75322046sp2.jpg

The sample is an aqueous solution of microbially generated products and thus its exact composition is unknown.
We use a jobin horiba fluorometer (double monochromators, 5nm bandpass, signal was corrected for wavelength response and normalized with reference signal to account for excitation flucturations)




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