Chromatography Forum

Dear all,

I am not familiar with fluorescence detection; my question is:

when I have a non-fluorescent matrix, a non-fluorescent sample solvent and a fluorescent analyte: does only an analyte peak appear in the chromatogram and no other peak (like the "water hole") ?

Thank you for your feedback

Kind regards

Florian

You've got it exactly right.

Best Regards

Sometimes one gets negative peaks via changes in light scattering, not in fluorescnce. Thus, negative peaks are possible even though the mobile phase does not fluoresce.

Scattered light by the mobile phase is actually not something one should worry too much about.
Unless the mobile phase comprises large molecules, particulate matter or tiny air bubbles. Such factors should be eliminated regardless of the detection mode (i.e. whether it is absorbance or fluorescence detection).
Even if there is some light scattering going on in the flow cell, it should not represent a big issue, because the scattered light has the same wavelength as the excitation ditto, while the light sent to/allowed to reach the PMT will have another/longer wavelength in fluorescence detection.
This is the theoretical part of the matter. In practise, some light cross-contamination can not be dismissed 100 %, but it shouldn’t cause large baseline disturbances.

Best Regards

Theorethically you should only see the fluorescent analyte.
Practically, depending on the wavelaengths, there is a good chance to see a lot of other stuff. We have observed fuorescence signals from starch and from other excipients. Plastic syringes and syringe filters can have fluorescent impurities, as can glass vials.


alex

It is all a matter of sensitivity with which one is working. At 100 units/page with 9000 the max. possible intensity units, one has enough scattering from most HPLC mobile phases to get a negative peak with H2O. Even H2O has a good scattering, havn´t any of you used the Raman peak of H2O for checking your instrument?? That is not scattering at the same wavelength as incidence light.
Furthermore, especially novices tend to underestimate the presence of tails of peaks reaching from excitation into emission. Worth than this are the wave length overtones which can produce light at emission wavelength.

Hi Florian,

Was your question a hypothetical one, or do you have a real-life experience you’d like to discus?
If the latter is the case, it might be useful if you elaborated a bit by describing the matter as well as sharing some details about the mobile phase, Ex/Em wavelengths and the nature of the matrix.
As you can see there are many things one can keep speculating about.

Best Regards