Chromatography Forum

Hello,

if I do not find excitation and emission wavelength in the literature and I would like to set up to 4 wavelength pairs for monitoring in order to detect peaks of compounds which I can spectrally examine in detail.

Which wavelength pairs would you recommend?

Thank you very much

Florian

Most fluorescence detectors will have a scanning option both for excitation and emission.
A poor man's way of collecting the excitation spectrum is to collect is UV-Vis absorption spectrum. The absorbance peak is usually the excitation peak. Secondly, emission wavelengths can be guessed by illuminating the sample under UV, note the color of fluorescence and use wavelength vs. color tables.

Quite agreed with M. Farooq, would add that the emission wavelength will always be less energetic (higher number) than the excitation wavelength...so direct your search toward emission wavelengths accordingly.

Best Wishes!

As an aside, did you try searching with ScienceDirect for the fluorescence conditions, Florian?

Based on my experience, if there are several maxima in the adsorption spectrum, then the one with the longest wavelength is going to be the excitation maximum that results in the highest fluorescence yield. This is true even if the molar extinction coefficient at that maximum is lower than for the maxima at shorter wavelengths. Of course, this puts the excitation light at a wavelength that may be uncomfortably close to the best wavelength for monitoring emission. What you can get away with depends on your slit width, but I'd pick wavelengths for excitation and emission that are at least 20-30 nm apart.

Florian, you did not mention the structures. Dr. Alpert raised a very useful issue; Often you will see that higher excitation wavelength usually gives the highest fluorescence yield. This is an artefact of the lamp in any fluorescence based measurements. Quinine is one good example of what is mentioned by Dr. Alpert; the absorption maximum of quinine is at 250 nm and another smaller peak is found around 350 nm. However, if emission spectra are collected for quinine, the 350 nm excitation apparently gives higher fluorescence yield than 250 nm because the Xenon lamp output is higher and more steady at longer wavelengths. In theory, 250 nm should give the highest emission intensity, and in this is indeed true when excitation spectra are corrected for wavelength dependency.

Hello,

thank you very much for all you answers:

- in literature, I found only fluorescence methods with a derivatization step before injecting
- this might be due to the lack of fluorescence at all or due to the low sensitivity for the particular application
- nevertheless, I intend to verify the possibilities
- my compound is lidocaine HCl, a simple aromatic amide w/o a significant UV max; this might be the reason for the absence of methods in the literature

Regards

Florian