How are the excitation and emission wave lengths determined for fluorescence detection?

Hmmm. OK, I'll bite. There are 3 closely related procedures to determine EX/EM for your HPLC method:
1). Simplest - look it up for the molecule in question. I'm not being flippant here, if the molecule is a known fluorescent molecule, you should be able to find a method. If it is similar to another molecule, you may get lucky with that molecules wavelengths. If you can't look up the molecule, how do you know it is fluorescent?
2). Significantly more difficult - use a scanning spectrophotometer, with a pure sample in a cuvette, to search for EX and EM. For a “typical” organic molecule, you can expect some absorption somewhere in the UV (you could also scan a cuvette sample with an absorption spectrometer to find the absorption max). Once you have a “good” absobtion wavelength to use as an excitation wavelength, you can scan for the “best” emission wavelength you can get with it. Then use the “best” emission wavelength to find the “best” excitation wavelength. These wavelengths may be the lambda max, and there may be more than one “good” EX/EM wavelength. You will have to check that the wavelengths you've discovered are actually the fluorescence and not the Raman emission. And you will have to check the solvent of choice to be sure you're not looking at it's fluorescence. This was done by somebody for every known fluorescent compound discovered, whether you want to do this work yourself depends on your needs.
3). Insanely more difficult - same as option 2, just use the HPLC detector. I suggest a good nights sleep, and a good supply of your favorite caffeinated beverage be on hand.

Hi.

Just to clarify, is there a way to do this with a spectrophotometer that isn't a flourescent spectrophotometer? Or is "scanning" chemist lingo
for "flourescent?" I guess you could find
the EX via regular absorbance, but how do you get
EM?

thanks!

Jon

Hi,

I have a Fluorescence detector that perform scans, Following R.C. coments my best EX wavelength is usually my UV max absorption ?

So I can -Theorecally- use it as a UV detector?

Thanks

Anonymous,

flourescence detectors are not always constructed
with the same light path used by UV detectors.
The excitation light must be reflected 90degrees,
for example, to be detected by the emission
detector. This reduces background, since only
reflected or emitted light can be detected. So
with such a detector, if you set EX and EM
to the same wavelength, you still get almost no light
transmitted unless it is reflected 90 degrees
by the sample. This means that your "UV detector"
would read extremely high absorbance all the time.

But of course in theory, you could add some sort
of light-scattering particles to your flow post-column.
Let me know how it works ;)

Jon

Jon H,
what sort of fluorescence detector do you have? With the Hitachi fluo spectrometer I use I get plenty of excitation wavelength through, simply via Rayleigh scattering.
To Anon Feb. 24: If your substance fluoresces your excitation spectrum will be parallel to the absorption spectrum (you could calibrate it and get at the absorption...). The problem is the mentioned scattering, it will limit the useful wavelengths.
To the original question: What R.C. suggests in his 2) is actually so simple that most, if not all, fluorescence spectrometers do it automatically for you.

HW Mueller,

That's neat! Did you get enough scattering to use
it as an absorbance detector? I was curious about
this myself but was cautioned against relying on
a fluorescence detector as our sole UV detector.

perhaps I am too gullible ;)

Since there is a lot of incident light, but only little absorption, I strongly doubt that the scattering is modulated "visibly" by the absorption. Even if the absorption did influence the scattering, how could you record it? You would have to change the ex and em wavelength together. You see Rayleigh scattering only when em is close to ex wavelength (the better the monochromater the narrower the scattering "peak"). This also holds for scattering overtones (double wavelength,etc.).