Chromatography Forum

I was trying to develop a method for the separation of an enzymatic reaction mixture, where the product was a fluorescent molecule (ex 362, em 448).

To my surprise, all of the buffers I tried seemed to fluoresce -- only water/MeOH seemed to be transparent.

In my silliness, I wasted a lot of time trying different buffers -- tris, phosphate, carbonate, etc. Glycine was the worst of all, completely saturating my detector at 10mM.

I finally gave up trying to achieve a separation, and am now not using a column at all -- just using the HPLC for its fluorescence detector since I don't have a fluorimeter.

The past is past, as they say, but where can I read more about the fluorescent properties of buffers, so that I don't make the same mistake again? Anything similar to UV cutoff tables available?

That's actually rather surprising.

What excitation/emission wavelengths were you monitoring? Depending on the design of the system, if the wavelengths are too close together you might simply be seeing scattered light.

I know, the results still confuse me. To clarify, the amount of fluorescence was relatively small when I used phosphate and carbonate buffers (compared to when I used glycine), but it would still interfere with my analysis.

I was using the excitation and emission maxima of the fluorescent product molecule, i.e. ex 362, em 448nm; the detector was a Waters 2475 Multi Channel fluorescence detector.

Glycine doesn´t fluoresce, especially not at 362nm, where it doesn´t absorb. It´s very easy to distinguish fluorescence from scattering: If you change the ex wavelength the em max changes if it is scattering, not if it is fluorescence (I am talking about position not intensity).
Part of your detection could be overtones "leaking" through, especially if a simple phosphate gave a sconsiderable signal. Overtones also move with the ex wavelength.
If it´s a real fluorescence your stuff is all crudded up.