Chromatography Forum

Does anyone know the optimal excitation/emission wavelengths for fluorescence detection of OPA-derivatized amino acids such as glutamate with the highest sensitivity? From what I have read, there seems to be a range of excitation wavelengths used, ranging from 330 to 400 nm....

Mobile phase I am using is 0.1 M sodium phosphate + 25% acetonitrile, pH=6.75, with a 80 x 3 mm C18 column and a Perseptive Biosystems 304 fluorescence detector.

Thanks,
Foster

It is somewhat hardware dependent because light sources and monochromator design affect the energy distribution and how it overlaps the real excitation spectrum of the derivative. For most detectors, 330 to 340 nm is the best range. You can run a couple quick runs at 5 nm intervals to home in on your best wavelength. I like the scanning capability of the Agilent FLD, too bad that feature is not more common.