Fluorescence of unknown samples

To understand the excitation wavelength, first record a spectrum (UV-Vis, full range as available).  From the spectrum, you could get the absorption maxima, to use as excitation wavelength.  You may prefer to test with the lowest wavelength (highest energy) maxima as the first try (if there are many absorption peaks).  Because, excitation energy is always higher compared to emission energy. Then, scan the emission spectrum. 

To determine the emission, the wavelength of maximum absorption is determined and the fluorochrome is excited at that wavelength.   A monochromator is used to scan the fluorescence emission intensity over the emission wavelengths. The relative intensity of the fluorescence is measured at the various wavelengths to plot the emission spectrum.

To determine the excitation, monitor fluorescence emission at the wavelength of maximum intensity while the fluorophore is excited through a group consecutive wavelengths. The emission maximum is chosen and only emission light at that wavelength is allowed to pass to the detector.