Resolution of two emission spectra for tryptophan using frequency-domain phase-modulation spectra

We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum.