A new method for decay-associated fluorescence spectroscopy. Application to the tryptophan zwitterion.

We describe a new method for decay associated fluorescence spectroscopy using synchrotron radiation as the excitation pulse and a photon counting technique. This method is based on the determination of the difference between the barycenters of the exciting pulse and of the fluorescence response at several wavelengths. It is applicable to the case where individual decay times are independent of emission wavelength. Coupled to the analysis of the decay curve at only one emission wavelength, this method reduces the time devoted to the numerical analysis and avoids the spectral distortion due to the lamp profile. The results obtained by this method on indole, the tryptophan zwitterion, and N-acetyl-tryptophan are presented. Results are compared to those obtained by two other methods: Determination of the fluorescence decay parameters by deconvolution analysis at several emission wavelengths. Photon counting of the fluorescence spectrum emitted during a selected time window after the excitation pulse.