Differential-wavelength deconvolution of time-resolved fluorescence intensities. A new method for the analysis of...

We describe a new procedure for the analysis of time-resolved decays of fluorescence intensity observed for excited-state reactions. This procedure is particularly valuable because it simplifies both determination of the rate constants of excited-state reactions and calculation of the emission spectra of the unreacted and the reacted species. These advantages are obtained without additional data acquisition, by the further analysis of data which would already be collected. As is usual for an excited-state process, time-resolved decays of fluorescence intensity are collected at wavelengths across the emission spectrum. Generally, one derives analytical expressions for the impulse response by deconvolution using the time profile of the exciting pulse. We suggest that in addition to the procedure just described, the response observed at longer wavelengths be deconvolved using the response observed on the blue side of the emission. Typically, this emission is from the initially excited state of the fluorophore (F). Then, the derived decay time is the decay rate of the reacted species (R). In addition, spectral overlap of the F and R states is revealed quantitatively as an apparent zero-decay-time component in the derived impulse response function. This is because the population of the initially excited state is considered to be the excitation function. This spectral overlap component is easily quantified, and allows the emission spectra of the F and R states to be calculated. This deconvolution procedure requires one favorable circumstance, which is the ability to choose a wavelength at which only the initially excited state is emitting. We tested our procedure on the excited state protonation of acridine and the excited-state deprotonation of 2-naphthol. The former reaction is essentially irreversible, whereas depending upon pH the dissociation of 2-naphthol can be reversible. We demonstrated that this procedure, which we call differential-wavelength deconvolution, revealed the individual spectra and simplified determination of the kinetic constants from the time-resolved decays. Without differential-wavelength deconvolution considerably more complex methods are required. We expect this procedure to greatly facilitate the use of pulse fluorometry methods in the analysis of excited-state processes.