ISSN:
1573-4994
Keywords:
Time-resolved
;
fluorescence spectroscopy
;
fluorescence lifetimes
;
Monte Carlo simulation
;
global analysis
Source:
Springer Online Journal Archives 1860-2000
Topics:
Physics
Notes:
Abstract In time-resolved fluorescence spectroscopy, the resolution of fluorescence species becomes increasingly difficult as their respective lifetimes get closer. For a biexponential decay, a factor of 1.4 between the two decay times is commonly accepted as the practical resolution limit. The goal of the present contribution is to characterize the fluorescence probe 5-carboxyfluorescein using frequency-domain time-resolved fluorescence spectroscopy (FD-TRFS). To resolve the different prototropic forms of this probe, the limit above had to be overcome. For this purpose, the standard global analysis method was used, and special emphasis was put on the errors associated with the recovered parameters. In particular, a Monte Carlo simulation was performed to estimate these errors and the results of this analysis were compared with those delivered by software packages widely used in the field. The lifetimes of the trianionic and dianionic forms of 5-carboxyfluorescein were 4.01 ± 0.06 and 3.03 ± 0.09 ns, respectively, and the pK a for this acid–base equilibrium was determined to be 6.9 ± 0.3.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1009466110802
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