| Abstract
| - The photophysical behavior of a new fluorescein derivative has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength, as well as total bufferconcentration, on both the absorbance and fluorescence has been investigated. The apparent acidity constantof the dye determined by absorbance is almost independent of the added buffer and salt concentrations. Asemiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-stateproton-exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer,inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces,collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis,yielding the following values of the rate constants describing excited-state dynamics: k01 = 1.29 × 1010 s-1,k02 = 4.21 × 108 s-1, k21 ≈ 3 × 106 M-1 s-1,= 6.40 × 108 M-1 s-1, and= 2.61 × 107 M-1 s-1. Thedecay rate constant values of k01, k21, and, along with the low molar absorption coefficient of the neutralform, mean that coupled decays are practically monoexponential at buffer concentrations higher than 0.02 Mand any pH. Thus, the pH and buffer concentration can modulate the main lifetime of the dye.
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