Experimental Design in Global Compartmental Analysis of Reversible Intramolecular Two-State Excited-State Processes with Added Quencher

Although reversible intramolecular two-state excited-state processes without a priori information are unidentifiable, bounds on the excited-state rate constants k ij can be specified when a quencher is used [Van Dommelen et al. J. Phys. Chem. 1993, 97, 11738]. These limits can be obtained by a scanning procedure consisting of a series of global compartmental analyses of a fluorescence decay surface in which one of the rate constants k ij is held fixed at different preset values. The theory requires that the rate constants of quenching ( k Q1, k Q2 ) for the two excited species have different values. In the present paper, computer-generated fluorescence decay surfaces are used to investigate the criteria under which reliable estimates of the bounds on the rate constants k ij can be obtained. If the values of k Q1 and k Q2 are substantially different, reliable estimates are obtained. If k Q1 and k Q2 are nearly equal in value, the quality of the estimates of the bounds depends on the combinations of the values of the rate constants k ij . It may happen that no reliable limits for the rate constants can be obtained so that another quencher is required. A test procedure based on a limited number of decay traces is described which allows one to assess the appropriateness of the quencher for the given excited-state process.