Fluorescence Quantum Yield Measurements.

Four molecular fluorescence parameters describe the behaviour of a fluorescent molecule in very dilute (~ 10^-6 M) solution: the fluorescence spectrum $F_M\left(\overline{v}\right)$ ;the fluorescence polarization P_M ;the radiative transition probability k_FM ; andthe radiationless transition probability k_IM .These parameters and their temperature and solvent dependence are those of primary interest to the photophysicist and photochemist. $F_M\left(\overline{v}\right)$ and P_M can be determined directly, but k_FM and k_IM can only be found indirectly from measurements of the secondary parameters,the fluorescence lifetime τ_M , andthe fluorescence quantum efficiency q_FM ,where k_FM =q_FM/τ_M and k_IM =(1-q_FM ) τ_M. The real fluorescence parameters $F\left(\overline{v}\right)$ , τ and ϕ_F of more concentrated (c > 10^-5 M) solutions usually differ from the molecular parameters $F_M\left(\overline{v}\right)$ , τ_M and q_FM due to concentration (self) quenching, so that τ > τ_M and ϕ_F < q_FM. The concentration quenching is due to excimer formation and dissociation (rates k_DMc and k_MD , respectively) and it is often accompanied by the appearance of an excimer fluorescence spectrum $F_D\left(\overline{v}\right)$ in addition to $F_M\left(\overline{v}\right)$ , so that $F\left(\overline{v}\right)$ has two components. The excimer fluorescence parameters $F_D\left(\overline{v}\right)$ , P_D , k_FD and k_ID together with k_DM and k_MD , and their solvent and temperature dependence, are also of primary scientific interest. The observed (technical) fluorescence parameters $F^T\left(\overline{v}\right)$ , τ^T and ${\varphi }_F^T$ in more concentrated solutions usually differ from the real parameters $F\left(\overline{v}\right)$ , τ and ϕ_F , due to the effects of self-absorption and secondary fluorescence. The technical parameters also depend on the optical geometry and the excitation wavelength. The problems of determining the real parameters from the observed, and the molecular parameters from the real, will be discussed. Methods are available for the accurate determination of $F^T\left(\overline{v}\right)$ and τ^T . The usual method of determining ${\varphi }_F^T$ involves comparison with a reference solution R, although a few calorimetric and other absolute determinations have been made. For two solutions excited under identical conditions and observed at normal incidence $\frac{{\varphi }_F^T}{{\varphi }_{FR}^T}=\frac{n^2\int F^T\left(\overline{v}\right)d\overline{v}}{n_R^2\int F_R^T\left(\overline{v}\right)d\overline{v}}$ where n is the solvent refractive index. Two reference solution standards have been proposed, quinine sulphate in N H₂SO₄ which has no self-absorption, and 9,10-diphenylanthracene in cyclohexane which has no self-quenching. The relative merits of these solutions will be discussed, and possible candidates for an "ideal" fluorescence standard with no self-absorption and no self-quenching will be considered.