Fluorescence Quenching

After excitation by absorption of light, a chromophore can lose the absorbed energy, and thus return from the excited state to the ground state, either by emitting a photon (observed as fluorescence) or alternatively by nonradiative processes, such as exchange of heat with the solvent. For a particular chromophore, the quantum yield of the observed fluorescence depends on the relative rates of fluorescence emission and the competing nonradiative processes. Under conditions where such nonradiative processes are very slow, one photon is emitted for every photon absorbed, and the quantum yield (the number of photons emitted per number of photons absorbed) approaches one. In practice, the observed quantum yields are often much smaller than one because nonradiative processes compete efficiently with the emission of light. Such a decrease in quantum yield is called fluorescence quenching . The collision of a chromophore with certain molecules in the solution is an effective means to quench fluorescence; such a process is called collisional quenching.

 Radiationless transitions can occur by two major routes: internal conversion and intersystem crossing. In internal conversion, the first excited electronic singlet state is converted to the ground singlet state by exchange of vibrational energy (ie, heat) with the solvent. In intersystem crossing, the first excited singlet state is converted to the first excited triplet state. This triplet state is more stable than the corresponding singlet state and is very long-lived, because transitions between triplet and singlet states are nominally forbidden. They are accompanied either by heat exchange or phosphorescence (ie, emission of light with low energy. (

Intersystem crossing is triggered by magnetic fluctuations in the system. It is thus greatly facilitated by collisions of the excited molecules with molecules or ions that contain unpaired electrons or loosely held electron clouds (such as acrylamide or iodide anions). The efficiency of collisional

quenching depends on the frequency of the collisions, ie, on the concentration of the added quencher and exposure of the fluorophore to the solvent. The susceptibility to collisional quenching can be used to measure the degree of exposure to solvent of a fluorescing group (such as a tryptophan residue in a protein). Experimentally, the ratio of the fluorescence in the absence and in the presence of a quencher is measured as a function of the concentration of the quencher and plotted in a Stern–Volmer diagram. The Stern–Volmer quenching constant is derived from this plot. It is a measure of the exposure of the fluorophore to the quencher. Static quenchers do not obey the Stern–Volmer relationship. They form nonfluorescent complexes with the fluorophore, and the binding constant can be derived from the quenching experiments.