Fluorescence Quenching and Stern-Volmer Plot: Estimation of the Quenching Constant from Stern-Volmer Plot
We know that there are many “intrinsic” pathways of de-excitation of an excited flurophore molecule. In addition, processes involving interaction of the excited molecule with another molecule (called quencher) competing with the intrinsic de-excitation can be observed. The intermolecular photophysical processes in the presence of quenchers change the fluorescence characteristics (intensity, fluorescence quantum yield, lifetime) as a result of the competition between the intrinsic de-excitation and these intermolecular processes. This process of decrease or loss of the fluorescence intensity in the presence of quenchers is known as “quenching of fluorescence”. A number of molecular interaction processes such as excited state reactions, ground-state complex formation, molecular rearrangements, energy transfer, etc. can decrease the fluorescence intensity of a fluorophore. Analysis of the quenching phenomenon provides qualitative and quantitative information on the surroundings of a fluorescent molecule. Therefore, the fluorescence quenching phenomenon has been exploited for many applications. For example, fluorescence quenching of tryptophan residues in proteins has been a very useful method to detect conformational changes of proteins in different conditions and protein-ligand interactions.
Quenching requires an interaction along with appropriate electronic orbital overlapping between a fluorophore and the quencher. Based on the nature of interactions, fluorescence quenching is broadly classified into two types: dynamic (or collisional) quenching and static (or complex formation) quenching. Dynamic quenching requires a collision between the quencher and fluorophore while the latter is in its excited state. On the other hand, the static quenching occurs because of a nonfluorescent ground state complex formation between the fluorophore and quencher. Shifts from the fluorophore absorption spectrum upon addition of the quencher provide evidence of such complex formation. In dynamic quenching, no permanent change in the flurophore occurs. The quencher diffuses to the fluorophore during the life time of excitation state. The fluorophore returns to the ground state without emission of photon after collision with quencher. This is a time-dependent process. Common quenchers include O2, I-, Cs+, pyridinium ion, acrylamide, hydrogen peroxide, nitric oxide, nitroxides, BrO4-, etc. Oxygen molecule is a well known collisional quencher that quenches almost all the fluorophores. The paramagnetic oxygen molecule causes the fluorophores to undergo intersystem crossing, i.e. crossing from the first singlet excited state S1 to the first triplet state T1. This leads to the decrease in the flouroscence intensity. Spin–orbit coupling favors intersystem crossing. The efficiency of this coupling varies with the fourth power of the atomic number, which explains why fluorescence intensity gets quenched by the presence of a heavy atoms, like bromine, iodine, silver, etc. Aromatic and aliphatic amines act as efficient quenchers for most of the unsubstituted hydrocarbons. The fluorophores accept electrons from the amines forming excited charge-transfer state (exciplex).