Fluorescence Quenching:

The fluorescence emission is quite sensitive to the presence of impurities and other species within the sample. These cause a decrease within the intensity of fluorescence emission. This decrease in the fluorescence intensity arising out of the interaction of the excited state of the fluorophore along with its surroundings is known as quenching.  For example, the quinine fluorescence is quenched through the presence of halide ions.  One of the mechanisms of quenching includes collisions among excited and ground state molecules leading to an increase in the amount of radiationless relaxation. It is known as self-quenching and it alters the ratio of excited molecules which relax through the fluorescence pathway. Because self-quenching depends on the rate at that collisions occur, it increases along with an increase in the concentration of the analyte. Because of self- quenching, the photoluminescence efficiency varies with the concentration.

The other mechanism which leads to the decrease in fluorescence intensity is known as self- absorption or the inner-cell effect. It is observed within the molecules in that the absorption band overlaps along with the wavelength of the emitted (fluoresced) photon. In such a condition some of the emitted photons are reabsorbed before they could escape the solution. This is known as self-absorption and same to the self-quenching, this is also important at high analyte concentrations. A significant portion of the excitation radiation is absorbed through the sample before it reaches the middle of the cuvette. Since self- absorption is a physical artifact, in principle it could be eliminated through the proper cell design and measurement conditions.