THEORY

Fluorescence is a two-step process that requires an initial absorption of light followed by emission. Fluorescence spectroscopy is an electronic spectroscopic method related to ultraviolet–visible–near infrared (UV–Vis–NIR) absorption spectroscopy. It is also a background-free method that involves light emitted from the sample in all directions, as is the case with Raman spectroscopy. The initial absorption of a photon by a molecule in the sample promotes an electron to an excited state. The excited electron returns to the ground electronic state by emitting a photon. If the emission arises from an “allowed” transition that typically has a short lifetime between 1 ns and 10 ns, then it is called fluorescence. If the emission arises from a “forbidden” transition that typically has a long lifetime between 1 ms and 1 s, then it is called phosphorescence. Under similar conditions phosphorescence usually is less intense than fluorescence. This general chapter discusses fluorescence spectroscopy, but many points raised here also apply to phosphorescence. The basic concepts behind fluorescence spectroscopy have been well established, but its applications and standardization are still expanding and progressing, making it a developing rather than a mature method.