Molecular absorption is a process in which light energy with certain wavelength is absorbed by a molecule during the interaction with light, promoting the molecule from ground state to higher energy excited states. The technique has found great application in organic chemistry, natural product chemistry, and fluorimetric reagent analysis. For absorption wavelengths of 250 nm or greater, fluorimetry is the method of choice for direct molecular quantitation at low concentrations. These systems can have one to four orders of magnitude better sensitivities than corresponding absorption techniques, though limitations of applicability occur since many chromophores do not undergo fluorescent relaxation. These instruments are inherently more sensitive than those based on absorbance since at low sample concentrations it is much easier to amplify electronically a small radiant signal superimposed on a dark background than measure a small difference in intensity due to absorption measured on a bright background. Observation of fluorescence occurs at 90° to the angle of source illumination, and an emission monochromator may precede the detector. The instrumental components of a spectrofluorimeter are very similar to those for the molecular absorbance technique described previously. Where K is dependent on the quantum yield, A is absorbance, and P 0 is the incident radiation power. The fluorescent intensity F is therefore a function of the chemical structure of chromophores and molecular interactions and, for dilute solutions with A <0.05, can be written as Ideally, the quantum yield would have a value of unity, but molecular interactions such as intermolecular collisions or intramolecular rotations in the sample matrix can reduce this dramatically. Competition between nonradiative and fluorescent energy loss occurs, reducing the quantum yield, which represents the ratio of energy released by radiative processes compared with total energy absorption. This phenomenon is often of very weak intensity and is not as common as fluorescence, thereby limiting its analytical potential. Phosphorescence occurs from a forbidden excited triplet state after inter-system crossing and may take a period of seconds. The closely related processes of fluorescence and phosphorescence are summarized in Fig. This is due to nonradiative energy losses by molecular vibration in the electronically excited state. Polyatomic molecules often reemit at wavelengths longer than those absorbed, producing a spectral feature known as a Stokes shift. Resonance fluorescence occurs when the absorbed radiation is reemitted at the same wavelength. Fluorescence is said to occur when the system relaxes to a lower energy state by the release of radiation within 10 −8 sec after absorption in a process that ceases in less than 10 −6 sec. Molecular absorption of ultraviolet or visible radiation results from a resonant effect between an incoming photon and the electrons of a chromophore, elevating the valence electronic configuration to a higher energy state.
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