Fluorescence spectroscopy
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Fluorescence spectroscopy or fluorometry is a type of electromagnetic spectroscopy used for analyzing fluorescent spectra. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. A complimentary technique is absorption spectroscopy.
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Theory
Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic states and vibrational states. Generally, the species being examined will have a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. Each of these electronic states has various vibrational states.
Photons of light are small "packets" of energy, each with an energy proportional to its frequency; photons of high frequency light have higher energy than those of low frequency light. These can be absorbed by molecules, with the molecule gaining the energy of the photon, or emitted by molecules, with the photon carrying some of the energy of the molecule away.
In fluorescence spectroscopy, the species is first excited, by absorbing a photon of light, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state.
The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of the vibrational levels of this ground state, the photons will have different energies, and thus frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescent spectroscopy, the structure of these different vibrational levels can be determined.
Fluorometer
A fluorometer measures fluorescence.
Types
- Filter fluorometers use filters to isolate the incident light and fluorescent light.
- Spectrofluorometers use diffraction grating monochromators to isolate the incident light and fluorescent light. In these devices, the spectrum can consist of the intensity of emitted light as a function of the wavelength of either the incident light (excitation spectrum) or the emitted light, or both.
Analysis of data
There is a relationship between concentration and fluorescence intensity.
Applications
It used in, among others, biochemical, medical, and chemical research fields, for analysing organic compounds. There has been report of its use in differentiating malign skin tumours from benign.