Fluorescence spectroscopy is a method of determining the composition of a sample. It excites a sample with electromagnetic radiation, causing it to emit characteristic radiation. This is a non-destructive method of analysing sample composition. Instruments used to perform fluorescence spectroscopy are known as fluorometers. Most commonly the sample is excited using ultraviolet light and the emitted light is in the visible spectrum. X-ray fluorometers are also used for elemental analysis. Fluorescence spectroscopy is used in many fields including biochemical, medical and water quality.
Fluorescence is a process by which substances absorb and re-emit radiation. Molecules have different energy levels determined by the position of electrons within shells and vibrational states. When a molecule is excited by absorbing a photon of electromagnetic radiation, it stores this energy momentarily by jumping to a higher energy state, with an electron moving to a shell further from the nucleus. Due to the electrical attraction between the electron and the nucleus, the electron quickly falls back to the lower energy state and the energy is released as a photon. These released photons are the re-emitted radiation — and they have a frequency which is characteristic of the unique vibrational state of the molecule.
Fluorescence spectroscopy may be carried out by filter fluorometers or spectrofluorometers. Filter fluorometers use filters to separate the excitation radiation from the emitted fluorescent light. Spectrofluorometers use a diffraction grating monochromator to separate the excitation and fluorescent light. In both cases, the excitation source emits radiation with a broad spectrum and the frequency band is then narrowed by a filter or monochromator before reaching the sample. The frequency of the fluorescent light is then determined by passing it through another filter or monochromator and photodetector. The sample will fluoresce in all directions, independent of the angle of incident excitation radiation. The detector is therefore normally positioned perpendicular to the excitation beam in order to minimize transmitted or reflected light reaching the detector.
The excitation light source may be a mercury-vapor lamp, xenon arc, LED, or laser. An advantage of using a laser is that the very narrow frequency band removes the need for an excitation monochromator or filter. However, it makes adjusting the frequency much more difficult and somewhat limited. A monochromator with a mechanically scannable diffraction grating and polarization filters can be adjusted to scan across a wide range of wavelengths.
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