Fluorescence Spectroscopy

aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. 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 complementary technique is absorption spectroscopy. Devices that measure fluorescence are called fluorometers or fluorimeters.


Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. Generally, the species being examined has a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. Within each of these electronic states are various vibrational states.

In fluorescence spectroscopy, the species is first excited, by absorbing a photon, 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 several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined.

In a typical experiment, the different wavelengths of fluorescent light emitted by a sample are measured using a monochromator, holding the excitation light at a constant wavelength. This is called an emission spectrum. An excitation spectrum is the opposite, whereby the emission light is held at a constant wavelength, and the excitation light is scanned through many different wavelengths (via a monochromator). An Emission Map is measured by recording a number of different emission spectra created from different excitation wavelengths, and combining them all together. This is a three dimensional data set, intensity of emission as a function of excitation and emission wavelengths, and is typically depicted as a contour map.

Perkin Elmer LS 55 FluorescencePerkin Elmer LS 55 Fluorescence

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