Fluorescence spectroscopy is a technique by which fluorescent substances can be identified and characterized. Fluorescence spectroscopy is usually performed with a fluorimeter. A fluorimeter is an instrument used to measure the intensity of fluorescence of a particular substance. The wavelengths for excitation and emission can be set.
Excitation and emission spectra are dust specific. Fluorescence spectroscopy can thus be used to identify substances.
The method can also be used for the quantitative determination of a component in a solution. This is usually called fluorometry. The method is sensitive and selective and therefore suitable for measuring various components in one and the same solution under certain conditions. Principe
A sample is irradiated with UV or visible light. The molecules to be analyzed absorb the photons from this light. Therefore, one or more electrons end up in an excited state; These are excited. Next, the electrons fall back to the ground state while transmitting photons. This emitted light (emission light) is measured.
The energy of the excitation light is greater than or equal to that of the emission light. The wavelength is thus inversely proportional; the excitation wavelength is therefore less than or equal to the emission wavelength. A substance can have multiple emission wavelengths at the same excitation wavelength.
Within a certain bandwidth, the measured fluorescence is directly proportional to the concentration of the solute. The quantitative determination of a substance in a sample is done by first measuring a calibration set with known concentrations. Instrumentation
A fluorimeter contains a lamp, a cuvette holder in which a cuvette is placed with the solution to be measured, monochromators and a detector. For samples in solution, glass or silica cuvettes are used.
A light source uses the (usually continuous) radiation of a deuterium or a xenon lamp of 150-800 W. The light first goes through a monochromator, which permits only one adjustable wavelength. Then the light reaches the cuvette with the sample.
The fluorescent light then diffuses in all directions. Perpendicular to the light path of the incoming light beam is a second, adjustable monochromator. Because it is perpendicular, only the fluorescence light is captured, not the light that has left the solution without being absorbed.
After the second monochromator, there is a photomultiplier or a photo diode detector. Using the exciting monochromator, the exciting spectrum can be determined and the fluorescent spectrum with the fluorescent monochromator. Also, a fluorimeter contains another linear amplifier and a recorder. Analysis
The signal can be processed both digitally and analogously. There is a large variation in systems and there must be different things.
Not all that the detector passes is a "real" signal, so a distinction must be made between signal and noise. This is possible by varying the filters and preventing light that has not passed through the filter to end up on the samples / detector. This must be noted because it is possible to have more noise than signal . There are already many faulty and inaccurate results (signal-to-noise ratio). obtained because the precise settings of the fluorimeter are not published.
The solvent, temperature and concentration are also important parameters that influence the intensity of fluorescence.
The sensitivity and detection limit must also be considered. Today, devices are available that can measure very low concentrations, so it's often not necessary to use large amounts of any expensive sample.
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