Molecules of several materials under certain conditions absorb radiant energy and after an interval of 10-7or 10-8 seconds, the excited electrons return to a lower energy level, and the energy being emitted as visible light.
This radiation is termed fluorescence. Under the conditions of biological and biochemical analysis, this energy has a greater wavelength that is lower energy than the exciting radiation. In Flourimetry, there is absorption and emission.
An instrument that is designed to measure the intensity of fluorescence is termed Fluorimeter.
Mode of Operation of the Instrument
In photoelectric Fluorimeters, a beam of ultraviolet light (< 400nm) of regulated identical intensity from a mercury vapour lamp passes through a diaphragm to fall on either an incident wavelength selector or an exciter filter. This filter or selector transmits only these UV wavelengths that are close to the absorption maximum of the fluorescent material to be approximated, and cuts off all other wavelength including those above 400nm. The UV wavelengths emerging from this selector or filter are focused via a lens system to the test solution in a transparent cuvettes or instance cell. The fluorescent solute in the solution releases long wavelengths of observable fluorescent light on absorbing UV rays. To avoid unabsorbed and undeflected UV rays being transmitted through the solution, long wavelength fluorescent rays, emerging from the example cell at only an angle to the path of the exciting beam, are made to pass through an emitted wavelength selector or a barrier filter. This filter cuts off any UV ray which might have been deflected from the sample cell, and transmits simply the precise long wavelengths of fluorescent rays to a lens system. The system focuses the fluorescent wavelengths to a photo cell that as a result generates a current in direct proportion to the intensity of fluorescence. This current pulse is enlarged and computed.
Fig: A Simple Fluorimeter design
When there is a reduction in the intensity of fluorescent due to the explicit consequences of constituents of the solution itself quenching happens. For instance, phosphate buffers quench fluorescence. When the fluorescent material is conscientious for the absorption of fluorescence, it is recognized as self-quenching. Quenching can as well be reasoned via the move of energy via the collision of the excited molecules of the fluorescent material through molecules of solvents or solute.
For extremely low concentration of materials, the sensitivity of fluorescence is proportional to concentration, provided the exciting energy continues constant. At higher concentrations of solutions, this linear association tends to be demolished.
Fluorescence is sensitive to these variables as solvent, pH, temperature, impurities and the presence of ions such as CNS-, I-, Br-, Cl-, which have all marked quenching consequence, for example Cl- quenches the fluorescence of quinine.
Nevertheless, through more sensitive equipment, fluorimetric methods can be more sensitive than spectrophotometric techniques via an order of magnitude of at least 103 to 104.
The instrument can be utilized for the examination of:
1. Thiamine, vit B1 and Riboflavin
2. Ruthenium in the presence of other platinum metals.
3. Quinine in dilute sulphuric acid solution.
4. Riboflavin (vitamin B2) using their native fluorescence.
5. Histamine and histidine by reacting with o-phthalaldehyde.
6. Magnesium by reacting with 8-hydroxyquinoline and sulphuric acid.
7. Amphetamines by reacting with formaldehyde and acetyl acetone.
Precautions for usage:
For practical purposes, the following precautions must be observed when using Fluorimeters:
1. The solvent used must be non-fluorescent and must not absorb radiation in the spectral regions used in the particular assay.
2. Pyrex or quartz cuvettes must be employed since of the slight fluorescence or ordinary glass.
3. Filters should be checked for contribution to blank readings.
4. Stopcock and other greases provide increase to difficulties and should be avoided.
5. pH and temperature must be watchfully controlled.
6. Solutions must be free from gas bubbles, suspended solids and turbidity.
7. Exciting radiant energy sometimes completed gradually transforms the colour or intensity of fluorescence reading and consequently must be made quickly.
8. Internal standards should be employed to detect quenching or alteration in colour of fluorescence - comparison should be made between an internal and external solution.
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