Fluorimetry

Introduction:

Fluorescence is based on the ability of certain molecules to absorb energy in the form of light and become excited.  On returning to the ground state, energy  is emitted in the form of light but because energy is also absorbed in the transition, the wavelength of the emitted light is always longer than the wavelength of the incident light.

The difference between fluorescence and phosphorescence is the time which light energy is emitted after exposure to the incident light beam.

Fluorescence intensity increases greatly for a small increase in concentration of the molecule.  The resulting high sensitivity is approximately ten thousand times greater than any ordinary spectrophotometry and is only linearly related to concentration in dilution solution. In addition to concentration, various other factors affect the linearity and intensity of fluorescence.

1. Temperature:  The constant K decreases with increasing temperature because of the increased frequency of molecular collisions.  This has the effect of lowering the fluorescence intensity.

2.  Solvent:  Energy transitions may by altered by the nature of the solvent.  Polar solvents tend to enhance fluorescence intensity.

3.  Influence of Other Substances: Decreased fluorescence occurs in solvents consisting of or containing particular elements such as halogens.

4.  pH:  The fluorescence of an aromatic substance is affected by pH, mainly as a result of alterations in charge and resonance forms of the chromophere.

5.  Oxygen:  Oxygen quenching decreases fluorescence and requires that solutions used in fluorimetry be deareted before analysis is performed.
6.  Path length of the Cell:  Fluorescence decreases along the length of the cell.

What type of molecules Fluoresce?
Compounds with conjugated double bonds without the presence of electron withdrawing groups such as COOH or -NO2 will fluoresce.  If electron donating groups such as _NH@ or -OH are present, they reduce fluorescence.
     

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