04-01-2013, 10:38 AM
SPECTROFLUORIMETRY
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THEORY
Spectrophotometry is a method of analyzing involving how light interacts with the atoms (or molecules) in a sample of matter.
Visible light is only a small portion of the entire electromagnetic spectrum and it includes the colors commonly observed (red, yellow, green, blue and violet).
The visible spectrum consists of electromagnetic radiation of wavelengths ranging from 400 nm to nearly 800 nm.
INTRODUCTION
A large no. of substances which absorb U.V. or Visible light energy loss excess energy as heat through collision with neighboring atom or molecule.
However a few number of these substances loss only a part of this excess energy as heat and emit the remaining energy as electromagnetic radiation of longer wavelength than that of absorbed. This process of emitting radiation is known as LUMISNESCENCE.
Fluorescence
When a beam of light is incident on certain substances, they emit visible light or radiations. This phenomenon is known as Fluorescence
Phosphorescence
When light radiation is incident on certain substances, they emit light continuously even after the incident light is cut off. This type of delayed fluorescence is known as phosphorescence.
ADVANTAGES
SENSITIVITY : It is more sensitive as concentration is low as µg/ml or ng/ml.
PRECISION : Upto 1 % can be achieved.
SPECIFICITY : More specific than absorption method where absorption maxima may be same for two compounds.
RANGE OF APPLICATION : Even non fluorescent compounds can also be converted to fluorescent compounds by chemical compounds.
SOLVENT EFFECT
Solvents characteristics have important effects on fluroescent behavior of molecules.
Three main effects can be recognized:
A. Solvent Polarity
A polar solvent is preferred as the energy required for the π−π* is lowered.
B. Solvent Viscosity
More viscous solvents are preferred since collisional deactivation will be lowered at higher viscosities.
C. Heavy Atoms Effect
If solvents contain heavy atoms, fluorescence quantum efficiency will decrease and phosphorescence will increase.
Factors affecting Fluorescence Intensity.
Conjugation
Nature of Substituent group
Rigidity of structure
Effect of Temperature
Viscosity
Oxygen
Effect of Ph
Effect of Concentration