Fluorimetry –Theoretical principles – Instrumental Methods of Analysis B. Pharma 7th Semester

Fluorimetry –Theoretical principles



At the end of the session, the student will be able to

       Identify Fluorescence as an important phenomenon for the determination of many pharmaceutical substances

       Discuss the phenomenon of photoluminescence

       Explain the factors affecting Fluorescence

       Discuss the methods used for converting non-fluorescent substances to fluorescent


       Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon.

It is of 2 types.



Ø  Chemiluminescence               


       When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence.

       Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off.

       The substances showing this phenomenon are known as flourescent substances.


       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 called phosphorescence.

       Substances showing phosphorescence are phosphorescent substances.

Theory of fluorescence and phosphorescence

       A molecular electronic state in which all of the electrons are paired is called singlet state.

       In a singlet state molecules are diamagnetic.

       Most of the molecules in their ground state are paired.

       When such a molecule absorbs uv/visible radiation, one or more of the paired electrons raised to an excited singlet state /excited triplet state.


From the excited singlet state one of the following phenomenon occurs:



       Radiation-less  processes

       Vibration relaxation

       Internal conversion

       External conversion

       Intersystem crossing


Jablonski energy diagram:



       Fluorescence is most commonly observed in compounds containing aromatic functional groups with low energy.

       Most un-substituted aromatic hydrocarbons show fluorescence – quantum efficiency increases with the number of rings and degree of condensation.

       Simple heterocyclic do not exhibit fluorescence.

       The n – π*singlet quickly converts to the n – π* triplet and prevents fluorescence.

       Fusion of heterocyclic nucleus to
benzene ring increases fluorescence.

       Substitution on the benzene ring shifts wavelength of absorbance maxima and corresponding changes in fluorescence peaks

        Fluorescence decreases with increasing atomic number of the halogen.

        Substitution of carboxylic acid or carboxylic group on aromatic ring inhibits fluorescence.

Structure rigidity:


       Fluorescence is favoured in molecules with structural rigidity.

       Organic chelating agent’s complexed with metal ion increases fluorescence.

Factors affecting fluorescence intensity:

       Nature of molecule

       Nature of substituent

       Effect of concentration

       Adsorption, Light

       Oxygen, pH


       Temp . &viscosity

       Intensity of incident light

       Path length


       Decrease in fluorescence intensity due to specific effects of constituents of the solution.

       Due to concentration, pH, pressure of chemical substances, temperature, viscosity, etc.

Types of quenching:


       Chemical quenching

       Static quenching

       Collision quenching

Self-quenching or concentration quenching:


Deviations at higher concentrations can be   attributed to self-quenching or self-absorption.

Chemical quenching:

       Here decrease in fluorescence intensity due to the factors like change in pH, presence of oxygen, halides & heavy metals.

       pH- aniline at pH 5-13 gives fluorescence but at pH <5  it does not exhibit fluorescence.

       Halides like chloride, bromide, iodide & electron withdrawing groups like NO2,COOH etc. lead to quenching.

       Heavy metals lead to quenching, because of collisions of triplet ground state.

Static quenching:

       This occurs due to complex formation.

     e.g.. caffeine reduces the fluorescence of riboflavin by complex formation.

Collisional quenching

       It reduces fluorescence by collision. Where no. of collisions increased hence quenching takes place.

Types of fluorescence:

Fluorescence mainly classified in to 2 categories.

       Based on wavelength of emitted radiation

       Stokes fluorescence

       Anti-stokes fluorescence

       Resonance fluorescence

       Based on phenomenon

       Sensitized fluorescence

       Direct line fluorescence

       Stepwise fluorescence

       Thermally assisted fluorescence.


       Fluorescence and Phosphorescence are the phenomena of Photoluminescence

       Fluorescence spectroscopy is an emission spectroscopic method

       The property of fluorescence can be predicted from chemical structure

       Fluorescence can be used for both qualitative and quantitative Pharmaceutical Analysis

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