Fluorimetry –Theoretical principles

Objectives

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

Fluorimetry

•       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.

•       Fluorescence

•       Phosphorescence

Ø  Chemiluminescence               

Flourescence:

•       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.

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 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:

•       Fluorescence

•       Phosphorescence

•       Radiation-less  processes

•       Vibration relaxation

•       Internal conversion

•       External conversion

•       Intersystem crossing

Jablonski energy diagram:

FLUORESCENCE AND CHEMICAL STRUCTURE:

•       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

•        Photodecomposition

•       Temp . &viscosity

•       Intensity of incident light

•       Path length

Quenching:

•       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:

•       Self-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.

Summary

•       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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