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

Fluorimetry –Theoretical principles

Objectives

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

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

       Discuss
the phenomenon of photo luminescence

       Explain
the factors affecting Fluorescence

       Discuss
the methods used for converting non fluorescent substances to fluorescent

Flourimetry

       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 complexd
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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