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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