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Principles
Interaction of photons with
molecules results in promotion of
valence electrons from ground state
orbitals to high energy levels.
The molecules are said to be in
excited state.
Molecules in excited state do not
remain there long but spontaneously
relax to more stable ground state.
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The relaxation process is brought
about by collisional energy transfer
to solvent or other molecules in the
solution.
Some excited molecules however
return to the ground state by
emitting the excess energy as light.
This process is called fluorescence.
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• The emitted light has two important
characteristics :
1. It is usually of longer wavelength
(lower energy) than the excited light.
This is because part of the energy
associated with S state is lost as heat
energy.
2. The emitted light is composed of
many wavelengths which results in
fluorescence spectrum.
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Quantam yield Q
The fluorescence intensity is
described in terms of quantum yield.
The quantum yield Q is the ratio of
the number of photons emitted to
the number of photons absorbed.
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Intrinsic Fluors
• Some biomolecules are intrinsic fluors ie.,
they are fluorescent themselves.
• The amino acids with aromatic groups eg
phenylalamine, tyrosine, tryptophan are
fluorescent. Hence proteins containing
these amino acids have intrinsic
fluorescence.
• The purine and pyrimidine bases and some
coenzymes eg NAD and FAD are also
intrinsic fluors.
• Intrinsic fluorescence is used to study
protein conformation changes and to probe
the location of active site and coenzymes in
enzymes.
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Extrinsic Fluors
These are fluorescent molecules that
are added in biochemical system
under study.
Extrinsic fluorescence has been used
to study the binding of fatty acids to
serum albumin, to characterize the
binding sites for cofactors and
substrates in enzyme molecules and
to study the intercalation of small
molecules into the DNA double helix.
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ANS, dansyl chloride, fluorescein are
used for protein studies.
Ethidium, proflavine and acridines
are used for nucleic acid
characterization.
Ethidium bromide has enhanced
fluorescence when bound to double
stranded DNA but not single
stranded DNA.
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Instrumentation
• The basic instrument is a
spectrofluorometer.
• It contains a light source, two
monochromators, a sample holder and a
detector.
• There are two monochromators, one for
selection of the excitation wavelength,
another for analysis of the emitted light.
• The detector is at 90 degrees to the
excitation beam.
• Upon excitation of the sample molecules,
the fluorescence is emitted in all directions
and is detected by photocell at right angles
to the excitation light beam.
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The lamp source used is a xenon arc
lamp that emits radiation in the UV,
visible and near-infrared regions.
The light is directed by an optical
system to the excitation
monochromator, which allows either
preselection of wavelength or
scanning of certain wavelength
range.
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The exciting light then passes into the
sample chamber which contains
fluorescence cuvette
A special fluorescent cuvette with four
translucent quartz or glass sides is
used.
When the excited light impinges on
the sample cell, molecules in the
solution are excited and some will
emit light.
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• Light emitted at right angles to the
incoming beam is analyzed by the
emission monochromator.
• The wavelength analysis of emitted
light is carried out by measuring the
intensity of fluorescence at
preselected wavelength.
• The analyzer monochromator directs
emitted light of the preselected
wavelength to the detector.
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A photomultiplier tube serves as the
detector to measure the intensity of
the light.
The output current from the
photomultiplier is fed to some
measuring device that indicates the
extent of fluorescence.
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