Molecular Fluorescence
Spectroscopy
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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 bio molecules are intrinsic fluors i.e., they
are fluorescent themselves.
• The amino acids with aromatic groups eg
phenylalanine, 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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Fluorophore:
A fluorophore (or fluorochrome, similarly to a chromophore)
is a fluorescent chemical compound that can re-emit light
upon light excitation.
Fluorophores typically contain several
combined aromatic groups, or plane or cyclic molecules with
several π bonds.
Fluorophores are sometimes used alone, as a tracer in fluids,
as a dye for staining of certain structures, as a substrate
of enzymes, or as a probe or indicator (when its fluorescence
is affected by environmental aspects such as polarity or
ions). But more generally it iscovalently bonded to
a macromolecule, serving as a marker (or dye, or tag, or
reporter) for affine or bioactive reagents (antibodies,
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peptides, nucleic acids). 12
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Fluorophores are notably used to stain
tissues, cells, or materials in a variety of
analytical methods, i.e.,fluorescent
imaging (Fluorescence imaging is the
visualization of fluorescent dyes or
proteins as labels for molecular processes
or structures. It enables a wide range of
experimental observations including the
location and dynamics of gene expression,
protein expression and molecular
interactions in cells and
tissues)and spectroscopy. 13
Light scattering
Rayleigh-Tyndall scattering
Incident radiation is not only absorbed or
transmitted by the sample but is also scattered
in all directions. The scattering takes place either
from the molecules themselves (Rayleigh
scattering) or from small particles in colloidal
suspension (Tyndall scattering).
Raman scattering
During the Rayleigh scattering process, some of
the incident energy can be abstracted and
converted into vibrational and rotational energy.
The resulting energy scattered is therefore of
lower energy and longer wavelength than the
incident radiation.
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 pre selection 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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Advantages of fluorescence spectroscopy:
Very sensitive
Can be used for quantitation of fluorescent species
Easy and quick to perform analysis
Disadvantages:
Not useful for identification
Not all compounds fluorescence
Contamination can quench the fluorescence and hence
give false/no results
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