3. FLUORESCENCE
SPECTROSCOPY
PRINCIPLE
• Fluorescence is an emission phenomenon where an
energy transition from a higher to a lower state is
accompanied by radiation.
• Only molecules in their excited forms are able to
emit fluorescence; thus, they have to be brought into
a state of higher energy prior to the emission
phenomenon.
• The energy conversions are based upon
Jablonski’s diagram.
6. Singlet excited state
Singlet ground state
Emission of radiation
Triplet excited state Singlet ground state
Emission of radiation
Fluorescence
Phosphorescence
7. •Two monochromators are used, one for tuning the wavelength of the exciting
beam and a second one for analysis of the fluorescence emission.
• Due to the emitted light always having a lower energy than the exciting light,
the wavelength of the excitation monochromator is set at a lower wavelength
than the emission monochromator.
8. • At low concentrations, the intensity of fluorescence
(If) is related to the intensity of the incident light (I0)
of appropriate wavelength by the relationship:
• If = I0 x 2.3eclq
where e is the molar absorption coefficient
c the molar concentration
l the length of the light-path
and q the quantum efficiency (i.e. the number of
quanta fluoresced divided by the number of quanta
absorbed).
9. EXAMPLES
• NADH and NADPH exhibit fluorescence, absorbing
light at 340 nm and reemitting it at about 460 nm.
• George Guilbault and David Kramer (1963) devised
an assay for triacylglycerol lipase based on the
following reaction:
dibutyryl fluorescein
(non-fluorescent)
fluorescein
(fluorescent)
Lipase
10. • Fluorescein diacetate may act as a substrate
for esterases
• Synthetic substrates that release a fluorescent
product are also available for the assay of
some enzymes.
• An example is the assay of b-D-glucuronidase
using 4-methylumbelliferyl-b-D-glucuronide
as substrate and assaying 4-methyl-
umbelliferone as the fluorescent product.
11. FLUORESCENCE RESONANCE
ENERGY TRANSFER
• It is the transfer of non-radiative energy from a
donor to an acceptor chromophore.
• The efficiency of this energy transfer is
FRET α 1/RO
6
showing that the FRET effect is inversely
proportional to the distance between donor and
acceptor chromophores , R0 (This makes FRET
extremely sensitive to small changes in distance).
12. • The FRET effect is particularly suitable for
biological applications, since distances of 10–
100A˚ are in the order of the dimensions of
biological macromolecules.
• Using fluorogenic substrates, assays of
enzymes like kinases , proteases and
phosphatases are practiced in large scales
(this process is done in-vivo using the principle
of FRET)
15. CONSTANT VOLUME MANOMETRY
• In constant volume manometry, the manometer is usually
connected to a reservoir, so that fluid may be added to or
removed from the manometer as required.
• The amount of fluid in the manometer is adjusted prior to
taking a reading so that the level in the limb nearest the
reaction vessel is at a fixed point.
• The difference between the fluid levels in the two limbs of
the manometer gives an indication of the pressure in the
reaction vessel, which changes as gas is taken up or evolved,
and so may be used to follow the course of the reaction.
16. CONSTANT PRESSURE MANOMETRY
• In constant pressure manometry , the volume of the
reaction chamber is changed by measurable amounts
sufficient to keep the manometer fluid level at a fixed
point as the reaction proceeds.
• The amount of fluid in the manometer is not changed, so
this procedure ensures that the levels in both arms
remain fixed, and no pressure changes take place.
• The course of the reaction may thus be followed in terms
of the volume changes required to maintain the constant
pressure
17. REFERENCES
• Principles and Techniques of Biochemistry and
Molecular Biology: By Wilson and Walker
• ENZYMES: Biochemistry, Biotechnology and
Clinical Chemistry by Trevor Palmer and Philip
Bonner.
• The Cell: A Molecular Approach Textbook by
Geoffrey M. Cooper
