Transcript L32

Molecular Luminescence
Spectroscopy
Lecture 32
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The shape of the emission spectrum is expected to be a
mirror image of the excitation spectrum since they
originate from opposite processes However, instrumental
artifacts result in excitation and emission spectra that are
not exactly mirror images.
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3. Fiber Optic Fluorescence
Sensors
These are the same as conventional
fluorescence instruments but the beam
from the excitation monochromators is
guided through a bifurcated optical
fiber to the sample container where
excitation takes place. The
fluorescence at the emission
wavelength is then measured and
related to concentration of analyte in
the sample.
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4. Phosphorimeters
Instruments that can measure phosphorescence are
called phosphorimeters. They are very similar to
fluorescence instruments but make use of the fact
that phosphorescence has a much longer lifetime
than fluorescence and thus can be time resolved
from the fluorescence signal. This can be achieved
by placing a rotating chamber with a hole directing
the beam to the sample. When the hole is aligned so
that the incident beam excites the sample, the
sample gives both fluorescence and
phosphorescence. However, as the chamber rotates
the incident beam becomes blocked and the
fluorescence ceases. Phosphorescence will
continue since it has a much longer lifetime and as
the hole faces the detector only phosphorescence
will be measured.
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As the chamber rotates,
phosphorescence will be detected as
the hole in the chamber becomes
aligned with the detector slit. No
fluorescence interfere as the
fluorescence lifetime is much shorter
than the time required by the rotating
chamber to align its hole with the slit of
the detector
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Applications of
Photoluminescence Methods
Fluorescence is the most widely used
luminescent technique for determination of
many metal ions that react with organic
ligands to form fluorescent molecules. On
the other hand, although phosphorescence
methods were used for analysis of a variety
of analytes, they are still rarely used because
of lower sensitivity and precision.
Furthermore, few chemical systems really
show good phosphorescence.
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In addition, too many precautions must
be followed for a successful
phosphorescent analysis preventing
widespread use of these methods.
Fluorescence methods are quantitative
techniques that are usually highly
sensitive. Either the analyte or a
reaction product of the analyte must be
fluorescent which makes the method
highly applicable to many systems that
can be made fluorescent.
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Chemiluminescence
This luminescence technique emerges in
systems where a chemical reaction produces
enough energy to excite an analyte or a
reaction product of the analyte. Upon
returning to ground state, the excited
molecule emits a photon and
chemiluminescence is observed. Several
systems show the phenomenon of
chemiluminescence where the
chemiluminescence intensity is proportional
to analyte concentration (in the nM to fM
range).
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This can be represented by the general
reaction:
A + B = C* + D
C* = C + hn
Analytical Applications of Chemiluminescence
Several reactions are known to produce
chemiluminescence under certain
conditions, some are described below:
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Determination of Nitrous Oxide
(NO)
Nitrous oxide reacts with
electrogenerated ozone to form an
excited nitrogen dioxide molecules
followed by emission of the excitation
energy as photons
(chemiluminescence). The intensity of
chemiluminescence is proportional to
concentration of nitrous oxide.
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NO + O3 = NO2* + O2
NO2* = NO2 + hn
A NO concentration down to 1 ppb was
determined using this method. On the
other hand, higher nitrogen oxides (NOx)
were also determined by this method; by
first reducing the oxide to NO followed by
reaction with ozone.
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Determination of Sulfur Dioxide
Sulfur dioxide reacts with hydrogen to form
an excited sulfur dimer species. The
excited sulfur dimer then relaxes to
ground state by emission of photons.
4 H2 + 2 SO2 = S2* + 4 H2O
S2* = S2 + hn
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Luminol Chemiluminescence
One of the most common chemiluminescent
reactions is that of luminal (5aminophthalhydrazide) with hydrogen
peroxide in basic medium.
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Luminol + H2O2 + OH- = (3-aminophthalate)* + N2
+ H2O
(3-aminophthalate)* = 3-aminophthalate + hn
This reaction is most important for
determination of many bioanalytical substrates
which produce hydrogen peroxide. Examples
include glucose, cholesterol, alcohol, amino
acids, lactate, oxalate, etc… which, in
presence of the respective oxidase enzyme,
produce hydrogen peroxide. The intensity of
chemiluminescence is proportional to
substrate concentration
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In addition, this system can be extended
for the analysis of many substrates that
can indirectly be made to produce
hydrogen peroxide such as cholesterol
esters which can be hydrolyzed to
cholesterol, using cholesterol
estearase, followed by oxidation of
generate cholesterol using cholesterol
oxidase where hydrogen peroxide is
produced.
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Instrumentation
The instrumentation used in
chemiluminescence is rather simple
and can be composed of a
photomultiplier tube and a readout.
However, the PMT should be of very
high sensitivity and a very low dark
current. A schematic of the instrument
can be shown below:
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