Molecular Luminescence
Spectroscopy
1
Molecular Luminescence
Spectroscopy
Luminescence spectroscopy is a
technique which studies the
fluorescence, phosphorescence, and
chemiluminescence of chemical
systems. The analyte or its reaction
product needs to be luminescent. The
relative luminescence intensity is
related to analyte concentration as will
be seen shortly.
2
Singlet and Triplet States
Electrons in molecular orbitals are paired,
according to Pauli exclusion principle. When
an electron absorbs enough energy it will be
excited to a higher energy state; but will keep
the orientation of its spin. The molecular
electronic state in which electrons are paired
is called a singlet transition. On the other
hand, the molecular electronic state in which
the two electrons are unpaired is called a
triplet state. The triplet state is achieved
when an electron is transferred from a
singlet energy level into a triplet energy level,
by a process called intersystem crossing;
accompanied
by a flip in spin
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In a singlet state, the spins of the two electrons are
paired and thus exhibit no magnetic field and called
diamagnetic. Diamagnetic molecules, containing
paired electron, are neither attracted nor repelled by
a magnetic field. On the other hand, molecules in the
triplet state have unpaired electrons and are thus
paramagnetic which means that they are either
repelled or attracted to magnetic fields. The terms
singlet and triplet stems from the definition of
multiplicity where:
Multiplicity = 2S + 1
Where, S is the total spin. The total spin for a singlet
state is zero since electrons are paired which gives a
multiplicity of one (the term singlet state).
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Multiplicity = (2 * 0) + 1 =1
In a triplet state, the total spin is one (the two
electrons are unpaired) and the multiplicity is
three:
Multiplicity = (2 * 1) + 1 = 3
It should also be indicated that the probability
of a singlet to triplet transition is much lower
than a singlet to singlet transition. Therefore,
the intensity of the emission from a triplet
state to a singlet state is much lower than
emission intensities from a singlet to a
singlet state.
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Energy Level Diagram for
Photoluminescent Molecules
The following diagram represents the
main processes taking place in a
photoluminescent molecule when it
absorbs and emits energy.
The different processes will be discussed
below:
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Absorption
The absorption of UV-Vis radiation is necessary to
excite molecules from the ground state to one of the
excited states. Absorption of radiation promotes
electrons in chemical bonds to be excited. However,
we have seen earlier that not all transitions have the
same probability and while certain transitions are
practically very important, others are seldom used
and are of either no or marginal importance.
Therefore, from information we have discussed in
Chapter 14 we concluded that there are four different
types of electronic transitions which can take place
in molecules when they absorb UV-Vis radiation. A
s-s* and a n-s* are not useful while the n-p*
transition requires low energy but the molar
absorptivity for this transition is low and transition
energy will increase in presence of polar solvents.
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The most frequently used transition is the p-p*
transition for the following reasons:
a.
The molar absorptivity for the p-p* transition is
high allowing sensitive determinations.
b.
The energy required is moderate, far less than
dissociation energy.
c.
In presence of the most convenient solvent
(water), the energy required for a p-p* transition is
usually smaller.
Therefore, best molecules that may show absorption
are those with p bonds or preferably aromatic nature
as discussed earlier. Absorption to higher excited
singlet states requires a very short time (in the range
of 10-14s).
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Vibrational Relaxation
Absorption of radiation will excite molecules to
different vibrational levels of the excited
state. This process is usually followed by
successive vibrational relaxations (VR) as
well as internal conversion to lower excited
states. In cases where transitions occur to
the first excited state, vibrational relaxation
to the main excited electronic level will take
place and/or an intersystem crossing (ISC) to
the triplet state can occur.
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Fluorescence
After vibrational relaxation to first excited electronic
level takes place, a molecule can return to the
ground state by emission of a photon, called
fluorescence (FL). The fluorescence lifetime is much
greater than the absorption time and occurs in the
range from 10-7 to 10-9s. As the lifetime in the excited
state is increased, the probability of fluorescence
will be decreased since radiationless deactivation
processes may take place. However, not all excited
molecules can show fluorescence by returning to
ground state and most return to ground state by
losing excitation energy as heat or through
collisions with other molecules or solvent.
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Internal and External Conversion
Internal conversion (IC) is a radiationless
deactivation process whereby excited
molecules return to the ground state without
emission of a photon. This process lacks
rigid understanding but seems to be the
most efficient deactivation process in
luminescence spectroscopy, since most
molecules do not show fluorescence.
However, molecules with close electronic
energy levels, to the extent that their
vibrational energy levels of ground and
excited states are overlapped, are believed to
cause efficient internal conversion.
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Dissociation and predissociation
Internal conversion can result in a phenomenon called
predissociation (PD) where an electron relaxes from
a higher electronic state to an upper vibrational
energy of a lower electronic state. When the
vibrational energy is large enough and is greater
than the bond senergy, bond rupture occurs in a
process called predissociation. Dissociation should
be differentiated from predissociation where
dissociation involves absorption of high energy so
that the molecule is directly promoted to a high
energy vibrational level where bond rupture directly
occurs.
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External conversion (EC) is a process
whereby excited molecules lose their
energy due to collisions with other
molecules or by transfer of their energy
to solvent or other unexcited
molecules. Therefore, external
conversion is influenced by
temperature, solvent viscosity, as well
as solvent composition.
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Intersystem Crossing
Electrons present at the first excited electronic
level can follow one of three choices
including emission of a photon to give
fluorescence, radiationless deactivation to
ground state, or intersystem crossing (ISC).
The process of intersystem crossing
involves transfer of the electron from an
excited singlet to a triplet state. This process
can actually take place since the vibrational
levels in the singlet and triplet states overlap.
However, crossing of the singlet state to the
triplet state involves a flip in electron spin in
order to satisfy the triplet state.
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Intersystem crossing is facilitated by presence
of nonbonding electrons as well as heavy
atoms. The presence of paramagnetic atoms
or species also enhances intersystem
crossing.
An electron in the triplet state can also cross
back to the singlet state and can result in a
photon as fluorescence but at a much longer
time than regular fluorescence. This process
is termed delayed fluorescence and has the
same characteristics as direct fluorescence
except for the large increase in lifetime.
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Phosphorescence
Electrons crossing the singlet state to the triplet state with a
flipped spin can also follow one of three choices
including returning to the singlet state (including a flip in
spin), relax to ground state by internal or/and external
conversion, or lose their energy as a photon
(phosphorescence, Ph) and relax to ground state with a
second flip in spin to satisfy the singlet ground state. As
can be rationalized from the processes involved in
collecting phosphorescence photons, this involves an
intersystem crossing and two flips in spin. This, in fact,
requires a much longer time than fluorescence (10-4s to
up to few s). Therefore, the probability of
phosphorescence, and hence the intensity of the
phosphorescence spectrum, is very low due to high
possibility of radiationless deactivation.
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Quantum Yield and Efficiency
The quantum yield or efficiency of
fluorescence is the ratio of the number of
fluorescing molecules to the total number of
excited molecules. For highly fluorescent
molecules, a quantum efficiency
approaching one can be obtained. The
quantum efficiency can be represented by
the relation:
F = kFL/(kFL + kISC + kIC + kEC + kPD + kdiss)
Phosphorescence quantum efficiency is
defined in the same manner
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The transitions most important in
luminescence spectroscopy are n-p* and pp*. However, fluorescence is encountered
more often in molecules having p-p*
transitions since this transition has a higher
quantum efficiency in terms of a higher
molar absorptivity and a shorter lifetime (10-7
– 10-9s) than the n-p* transition which has a
longer lifetime (10-5 – 10-7s). Once again, a
longer lifetime means a lower luminescent
probability due to increased possibility of
radiationless deactivation.
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Variables That Affect Fluorescence and
Phosphorescence
Factors affecting fluorescence and phosphorescence
include both environmental and structural factors.
Some of the important factors are discussed below:
Fluorescence and Structure
As indicated earlier, best luminescence is observed for
molecules with p bonds and preferably those having
aromatic rings due to presence of low energy p-p*.
However, some heterocyclic aromatic rings do not
show fluorescence. These include pyridine, furan,
pyrrole, and thiophene
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The lack of fluorescence in such molecules
is largely believed to be due to existence of a
low lying n-p* transition that rapidly converts
the excited molecule to the triplet state and
prevent fluorescence. However, fusion of a
phenyl ring to any of the above molecules
increase the possibility of the p-p*
transitions
and
thus
increase
the
fluorescence quantum efficiency.
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Substitution of halogens to the aromatic ring
has important influence on the fluorescent
signal where a decrease in fluorescence is
observed with an increase in the atomic
weight of the halogen and a subsequent
increase in phosphorescence. This is
referred to as the heavy atom effect where
promotion of intersystem crossing takes
place. In addition, substitution of a carbonyl
or carboxylic acid groups decreased
fluorescence due to enhancement of
intersystem crossing.
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Effect of Structural Rigidity
The nature of the chemical structure of a
molecule in terms of flexibility and rigidity is
of major influence on the fluorescence and
phosphorescence signal. Molecules that
have high degree of flexibility will tend to
decrease fluorescence due to higher
collisional probability. However, more rigid
structures have lower probability of
collisions and thus have more fluorescence
potential. Biphenyl has very low
fluorescence quantum efficiency (~ 0.2) while
fluorine has a quantum efficiency close
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In addition, some metal complexes have
higher fluorescence efficiency than do
the ligands; also as a result of
increased structural rigidity. For
example, the fluorescence intensity of
8-hydroxyquinoline is increased to a
large extent in presence of zinc ions,
due to more rigid complex formation.
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Effect of Solvent Nature
Solvents characteristics have important effects on
luminescent behavior of molecules. Three main
effects can be recognized:
a. Solvent Polarity
A polar solvent is preferred as the energy required for
the p-p* is lowered.
b. Solvent Viscosity
More viscous solvents are preferred since collisional
deactivation will be lowered at higher viscosities.
c. Heavy Atoms Effect
If solvents contain heavy atoms, fluorescence quantum
efficiency will decrease and phosphorescence will
increase.
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Effect of Temperature
Higher temperatures result in larger collisional
deactivation due to increased movement and
velocity of molecules. Therefore, lower temperatures
are preferred.
Effect of pH
The pH of the solution is a very important factor that
influences luminescence. For example, aniline
shows fluorescence while aniline in acid solution
(anilinium ion) does not. Most compounds luminesce
in basic or slightly basic solutions while some show
fluorescence in acidic medium. It is therefore
important to adjust the pH so that maximum
luminescence intensity is obtained. The pH also
affects the emission wavelength where usually a
longer emission wavelength is observed at higher
pH.
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Effect of Dissolved Oxygen
Dissolved oxygen largely limits fluorescence since it
promotes intersystem crossing because it is
paramagnetic. However, dissolved oxygen affects
phosphorescence more than it does to fluorescence.
Although one would think that as far as intersystem
crossing is increased in presence of oxygen,
phosphorescence is expected to increase. On the
contrary, phosphorescence is completely eliminated and
quenched in presence of dissolved oxygen. This may be
explained on the basis that the ground state of oxygen is
the triplet state and it is easier for an electron in the triplet
state to transfer its energy to triplet oxygen rather than
performing a flip in spin and relax to singlet state.
Therefore, oxygen will be excited and what we really
observe is oxygen emission rather than phosphorescence.
It is for this reason that oxygen should be totally excluded
to be able to detect phosphorescence.
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Effect of Concentration on
Fluorescence
It can be undoubtedly said that the
fluorescence is directly proportional to the
amount of absorbed radiation where:
F = k(Po-P)
P = Po 10-A
Substitution gives:
F = kP0 (1-10-A)
This relation can be expanded by Mc Lauren
series giving:
F = kP0 (2.303 A – (2.303 A)2/2! + (2.303 A)3/3! (2.303 A)4/4! + ….)
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Only the first term is important at the
very low concentrations used. The
relation simplifies to:
F = kPo (2.303 A)
F = KPoebc
Where K = 2.303k
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The relation F = KP0ebc implies the following
a.
The fluorescence signal can be increased if the
radiant power of the incident beam is increased.
Therefore, always use more intense sources.
b.
The fluorescence signal is directly related to the
molar absorptivity and thus molecules of higher
molar absorptivities are better fluorescers.
c.
Fluorescence signal is directly proportional to
path length.
d.
Fluorescence signal is directly proportional to
concentration. This is different from relation between
absorbance and concentration which is logarithmic.
e.
The linear correlation between fluorescence and
concentration is only true when the absorbance is
less than 0.05.
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Deviation from Linearity between
Fluorescence and Concentration
Negative deviations from the linear relation between
fluorescence and concentration may be observed in
the following cases:
a.
At absorbances higher than 0.05.
b.
Self-quenching whereby excited molecules lose
their energies by collision with other molecules or
solvent
c.
Self-absorption which occurs when an emission
band overlaps with an excitation (absorption) band.
In this case, emitted photons excite other molecules
in the ground state which results in no net emission.
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Fluorescence Instruments
Like any emission instrument, a fluorescence
or phosphorescence instrument consists of
a source, wavelength selectors, a sample
cell, a detector, as well as a signal processor.
Two wavelength selectors are used, the first
is the excitation filter or monochromator
which excites the sample while the other is
the emission filter or monochromator that
separates the fluorescence or
phosphorescence wavelength.
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Sources
We have seen earlier that the
fluorescence signal is proportional to
the radiant power of the source.
Therefore, it is very important to select
a source of as high radiant power as
possible. In most instruments, a xenon
arc lamp is usually the source of
choice. However, some instruments
use lasers.
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Xenon Arc Lamp
A quartz envelope hosting two electrodes and xenon at
high pressure (5-10 atm). The discharge which takes
place between the two electrodes excites xenon and
produces a continuum in the range from 200-1000
nm. The wavelength maximum occurs at about 500
nm. The radiant power produced by a xenon arc
lamp is very high which makes the lamp suitable for
luminescence analysis. Also, the coverage of the
whole UV-Vis range adds to the assets of the lamp.
However, a very good regulated power supply is
essential since any fluctuations of the radiant power
will be directly reflected on the fluorescence signal.
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Lasers
We have seen examples of lasers early in this
course. However, it should be indicated that
since lasers may have very high intensities,
some molecules, which are otherwise non
fluorescents, show good fluorescence and
can thus be determined by fluorescence
spectroscopy. This is called laser induced
fluorescence (LIF) and is a common
technique in fluorescence spectroscopy. In
addition, one should remember that as the
radiant power is increased,
photodissociation may become a problem
especially at shorter excitation wavelengths.
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1. Fluorometers
When the wavelength selectors are filters, the
instrument is called a fluorometer. It is a
simple instrument that is usually used for
quantitative analysis. The use of
fluorometers implies that the excitation and
emission wavelengths are predetermined by
other means or are known from literature.
The detector is usually a sensitive
photomultiplier tube. A schematic of a simple
fluorometer is shown below:
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Unlike the cell used in UV-Vis absorption
studies, the cell used in luminescence
studies has the two transparent faces
perpendicular to each other, rather than
parallel. This is important since
luminescence is collected 90o to the
incident beam. This configuration
decreases the noise and fully excludes
interferences from the incident beam.
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2. Spectrofluorometers
In this case, the excitation and emission
wavelengths are selected using dispersive
elements like gratings or prisms. The
same instrumental configuration as
fluorometers is usually used. The following
schematic represents a basic configuration
of a spectrofluorometer:
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Excitation and Emission Spectra
The first step in a successful fluorescence
measurement is to determine the excitation and
emission wavelengths. The excitation spectrum
is determined by adjusting the emission
monochromator at an arbitrary, but well selected,
wavelength which is thought to be longer than
the anticipated wavelength. Keeping the
emission monochromator at the preset
wavelength, the excitation spectrum is recorded
by scanning the excitation monochromator in a
range less than the preset wavelength of the
emission monochromator.
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After obtaining the excitation spectrum, the
lex (resulting in highest absorbance) is
determined. To obtain the emission
spectrum, the excitation monochromator is
adjusted at the determined lex and the
emission monochromator is scanned so
that the emission spectrum is recorded.
lem is the wavelength with highest
fluorescence signal. At this point we are
ready to perform a fluorometric
determination using the defined excitation
and emission wavelengths.
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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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