(Instrumental Analysis)

(Code: SOU30270)

4th Stage Chemistry Department

Semester 7/Level UGIV

Lecturer: Dr. Vian S. Ismail

Email: vian.esmaeil@soran.edu.iq

Week 8/Lecture No. 8

Lecture time: Sunday: 11.30 -2.30

(13/ 11/2022 ) 1
Chemiluminescence and bioluminescence.

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 Other Luminescent Methods
Two other important forms of luminescence are chemiluminescence and
bioluminescence.
Chemiluminescence refers to a process in which a chemical reaction forms a
product molecule that is in an excited state. The excited state product then
emits radiation. The classic example of a chemiluminescent process involves
the reaction of luminol with hydrogen peroxide (H2O2) in the presence of a
catalyst as shown below:

The reaction generates 3-

aminophthalate in an
excited state and it emits a
bluish light. The luminal
reaction is used in forensics
to detect the presence of
blood. In this case, the iron
from the hemoglobin serves
as the catalyst.
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Bioluminescence refers to a situation when living organisms use a
chemiluminescent reaction to produce a luminescent emission. The
classic example is fireflies. There are also a number of
bioluminescent marine organisms.

Firefly luciferin
Firefly luciferin is the luciferin, or light-
emitting compound, used for the firefly,
railroad worm, starworm, and click-beetle
bioluminescent systems. It is the substrate
of luciferase, which is responsible for the
characteristic yellow light emission from
many firefly species

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 Example,
1. The glow of phosphorus and its oxides.
2. Oxidation-reduction of hydrazine.
3. Cold light emission by glow worms – Enzyme catalyzed oxidation of
protein (Luciferin).

4. Reaction between alkaline solutions of H2O2 and either Cl2 or

hypochlorite ion which produces a red glow at a number of wavelengths.
One of the glows has been identified as resulting from the excitation of
the oxygen produced in the reaction.
5. Electron transfer reaction.
6. Interaction between the aromatic anions Ar- and cations Ar+
generated during electrolysis of large polycyclic hydrocarbons.
Ar- + Ar+ → Ar* + Ar Ar* → Ar + he
7. The luminescence observed at night in the ocean due to the decay of
the luminous jelly-fish.

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In general, a chemiluminescent reaction can be generated by two
basic mechanisms.

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 In a direct reaction, two reagents, usually a substrate and an oxidant in the
presence of some cofactors, react to form a product or intermediate, sometime in
the presence of a catalyst . Then some fraction of the product or intermediate will
be formed in an electronically excited state, which can subsequently relax to the
ground state with emission of a photon.
The catalyst, enzyme or metal ions, reduces the activation energy and provide an
adequate environment for producing high CL efficiency out of the process.
Cofactors, sometimes are necessary to convert one or more of the substrate into
a form capable of reacting and interacting with the catalyst, or to provide an
efficient leaving group if bond cleavage is required to produce the excited emitter.

Indirect or sensitized CL, is based on a process of transfer of energy of the excited

specie to a fluorophore. This process makes it possible for those molecules that are
unable to be directly involved in CL reactions to transfer their excess of energy to a
fluorophore that in turn is excited, releasing to its ground state with photon
emission. All of these paths lead to a great variety of practical uses of CL in solid, gas ,
and liquid phases.

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Requirements for Chemiluminescence Emission
For a chemical reaction to produce light, it should meet some
essential requirement:
1. The reaction must be exothermic to produce sufficient energy
provided by a chemical reaction to form the electronically excited
state.
2. Formation of products capable of forming an excited state multiple
bonds, conjugation aromatic systems
3. Presence of an emitter, (doesn't have to be a product) multiple
bonds conjugation aromatic systems.
4. Rapid kinetics of the chemical reaction, the rate of reaction is more
important than high yield fCL
5. Reaction coordinate system must favour formation of an excited
state over ground state of products.

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For chemiluminescence, the radiant intensity ICL (photons emitted per second)
depends on the rate of the chemical reaction (d[C]/dt) and the
chemiluminescence quantum yield fCL (photons per molecule reacted).
The latter term is equal to the product of the excitation quantum yield fEX (excited
states per molecule reacted) and the emission quantum yield fEM (photons per
excited state).
These relationships are described by the equation:

Chemiluminescence systems useful in analytical chemistry generally

have values of fCL of 0.01 to 0.2.

In general ICL decrease with time as reactant is consumed.

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Factors Influencing Chemiluminescence Emission
CL measurements are strongly modified by experimental factors that
affect quantum yield and rate of reaction, such :
1-The chemical structure of the CL precursor, not only the central
portion containing the electronically excited group.
2-The nature and concentration of other substrates affecting the CL
pathway.
3-The selected catalyst.
4-The presence of metal ions, especially transition metals involved in
the processing of the oxidant.
5-the Temperature.
6-pH and ionic strength.
7-The hydrophobicity of the solvent and solution composition
[example, the ɸCL of luminol oxidized in dimethylsufoxide[DMSO]is
0.05 compared with 0.01 in water, the colors being blue –violet (425
nm ) and blue green (480 –502 nm ) respectively ].
8-the presence of energy transfer acceptors.
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Types of chemiluminescence depending on the phases
1-liquid phase chemiluminescence:
Chemiluminescence in liquid phase occurs most frequently in oxidation reactions.
Only few organic substances have particular importance.
Lucigenin: a blue-green CL arises during alkaline oxidation of lucigenin ( N, N” –
dimethyl, diacridinium dinitrate) with hydrogen peroxide. The emitting substance is
believed to be N-methylacridone.
Note: The lucigenin
reaction is strongly
influenced by catalysts
and inhibitors. It
enhanced by Pb(II) ,
Bi(III) , TI (III) and Hg(I)
ions.

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2- Gas- phase chemiluminescence:
Gas phase found wide application in monitoring of air pollution and has been
studied in simple gases, flames , metal vapors.

Example of simple gases:

A- (gas) determination of nitrogen oxides based on the reaction with ozone.

NO + O3 NO2* + O 2

NO*2 NO2 + hv (2800 -600 (nm

This is a important example of a chemiluminescent reaction that involves the

reaction of nitric oxide (NO) with ozone (O3) to produce excited state nitrogen
dioxide (NO2*) and oxygen gas. Nitric oxide is an important compound in
atmospheric chemistry and, with the use of an ozone generator, it is possible to use
the chemiluminescent reaction as a sensitive way of measuring NO.
An important feature of both chemiluminescent reactions above is that peroxide
and ozone, which are strong oxidants, have an unstable or energetic chemical bond.
Chemiluminescence is a rare process only occurring in a limited number of chemical
reactions.
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B- (gas) CL reaction between sulfur dioxide and oxygen atoms.

O· + O· + SO2 O2 + SO2*
SO2* SO2 + hv ( 280 nm)

The main difficulty encountered in this type of analysis consists in

finding a stable oxygen atoms source.
This reaction is used for determination of sulfur dioxide with a
sensitivity of 0.001 ppm is reported

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C- (flame) Most application of flame CL is determination of sulphur
and phosphorus when they burned in a hydrogen rich flame

SO2 + 2H2 S2+ H2O

S + S S*2

S2* S2 + hv 425 -300nm

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D-Metal vapor:
Alkaline metal:

K + SO2 [K+SO2-] SO2 + K* K + hv

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3- Solid phases Chemiluminescence:
The first best illustration of this type of reaction is the CL produced
from the oxidation of siloxenes .The compound and its derivatives are
highly polymerized with permuted structure forming isolated network
that a thickness the size of a molecule.

The basic formula is (Si6H6O3)n.

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 This compound is produced readily from calcium silicate and HCl.
The CL intensity varies with the number of hydrogen atoms
replaced; this has formed the basis of several applications.

Example: Chlorine and hydroxide have been determined based on

the replacement of the siloxene hydrogen atoms or hydroxide
group.

-oxidation of siloxene with H2O2 , KMnO4 , CeSO4 , Chromic

acid, Nitric acid or other strong oxidant result in a red CL.

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Applications:
1. Chemiluminescence is used in the study of firefly
reaction in which luciferin, LH2 reacts with ATP in
presence of Luciferase enzyme to yield a complex.
The amount of h emitted is proportional to the
concentration of ATP
This method is used to monitor the effectiveness of
biocides, effect of drugs and detection of microbes in
food.
2. Chemiluminescence is the standard method for
monitoring NOX in the atmosphere.
3. It is also useful for the measurement of O3.
4. Forensic science
5. Food analysis
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ATOMIC SPECTROSCOPY
Atmoic absorption and atomic emission
It is likely that most people studying chemistry have seen a
demonstration where a solution of a metal salt was sprayed into a
Bunsen burner and gave off a color that depended on the particular
metal in the salt. Metal salts are used to create the different colors
observed in firework displays.

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Earlier we discussed the difference between atomic spectra, which
only consist of electronic transitions and therefore appear as sharp
lines, and molecular spectra, which because of the presence of lower
energy vibrational and rotational energy states appear as a broad
continuum. Provided we have atoms present in a sample, it is
possible to analyze them spectroscopically using either absorption or
emission measurements. One problem is that most samples we
analyze do not consist of atoms but instead consist of molecules with
covalent or ionic bonds. Therefore, performing atomic spectroscopy
on most samples involves the utilization of an atomization source,
which is a device that has the ability to convert molecules to atoms.

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It is also important to recognize that the absorption or emission
spectrum of a neutral atom will be different than that of its ions (e.g.,
Cr0, Cr3+ , Cr6+ all have different lines in their absorption or emission
spectra). Atomic absorbance measurements are performed on
neutral, ground-state atoms. Atomic emission measurements can be
performed on either neutral atoms or ions, but are usually performed
on neutral atoms as well. It is important to recognize that certain
metal species exist in nature in various ionic forms. For example,
chromium is commonly found as its +3 or +6 ion. Furthermore, Cr3+ is
relatively benign, whereas Cr6+ is a carcinogen. In this case, an analysis
of the particular chromium species might be especially important to
determine the degree of hazard of a sample containing chromium.
The methods we will describe herein cannot be used to distinguish
the different metal species in samples. They will provide a
measurement of the total metal concentration.

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Table 28-1 lists several methods that are used to atomize samples for atomic
spectroscopy. Inductively coupled plasmas, flames, and electrothermal atomizers are
the most widely used atomization methods. Flames and electrothermal atomizers are
found in atomic absorption (AA) spectrometry, while the inductively coupled plasma
is used in optical emission and in atomic mass spectrometry.

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Atomic Absorption Spectroscopy

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Processes leading to atoms, molecules, and ions with continuous sample introduction into a plasma or
flame. The solution sample is converted into a spray or mist by the nebulizer. The high temperature of
the flame or plasma causes the solvent to evaporate leaving dry aerosol particles. Further heating
volatilizes the particles producing atomic, molecular, and ionic species. These species are often in
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equilibrium at least in localized regions.
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