1. Powered by TCPDF (www.tcpdf.org)

2. CHEMISTRY HONOURS LIBRARY PROJECT
CHEMILUMINESCENCE
A DETAILED STUDY OF REACTIONS THAT EMIT LIGHT
AUTHOR:
MEGHOMITA DAS
FYBSC-384
UID-132161

3. ABSTRACT
We come across various kinds of exothermic and endothermic reactions in our daily life. These
reactions involve the emission or absorption of heat. However, there is another kind of reaction
which gives out light instead of heat. These spontaneous light-emitting reactions are termed as
Chemiluminescent Reactions.
Any Luminescent reactions involve the emission of light by a substance which is produced not
due to heating. Chemiluminescence is the emission of light due to a chemical reaction. These
reactions follow a slightly different kind of mechanism. The light produced in a
Chemiluminescent reactions is also called cool light as it produced without heat.
This project primarily focusses on the various kind of Chemiluminescent reactions that we come
across in our everyday lives. It also tries to explain the possible causes for Chemiluminescence
and gives a general idea about its applications in various fields.
One of the applications is explored in detail: Chemiluminescent Chemistry of a Glowstick. A
glowstick is a light device primarily used by armed personnel during warfare.
Before I start the discussion on Chemiluminescence, I would like to establish the difference
between Chemiluminescence, Phosphorescence, Fluorescence, Radioluminescence and
Triboluminescence.[1]
Phosphorescence is the process when a material gives out light after it absorbs energy from
another light source (usually in the UV range). The energy absorbed causes the electrons to
change their energy levels and they give out a radiation which is of a different wavelength in the
process. This output radiation is usually observed in the visible light region of the
electromagnetic spectrum. Fluorescence also follows the same process. The difference between
the two is that in Phosphorescence, the material continues to glow even after the original light
source is cut off. The material has the property of “storing” the incident light energy. In
Fluorescence, the material stops glowing after the incident light is extinguished.
Chemiluminescence and Bioluminescence rely on chemical energy for production of light
when two chemicals are reacted together. Chemiluminescence is termed as Bioluminescence
when it is observed in living organisms. One example of Bioluminescence would be the firefly’s
glow or some species of glowing Jellyfish- Aequorea victoria.
Radioluminescence[2] is a phenomenon in which a substance emits light on bombardment by an
ionizing radiation like beta rays. It has a low light intensity however, the light emission lasts for
considerable time. Tritium is a radioisotope which is used as a Radioluminescent light source.
Triboluminescence is the property exhibited by certain minerals or crystals which produces
light when its chemical bonds are broken due to rubbing, scratching or pulling apart. One
example of Triboluminescence is the crushing of sugar crystals producing a faint blue light.
We will now proceed further into the discussion of Chemiluminescent phenomena.

4. INDEX:-
1. What is Chemiluminescence?
2. Examples of Chemiluminescent Reactions :
 NO+O3 [Ozone] → NO2*
 Luminol Reaction
 Phosphorus oxidizing
 Firefly reaction
3. Enhanced Chemiluminescence using Horseradish Peroxidase
4. Causes/ Mechanisms of Chemiluminescence
5. Applications of Chemiluminescence
 ELISA and Western Blots
 Pyrosequencing of DNA
 Forensic studies
 Cancer Research
 Combustion Analysis
6. Chemistry behind a GLOWSTICK
 Making TCPO and Glowstick [ Demonstration Experiment]
7. Acknowledgements
8. References

5. WHAT IS CHEMILUMINESCENCE?
Chemiluminescence[3]
is the phenomena of emission of light due to a chemical reaction. The
reactants produce an unstable Chemiluminescent intermediate that decomposes to produce a non-
Chemiluminescent product and in the process it emits light. The emitted light, can be in theory
belong to UV, infrared or visible region of the electromagnetic spectrum. But those that fall in
the visible region category are considered the most useful.
The Chemiluminescent intermediate represents an excited electronic state. It rapidly decomposes
to a lower ground state by emission of a photon. If the wavelength of the emitted photon
corresponds to that of the visible region, we can see the light produced. In theory, one photon of
light should be emitted for every molecule of reactant. Therefore Avogadro’s number of photon
should be given off per mole of reactant. Quantum yield in Chemiluminescence is defined as the
probability of photon emission when a single substrate molecule reacts. The value of this
quantum yield determines the efficiency of the Chemiluminescent reactions.
The difference between other chemical reactions and Chemiluminescent reactions is that in other
reactions the difference between the energy of the reactants and that of the products manifests
itself as heat. The difference in energy of reactants and products is denoted by ΔH. Depending on
this, we classify the reactions as exothermic or endothermic. If the value of ΔH is negative, it is
an exothermic reaction. If the value is positive, then it is an endothermic reaction. In
Chemiluminescent reactions, the difference in energy manifests itself as light with limited
production of heat.
Chemiluminescent reactions can be grouped into three types:
1. Light-emitting reactions involving synthetic compounds and highly oxidizing agents like
Hydrogen Peroxide. These are called Chemiluminescent reactions
2. Light-emitting reactions observed in living organisms such as jellyfish or fireflies. These
reactions are termed as bioluminescent or bio-Chemiluminescent reactions
3. Light-emitting reactions which involve the use of electric discharge are termed as electro-
Chemiluminescent reactions.
We will be focusing on Chemiluminescent reactions and one particular example of
Bioluminescent reaction in this project.
A general Chemiluminescent reaction can be represented as follows:
[A] + [B] → [INTERMEDIATE]* → [PRODUCT] + LIGHT
[A] and [B] are reactants and [INTERMEDIATE]* represents the excited electronic state.

6. EXAMPLES OF CHEMILUMINESCENT REACTIONS
The Chemiluminescent reactions are classified based on the phase of the reactants. Accordingly,
we have liquid-phase and gas-phase Chemiluminescent reactions.
Some examples of Liquid-phase Chemiluminescent reaction would be:
Luminol (C8H7N3O2) is a compound which produces a blue glow when it reacts with an
appropriate oxidizing agent like Hydrogen Peroxide. Usually for this reaction the Luminol is
used in an alkaline solution.
A typical Glowstick reaction can be classified as a Liquid-phase reaction. It involves a reaction
between an oxalate and an oxidizing agent in the presence of a dye.
Some examples of Gas-phase Chemiluminescent reactions would be:
Nitric Oxide detection of environmental air-quality testing: An extensively studied reaction
where the reaction produces excited nitrogen dioxide (NO2).
Elemental Phosphorus oxidizing in air is considered a gas-phase reaction as it occurs between
phosphorus vapor that is present above the solid and oxygen. It gives out a greenish glow.
The light emitted by each of this reactions falls under a different wavelength but all under the
visible region of spectrum. Particularly, in the Glowstick reaction varying colors can be
produced by using different kinds of fluorophores.
Many biological assays involve Liquid-phase Chemiluminescent reactions where the protein
(substrate) is taken in liquid form.
Based on the light emitting efficiency of these reactions, they find wide-scale applications in the
industry. Sometimes the natural reactions are mimicked in the laboratory and used for cancer
research or HIV/AIDS detection.
Almost all Chemiluminescent reactions are oxidizing reactions where the oxidized intermediate
is an excited electronic state. They are also irreversible reactions and involve the participation of
catalysts. The light produced can lasts for a few seconds or even days. Some particular dyes used
in the Glowsticks continues to glow for days.
Another example that will be discussed in this project that belongs to the realm of
Biochemiluminescence or Bioluminescence is the Firefly-Luciferin reaction used by male
fireflies to attract mate.

7. Nitric Oxide (NO) Detection using Ozone (O3)[4]
This reaction is generally used in analytical instruments that detect the level of Nitric Oxide that
is emitted from automobile exhausts. It was developed in the 70s as an instant and quantitative
method for detecting Nitric Oxide levels in the atmosphere. Nitric Oxide (NO) is a secondary air
pollutant that contributes to ozone layer depletion and Nitric Oxide can convert into Nitric acid
which is a major component in acid rain.
Nitric Oxide is reacted with Ozone and the reaction produces Nitrogen dioxide (NO2) in its
excited state. The excited Nitrogen dioxide luminesces between infrared and visible region of the
spectrum.
The reaction can be represented as follows:
NO + O3 → [NO2]]* → NO2 + Light.
[NO2]* represents the excited state.
The amount of photon produced is proportional to the amount of Nitric Oxide (NO) present in
the sample.
This reaction is shown only by Nitric Oxide and not by Nitrogen dioxide. However, Nitrogen
dioxide (NO2) is a secondary air pollutant as well. In case the sample contains Nitrogen dioxide,
the sample is first converted to Nitric Oxide by a converter and then the Ozone reaction takes
place. The photon count produced in this reaction is proportional to the amount of Nitric Oxide
which in turn is proportional to the amount of Nitrogen dioxide present in the sample initially. In
case the mixture consists of both NO and NO2, the ozone reaction will be carried out directly.
The ozone reaction will not activate the NO2 present in the sample. Photons will be emitted only
by the excited NO2 which is the product of the NO and O3 reaction. Thus the output will consists
of both activated NO2 and non-activated NO2. Since the amount of activated NO2 is proportional
to the amount of NO, we can take this as a measure of NO present in the sample. By subtracting
the amount of NO from the original sample (NO+NO2), we get the amount of NO2 present in the
sample originally.
Ozone is produced from dry, cold oxygen by silent, electric discharge in the Ozonizer. This
ozone stream and the NO sample is mixed in a dark chamber where a photomultiplier (Light
detector) amplifies the Chemiluminescent signal. These photomultipliers are highly sensitive
instruments and thus are able to detect Nitric Oxide levels in parts per trillion range. Some
detectors can measure the Nitric Oxide levels to six orders of magnitude.

8. LUMINOL REACTION[5]
Luminol (C8H7N3O2) is a whitish-yellow compound that shows the property of
Chemiluminescence in the presence of an oxidizing agent. It is soluble in polar organic solvents
but insoluble in water.
Luminol is used by forensic analysts to detect microscopic traces of blood present at a crime
scene. The compound reacts with iron present in haemoglobin and in the presence of an oxidant,
emits a blue glow. It is also used during biological assays to detect copper, iron, cyanides and
specific proteins.
The structure of Luminol:
Figure 1: 5-Amino-2,3-dihydro-1,4-phthalazinedione -Luminol
This is a catalyzed reaction, the catalyst being a base. Luminol is dissolved in a basic aqueous
solution to increase the light emission or the rate of oxidation.
Luminol is synthesized from 3-nitrophthalic acid and hydrazine (NH2.NH2). It is reduced from
nitro group to amino group using sodium dithionite (Na2S2O4).
Figure 2: Synthesis of Luminol
Luminol shows Chemiluminescence when it is activated by an oxidant. In most cases, the
oxidant is a mixture of Hydrogen Peroxide (H2O2) and hydroxide ions in water. For forensics,
iron acts as the catalyst and causes the decomposition of Hydrogen Peroxide to water and
oxygen. Various kinds of enzymes, potassium ferricyanide, metal ions like copper (Cu), cobalt
(Co) acts as catalysts for the decomposition of hydrogen peroxide. Luminol reacts with the
hydroxide ions and forms a dianion. The Luminol dianion then reacts with the oxygen formed
from the decomposition of Hydrogen Peroxide and produces an unstable organic peroxide and

9. nitrogen. Electrons present in the peroxide moves to the ground state from excited state and
emits a photon of light. The emission has an approximate wavelength of 425-445 nm which
produces a blue glow.
The reaction can be represented as follows:
2 H2O2 → O2 + 2 H2O (catalyzed reaction; catalysts: iron, copper, cobalt, potassium ferricyanide)
Luminol (C8H7N3O2) + H2O2 → 3-APA*→ 3-APA + Light (general reaction)
*-represents the electronic excited state
3-APA- 3-aminopthalate
Figure 3: Luminol reaction with H2O2
Figure 4: Overall reaction of Luminol with H2O2

10. An experiment conducted by University Of California, Davis[6]
tried to measure the maximum
wavelength produced by the reaction as well as the decay rate of the emission. They used 15ml
of Luminol, Copper [Cu(II)] catalyst as sulfate salts and pH controllers with aqueous solution of
0.25% Hydrogen Peroxide added dropwise. They conducted this experiment at 25⁰C using a
fluorescence spectrometer and recorded the light intensity-time data for 60 seconds. They
observed that the peak value of wavelength was at 445 nm and the light emission was almost
instantaneous and reached a maximum within seconds. At about 8 seconds, the light intensity
reduced to about 50% of its original value. The scientists found that this decay rate depended on
the content of Cu(II) present in the solution. Concentrations of the components are: 1 x 10-3
M
luminol; 0.05 M sodium carbonate; 0.3 M sodium bicarbonate; 5 x 10-3
M ammonium carbonate;
1.5 x 10-3
M Cu(II) added as sulfate salt.
Figure 5(A) Wavelength emission (B) Light decay rate for Luminol reaction

11. ELEMENTAL PHOSPHORUS OXIDIZING IN AIR[7]
The greenish glow around elemental Phosphorus when it was exposed to damp air was the first
ever observed examples of Chemiluminescence. It was accidentally discovered by Hennig
Brand, a German alchemist in 1669 when he attempted to retrieve gold from human urine using
heat. He ended up extracting a white, waxy substance that glowed green in damp air.
The reaction occurs between oxygen in air and Phosphorus vapor that is absorbed into the air
around the solid Phosphorus. It is an oxidation reaction where P4 vapor evaporating from solid
Phosphorus gets oxidized and produces PO and O3 as intermediates. A further reaction between
PO and Ozone takes places that gives HPO and (PO2) in their excited states. As PO2 molecules
get back to their ground states by emitting a photon, they produce a greenish glow.
The reaction can be represented as follows:
P4 + O2 → PO + O3 → HPO + [PO2]* → HPO + [PO2] + LIGHT
[PO2]* represents excited electronic state of PO2.
This reaction is often confused with Phosphorescence. However, in Phosphorescence no
chemical reaction is involved. It includes light emission from a material that has absorbed energy
from another light source. The above mentioned reaction is purely a Chemiluminescent reactions
as it involves an oxidation reaction between Phosphorus vapor and Oxygen. Common examples
of Phosphorescence would be “glow-in-the-dark” paints and toys which have Phosphorescent
pigments like Zinc Sulfide and Calcium Sulfide present in them.
FIREFLY REACTION (LUCIFERIN)[8]
Firefly Luciferin or D-Luciferin (C11H8N2O3S2) [4S)-2-(6-hydroxy-1,3-benzothiazol-2-yl)-4,5-
dihydrothiazole-4-carboxylic acid] is a light emitting compound commonly found in various
firefly species. It is a substrate of the Luciferase protein. The Bioluminescent reaction observed
in fireflies requires oxygen as well as ATP and Magnesium to activate the Luciferin.
D-Luciferin reacts in the presence of Luciferase catalyst and ATP (adenosine triphosphate) +
Magnesium ions (Mg2+
) to give D-Luciferyl Adenylate with an AMP (adenosine
monophosphate) addition. A particular protein (PPi) is removed in the process. The Adenylate
produced further reacts with oxygen and produces a Dioxetanone ring with the loss of AMP. The
Dioxetanone ring loses a molecule of carbon dioxide to give excited Oxyluciferin in keto-form.
This excited molecule rapidly falls back to its ground state by emitting a photon of light. Usually
the glow produced is in the yellow region of the spectrum but it varies greatly from one species
to another depending on the pH changes or difference in primary structures.
The reaction proceeds in steps:
1. D-Luciferin + ATP + Mg2+ → D-Luciferyl Adenylate + PPi (catalyst: Luciferase)
2. D-Luciferyl Adenylate + O2 → Dioxetanone ring + AMP

12. 3. Dioxetanone ring → Oxyluciferin* (keto-form) (Loss of Carbon dioxide)
4. Oxyluciferin* → Oxyluciferin + Light [*-excited electronic state]
Reaction Mechanism: Bioluminescent pathway
Figure 6: Bioluminescent pathway for Luciferin Reactions in Fireflies.

13. Figure 7: Glowing lower body of a male Firefly

14. ENHANCED CHEMILUMINESCENCE (Using Horseradish Peroxidase)[9]
Enhanced Chemiluminescence is a common technique used in various biological assays for
detecting minute quantities of specific biomolecules. The process generally involves the use of
Horseradish Peroxidase (HRP) as the enzyme has the ability to amplify weak signals and
increase the detection probability of a particular target molecule. It is derived from the roots of
plant Horseradish (Armoracia rusticana). HRP oxidizes a particular substrate using Hydrogen
Peroxide as the oxidizing agent and produces light that can be detected using spectrophotometric
methods.
For biological assays, HRP attaches itself to an antibody that recognizes a specific molecule. The
enzyme complex acts as a catalyst in the conversion of a Chemiluminescent substrate into a
sensitive reagent near the target molecule. The reagent undergoes oxidation with Hydrogen
Peroxide to form a triplet carbonyl (C=O) in excited state that decays to a singlet carbonyl
(ground state) by emitting a photon. The light produced in the vicinity of the specific
biomolecule indicates the presence of that particular target molecule. The emitted light is
detected by spectrophotometric instruments. Since the HRP enzyme acts as a signal amplifier,
the technique of Enhanced Chemiluminescence helps in the detection of minute quantities of
biomolecules like proteins that can be detected down to femto-quantities (10-15
).
This reaction is molecule-specific and varies between different types of biomolecules found in
plants and animals.
General Processes involved are:
1. HRP + Antibody → HRP-Antibody complex
2. Chemiluminescent Substrate → Sensitized Reagent (Catalyst: HRP-Antibody
complex)
3. Sensitized Reagent + H2O2 → Triplet Carbonyl* (Oxidation)
4. Triplet Carbonyl* → Singlet Carbonyl + Light
*-represents excited electronic state

15. CAUSES/MECHANISMS OF CHEMILUMINESCENCE[10]
The exact cause for some reactions to show Chemiluminescence has not been explained. Several
theories have been proposed but they share one common link: the excited electrons of the
product decaying to ground level by emitting a photon as the last step.
One theory suggests that the role of the oxidizing agent is crucial for the light emission as the
process of oxidation provides energy to the product and excites it. Since most of the
Chemiluminescent reactions are oxidation reactions, this theory holds true in such cases.
Another theory involves blocking a particular pathway and enhancing the Chemiluminescent
pathway. If a chemical reaction can produce products using multiple pathways, then by blocking
one pathway (say, exothermic one) we will be able get a Chemiluminescence reaction. The
blocking can be done mechanically or chemically by using different chemical environment or
different catalysts. The energy evolved in a reaction needs to be given out to the surrounding
environment. If we block the thermal pathway of energy transfer, then the reaction may re-orient
itself and use a luminescent pathway for giving out its energy. Dissipation of energy as heat
(exothermic) is termed as “non-radiative relaxation” whereas dissipation of energy involving
radiation is termed as “radiative relaxation”.
Another theory involves the breaking of the bond. The amount of energy associated with a
chemical bond also depends upon (besides other factors) on the way it is arranged in space.
There are situations when a particular way of bond-breaking will give rise to a
Chemiluminescent reaction. The amount of energy released during bond-breaking gets
transferred to the product and excites it.
As we have seen in the examples of Chemiluminescent reactions, most of the reactions involve
the fragmentation or breaking of the O-O bond of an organic peroxide compound. In the luminol
reaction, the dianion converting into a triplet dianion and in the firefly reaction, the dioxetanone
ring converting into oxyluciferin, involve the fragmentation of the O-O bond present in an
organic peroxide compound. Cyclic peroxides are prevalent in most Chemiluminescent reaction
because the O-O bond can be cleaved easily as it is a relatively weak bond. After the O-O bond
breaks, the molecule reorganizes itself releasing a large amount of energy which excites the
product. The excited product comes back to the ground level by emitting a photon of light.
A sensitive Chemiluminescent reaction is the one which has the maximum efficiency in
generating photons of light. As mentioned earlier, one Chemiluminescent compound or molecule
can produce only one photon of light. A perfectly efficient reaction is one which has a
Chemiluminescent Quantum Yield of 1, that is, one photon released per molecule reacted. The
Chemiluminescent Quantum Yield is denoted by ΦCL.
The equation for Chemiluminescent Quantum Yield is given by:
ΦCL= ΦCE x ΦF x ΦR.

16. Where, ΦCE represents Chemiexcitation Quantum Yield which is the probability of generating
an electronic excited state and has a value between 0 and 1. (0 would represent a completely dark
reaction, 1 would represent all products molecules are generated in the excited state.) It has been
observed that the most efficient Chemiluminescent reactions have a value of 10-3
or greater for
ΦCE.
ΦF represents Fluorescence Quantum Yield which is the probability of the excited state emitting
a photon by Fluorescence rather than decaying by some other processes such as heat or
Phosphorescence. This also has a value between 0 and 1. In most cases, ΦF has a value of 0.1.
(By definition, ΦF is the number of photon emitted to the number of photon absorbed.)
ΦR represents Reaction Quantum Yield which is the number of reactant molecules at the start of
the reaction that undergo the Chemiluminescent reaction rather than a side reaction. The value of
this factor is usually 1.
There is one more kind of Chemiluminescence that follows a slightly different kind of
mechanism. It is called “sensitized Chemiluminescence” in which the excited electronic energy
is transferred to a fluorophore. In the Glowstick reaction which we will discuss later, we will see
this type of mechanism where the initial energy produced gets transferred to a sensitized
molecule i.e. the fluorophore, which becomes the light emitter.

17. APPLICATIONS OF CHEMILUMINESCENCE[11]
The phenomenon of Chemiluminescence finds many applications especially in the biological
field. The process of light-emitting chemical reaction is used in the detection of various cancers
and other diseases like AIDS. It is also used in detecting trace quantities of several inorganic ions
as well. Some pharmaceutical companies use this process to test a particular drug’s ability to
bind to the molecule of interest. The light signals are usually amplified and the site of the
reaction is analyzed to improve the reactivity of the drug.
Nowadays with advanced electronics, it is possible to amplify minute amounts of light signals.
This expands the application range for Chemiluminescence as the principle can now be applied
to various sensitive analytical or bio-analytical techniques or assays that quantify particular
compounds in a sample. All such analytical assays or techniques are mentioned in the book,
Journal of Bioluminescence and Chemiluminescence.
Some new research in the field of Chemiluminescence also includes extracting enzymes and
proteins found in fireflies and other light-emitting organisms like deep-water jellyfish to quantify
certain levels of ions in the human body.
Chemiluminescence is extensively used in forensics study to detect trace amounts of blood left at
a crime scene. Another application of Chemiluminescence includes Combustion Diagnostics
where the principle is used to understand unsteady heat release in an unstable combustion
system.
Scientists are also using this technique to artificially create a sequence of DNA which is then
used to create genes for vaccine research or optimize protein expression. The technique is also
used to identify the presence of a particular compound if the reactant or the substrate is non-
Chemiluminescent but the product is. This principle is used for detecting both inorganic and
organic species present in a sample where the light emitted by the species, in some cases, is a
characteristic property of the species. At times, the light-emitted by a particular species indicates
the amount of energy present in the species. However, light-emitted by a particular species can
be varied by using different reaction conditions.
Chemiluminescence is also used to detect the levels of NO2 and NO in air as mentioned earlier
where [NO2]* produces a glow before decaying to NO2 in the non-excited state.
Other than the above mentioned applications, Chemiluminescence forms the basis in making
lighting objects like emergency glow-sticks which are used by the armed forces for signaling in
combat situations.
Some of the applications that we will be discussing in detail are: ELISA and Western Blot
detection methods, Pyrosequencing of DNA, Combustion Diagnostics, Chemiluminescence in
forensics and Emergency glow-sticks.

18. ELISA BLOT TECHNIQUE[12]
ELISA stands for Enzyme Linked Immuno-Sorbent Assay. It is a bio-chemistry assay that uses
the principle of Chemiluminescence to identify a substance where the enzyme-substrate reaction
emits light. It involves the use of primary antibody and secondary antibody to detect the presence
of a particular type of antigen. It was first developed in 1971 by Peter Perlmann, Eva Engvall,
Anton Schuurs and Bauke van Weemen. (The immune-sorbent method was developed by Wide
and Jerker Porath in 1966). It is a form of wet-test where the analyte is kept in wells to avoid
mixing of samples. This assay technique is used for the detection of HIV virus and several food
allergens. It is also used in toxicology to detect a certain class of drugs.
In this technique, the reaction well is washed after every step to remove unbound Ag/Ab and
increase the sensitivity of the test.
There are three types of ELISA techniques:
1. Competitive ELISA- In this method, the unlabeled antibodies are developed in the
presence of unlabeled antigens (sample). These Antigen-Antibody (Ag-Ab) complexes
are then added to an antigen-coated (labeled) well. The test plate is washed to remove the
unbound antibodies. A secondary antibody, coupled with an enzyme is added to the
primary antibody. A substrate is added that reacts with the enzyme and undergoes a color
change or emits a fluorescent signal. This indicates the presence of the primary antigen in
the sample. It is called Competitive ELISA because the Antigen present in the sample
competes with the Antigen coating the well for Antibody binding sites. The more
antigens present in the sample, the more Ag-Ab complexes will be formed, which means
that less unbound antibodies will be available for the antigens coating the well.
The commercially available kit includes enzyme-linked antigen in which the labelled
antigen (labelled with a reporter like alkaline Phosphatase) competes with the unlabeled
antigen present in the sample. Less the number of antigen present in the sample, more
labelled antigen will be retained in the well and thus the output signal will be stronger.
2. Sandwich ELISA- In this method, a “capture” antibody is added to the plate/well first.
Before adding the antigen-containing sample, all non-specific binding sites are blocked.
After the antigen is added to the plate, it is washed to remove unbound antigens. A
specific primary antibody is then added that binds to the antigens present on the plate.
This is why it is called Sandwich ELISA as the antigen is sandwiched between two
antibodies. A secondary antibody (specific to the primary antibody) coupled with an
enzyme is added to the plate. A substrate is added that reacts with the enzyme and
produces Chemiluminescent signals that can be detected using a spectrometer.
3. Indirect ELISA- This method is essentially the same as the Sandwich ELISA method,
however, no “capture” antibody is used in this method. The antigens are added to the
well, followed by the addition of primary antibodies. Secondary antibodies coupled with
the enzyme is added to the well. A substrate reacts with the enzyme producing a light
signal or a color change and indicating the presence of that particular antigen in the
sample. Sandwich ELISA method is preferred over this method, as the sample may

19. contain multiple proteins that may coat the well and the antigen has to compete with
these proteins to bind to the well surface.
In all the methods, the Chemiluminescent signal produced will be more and faster if more
number of primary antibodies are present in the sample as that would mean that more number of
secondary antibodies coupled with enzymes have bound to the primary antibodies. This
technique involves the use of serum as the source antigen.
For the HIV test: The person’s serum is diluted 400 times and then tested. It is added to an
already treated HIV antigen plate. If the person is infected with the virus, his serum will contain
the HIV antibodies. These antibodies will bind to the plate containing the HIV antigens and will
show color change or emit a Chemiluminescent signal when the secondary antibody attaches
itself to the HIV antibody (primary) and the enzyme coupled with the secondary antibody reacts
with the substrate. The results are quantified depending upon the signal obtained to measure the
concentration of HIV antibody present in the blood.
ELISA technique is a highly-sensitive detection assay and recent advancement in the field
includes using nanoparticles to detect the color change.
Figure 8: Testing wells for ELISA blot
Figure 9: Different types of ELISA blots

20. WESTERN BLOT TECHNIQUE[12]
The western blot technique is used for analytical detection of specific proteins present in a
sample of mostly tissue extract. This technique was developed by Harry Towbin and is also
called protein immunoblot. The results from this test is analyzed using various detection
methods. We will be dealing with the Chemiluminescent Detection method.
This technique is carried out in multiple steps. They are:
1. Tissue Preparation- Cellular samples or tissue samples are taken from cell culture or
whole tissues. Solid tissues are broken down using mechanical methods such as blender
or homogenizer.
2. Gel Electrophoresis- Protein is separated using gel electrophoresis which is a method of
separating and analysis of macromolecules based on their size and shape. The gel used in
this process is a polyacrylamide gel with sodium dodecyl sulfate acting (SDS-PAGE-
sodium dodecyl sulfate polyacrylamide gel electrophoresis) as the buffer. The proteins
which gets covered by the negative SDS gel migrates towards the positive electrode
through the acrylamide mesh of the gel.
3. Transfer- After the required protein is obtained, it is transferred to a membrane for
detection by the antibody. The membrane used is either nitrocellulose or polyvinylidene
difluoride (PVDF). The proteins are transferred using a process called electroblotting that
uses an electric current to transfer the proteins from the gel to the membrane. The
nitrocellulose membrane is a sticky membrane that immobilizes the protein and PVDF is
resistant to solvents so it can be stripped and re-used to analyze other proteins.
4. Blocking- The membrane is used for binding proteins and since the antibody itself is a
protein, the membrane is treated so that only the target protein can be detected and
interactions are avoided between the membrane and the antibody. The membrane is
placed in a dilute protein solution such as Bovine Serum Albumin. This protein covers all
the free spaces on the membrane and thus the antibody can bind to the protein only.
5. Detection- The membrane containing the protein is treated with a modified antibody
coupled with a reporter enzyme. Different substrates are used for different methods of
detection and analysis. This detection process is usually carried out in 2 steps: (A)
Primary Antibody as part of immune response on exposure to the protein. (B) Secondary
Antibody with the reporter enzyme attached to it that binds to the primary antibody. The
reporter enzyme can be alkaline phosphatase or horseradish peroxidase (Enhanced
Chemiluminescence).
6. Analysis- Chemiluminescent Method: The western blot now contains probes that are
labelled and bound to the protein. For the Chemiluminescent method, the substrate is
chosen in such a way that it will emit light when it reacts with the reporter enzyme. The
light is either detected using photographic film or CCD cameras. The image is analyzed
by densitometry that analyzes and evaluates the relative staining of the protein and it
quantifies the result in terms of optical density.
Since this method extracts only the protein of interest, the sensitivity of the system increases.

21. Figure 10: Illustration of Western Blot
Figure 11: Western Blot

22. PYROSEQUENCING OF DNA[13]
Pyrosequencing of DNA (deoxyribo-nucleic acid) basically involves a procedure where a
complementary strand of DNA is developed by using the original DNA as the template. It helps
in determining the order of the nucleotides in the DNA sequence by synthesizing its
complementary strand. It is based on the “sequencing by synthesis” principle.
It was first developed by Mostafa Ronaghi and Pål Nyrén at the Royal Institute of Technology in
Stockholm in 1996.
Figure 12: Pyrosequencing of DNA
The process involves the use of Luciferin for the emission of the Chemiluminescent signal. At
every step one of the four nucleotides- A, T, C and G are added and based on the intensity
obtained, we can predict if one of these nucleotides appear in a row in the sequence. This
methods tries to detect the activity between DNA polymerase (DNA synthesizing enzyme) and a
Chemiluminescent enzyme (in most cases it is Luciferin). Since this method synthesizes one base
pair at a time, we can detect which base pair was actually added to the strand. To carry out the
process, the template DNA is made immobile, and solutions containing A, G, T and C are added
and removed sequentially from the reaction. A Chemiluminescent signal is given out when the
nucleotide solution matches with the unpaired base of the template DNA. Based on the
Chemiluminescent signals obtained we can determine the sequence of the template DNA.
First, the template DNA is converted into a primer (which is a starting point for the DNA strand)
and is incubated with enzymes like DNA polymerase, ATP sulfurylase, luciferase, apyrase and
substrates- Luciferin and adenosine 5’phosphosulfate. DNA polymerase is responsible for
copying the DNA sequence. It looks at the template DNA and picks up the complementary
nucleotide from the solution and adds it to the DNA strand.
One of the four deoxynucleoside triphosphates [dNTP] (dATPαS, dCTP, dTTP and dGTP) is
added to the solution containing the template DNA. The DNA polymerase identifies the right

23. dNTP and adds it to the chain. This releases pyrophosphate (PPi) into the solution. The ATP
sulfurylase present in the solution picks up the PPi and converts it into ATP in the presence of
adenosine 5’phosphosulfate. This ATP converts Luciferin into Oxy-Luciferin with Luciferase
acting as a catalyst. The Oxy-Luciferin in its excited state emits a photon of light before
decaying to the ground level. This emitted light is picked up by cameras and analyzed. The other
non-attached dNTPs are broken down by apyrase and the reaction re-starts with the attachment
of the next dNTP. The amount of light produced is directly proportional to the amount of ATP
produced. In case the dNTP solution taken does not match with the template DNA, then no light
will be produced as PPi won’t be released into the solution.
Genome assembly is difficult using this process as the process restricts the length of the DNA
chain. Nowadays, Pyrosequencing is carried out in two ways- (A) Liquid-Phase Pyrosequencing
with apyrase and exonuclease [enzymatic]. (B) Solid-Phase Pyrosequencing with streptavidin
coated magnetic beads.
Figure 13: Pyrosequencing

24. FORENSIC STUDIES[14]
As we saw above that Luminol produces a blue glow when it reacts with Hydrogen Peroxide in
the presence of a base (hydroxide ions). The decomposition of Hydrogen Peroxide is usually
catalyzed by iron compounds. This principle is used by forensic specialists to detect minute
traces of blood present at a crime scene.
The haemoglobin present in the blood contains iron. The iron catalyzes the decomposition of
Hydrogen Peroxide leading to the production of oxygen. The Luminol dianion (Luminol +
hydroxide ions) reacts with this oxygen and produces the excited triplet state that decomposes to
produce the blue glow. Since this is a very sensitive reaction and requires only a small quantity
of iron to catalyze the decomposition reaction, it can pick up even trace quantities of blood, even
if the blood is cleaned or removed. The blue glow is observed better if it is a very dark room. For
trace quantities of blood, the glow lasts for about 30 seconds.
Figure 14: Blue glow of Luminol at a crime scene
This method was first proposed by a German chemist H. O. Albrecht in 1928 and later developed
by Karl Gleu, Karl Pfannstiel and German forensic scientist Walter Specht. However this method
has certain drawbacks. Luminol will give a blue glow when it reacts with copper-containing
substances and bleaches. Thus if the crime scene was cleaned using a bleaching agent, the
Luminol test will come positive. Plus, Luminol reacts with iron present in fecal matter as well as
urine (if it contains small amount of blood), therefore in such cases the forensic analysis will get
distorted.
CANCER RESEARCH[15]
Chemiluminescence is used for cancer research in two ways-(A) Detection of the malignant
tumor and (B) drug testing.

25. For detection of malignant tumor, scientists use several fluorescent materials or reporter enzymes
that attach themselves to the lipids and proteins associated with that tumor. They use suitable
substrates that causes the reporter enzyme or the fluorescent material to luminesce. This is
detected under the microscope and confirms the presence of the tumor.
For drug testing, scientist extract the gene responsible for the glow of firefly along with the
protein Luciferin and use these as the Chemiluminescent material to test how a cancer drug
chokes off a tumor’s blood supply. The Chemiluminescent signals are detected using
Bioluminescence imaging. Pharmaceutical companies use this process extensively while testing
the drug in its pre-clinical stages as it an effective and economical method.
COMBUSTION ANALYSIS[16]
Combustion analysis is a method used to study and explain the unsteady heat release of an
unstable combustion system. Chemiluminescent methods help in quantifying the data obtained
from a combustion system and new models have been developed to increase the accuracy and the
sensitivity of the process.
In a Chemiluminescent reaction, the emission produced is a characteristic property of the
molecule as well the path it undertook for that transition. Depending on the nature of the
molecule, we get different types of spectrum- continuous or simple. More complex molecules
will produce a continuous spectrum, example-CO2; whereas molecules like OH, CH and C2
produce simple spectrums with major peaks at 308nm, 431nm and 513 nm respectively.
Flame Chemiluminescence is the Chemiluminescence observed in flames. It is seen that the
concentrations of excited molecular species seen in flames exceeds the normal concentration of
species at equilibrium when no chemical reactions take place by many orders of magnitude. This
helps us to conclude the fact that excited species are produced by thermal agitation as well as
chemical reactions. Since amount of radiation/emission observed in a flame at a particular
wavelength is directly proportional to the concentration of the species present, we can use Flame
Chemiluminescence to find out the concentration of the species. We can also use this method to
detect the nature and identity of the species present by analyzing the radiation obtained. By
calculating the wavelength of the radiation obtained, we can figure out the identity of the species
using the emission spectra literature available. Thus, intensity of the radiation is used to calculate
the concentration of the species whereas the wavelength of the radiation is used to detect the
identity and nature of the species.
One factor however needs to be taken into account- quenching efficiency. Some of the excited
molecules lose their energy not by emission but by non-reactive collisions. Such a collision
produces no light and it depends on the colliding molecule. Quenching efficiency is defined as
the ability of a non-excited molecule to remove the excess energy present on an excited molecule
during the collision process. It varies greatly from molecule to molecule and is strongly
dependent on temperature.

26. Photoelectric Flame Photometer is used to for detecting the identity of the species as well as the
intensity of the flame emission obtained when unknown radicals and ions are introduced into the
flame. The ions or radicals are passed through the flame at a constant rate where they vaporize
by absorbing energy. This energy controls the intensity of the color obtained. It is a highly
controlled flame test. The intensity of the flame is quantified using a photoelectric circuitry and
several filters are introduced in to the apparatus to increase the accuracy of the results as well as
eliminate the interference of some ions and radicals that can distort the result.
Large elaborate reaction mechanisms that helps in explaining the behavior of reaction
intermediates are used for Flame Chemiluminescence to understand the concentrations as well as
role of intermediate Chemiluminescent species. This also helps us in understanding the particular
pathway chosen by the reaction and its kinetics. To understand such chemical kinetics and
mechanisms Combustion Modelling is developed that simulate all the process taking place in a
Flame Chemiluminescence system.

27. CHEMISTRY BEHIND A GLOWSTICK[17]
A Glowstick is a self-sustaining short-term light source that contains two sets of chemicals in a
tube. It produces light when these chemicals mix inside the tube. The reaction is irreversible and
the Glowstick can be used only once. The reaction that occurs inside the Glowstick is a
Chemiluminescent reaction where the energy from the chemical reaction gets transferred to a
fluorescent dye present inside the tube. This kind of Chemiluminescent reaction is also called a
sensitized Chemiluminescent reaction as the energy from the original reaction is transferred to
the dye which gets excited to a higher energy level. The illumination produced by the Glowstick
is not bright, however depending on the fluorescent dye used, the glow can last for hours to days.
It is used extensively by military, navy and air forces as it an inconspicuous and easily shielded
illumination device which can be used during Special Operations. It is also used during
emergency medical services and by divers as a navigation aid in muddy waters. As it useful in
highlighting someone’s position in the dark, it is used during situations like after an earthquake
or nighttime scuba diving where electricity cannot be used.
The Glowstick was first invented for the US Navy and the main chemical: Cyalume was
developed by Frank Arthen and Laszlo J. Bollyky of the American Cyanamid Company. Other
researchers include Herbert Richter of China Lake Naval Weapons Centre.
Glowstick has several advantages over flashlights. It is waterproof, doesn’t require a battery,
generates negligible amount of heat, are inexpensive and can be disposed. Rescuers recommend
the use of Glowsticks during catastrophic emergencies.
The Glowstick contains an inner glass vial and an outer plastic tube. For illumination, the
Glowstick is snapped. The chemicals present inside the glass vial mixes with the chemicals
present inside the plastic tube and a bright glow is produced. For better illumination, the
Glowstick is thoroughly shaken after snapping to aid the mixing of the chemicals.
The basic reaction is an oxidation reaction between a Diphenyl Oxalate and an oxidant.
Hydrogen Peroxide is commonly used as the oxidant because the O-O bond is easily cleaved and
this releases enough energy to excite the dye. Cyalume produces phenol on reaction with
Hydrogen Peroxide. The phenol which is produced is harmful and it is advised to keep it away
from skin as it can cause irritation and burns. Some of the fluorophores used are carcinogenic.
Thus, care needs to be taken in case the Glowstick tube is cracked or broken.
In this project, we will discuss the parts of the Glowstick, Mechanism of the reaction and its
Chemical kinetics along with the synthesis of TCPO (a Cyalume) and a working Glowstick. We
will also discuss how the reaction gets affected by temperature and buffers used.

28. PARTS OF A GLOWSTICK
The Glowstick contains two tubes: inner glass vial and outer plastic tube. The inner glass vial
contains the oxidant whereas the outer plastic tube contains the Diphenyl Oxalate and the
fluorescent dye.
1. Diphenyl Oxalate (Cyalume) – This is the main chemical responsible for the glow. It is a
symmetric diester. It is to be noted that Cyalume itself is a non-Chemiluminescent
substance. The reaction between the Cyalume and the oxidant doesn’t produce light. It
releases sufficient energy to excite the fluorophore. Most commercially available
Glowsticks uses TCPO [bis-(2, 4, 6-trichlorophenyl) oxalate]. This is usually mixed with
the fluorophore.
2. Hydrogen Peroxide (H2O2) – This is the oxidant that oxidizes the Cyalume and releases
energy. As mentioned above, Hydrogen Peroxide is used because the O-O bond can be
easily cleaved which releases sufficient amount of energy. It is kept inside the glass vial.
3. Fluorophore- This absorbs the energy released from the reaction between Cyalume and
H2O2. It shifts to an excited state and readily decays to the ground state by releasing a
photon of light. Depending on the fluorophore used, we get different colors of light. The
fluorophore used in the Glowstick is usually an anthracene derivative which has
chromogenic properties.
4. Solvent- The role of the solvent is to dissolve and bind the Cyalume to the dye.
Depending on the solvent used, the glow can last for several minutes to several hours.
The efficiency of the solvent in binding the Cyalume will decide the glow duration. For
example: Ethyl acetate causes the glow to last for several minutes whereas diethyl
phthalate causes the glow to last for hours.
A basic condition is favored because it helps in transforming the Cyalume into a dione as the
intermediate formed is peroxyacid ester. The reaction is also temperature-dependent.
Figure 15: Using a Glowsticks.

29. REACTION OF THE GLOWSTICK
The Glowstick reaction takes place between two sets of chemicals. One is the chemical that is
responsible for the glow which is mixed with the fluorophore and the other is the oxidant which
oxidizes the chemical and releases energy. Generally the plastic tube contains the fluorophore
and the chemical whereas the glass vial contains the oxidant, in most cases it is Hydrogen
Peroxide. The chemical used is a Diphenyl Oxalate which on oxidation with Hydrogen Peroxide
gives an unstable dione that readily decomposes to carbon dioxide and in the process ends up
exciting the dye that emits the glow.
The reaction between Diphenyl Oxalate and Hydrogen Peroxide produces two molecules of
phenol and one molecule of peroxyacid ester (1, 2-dioxetanedione). This is a highly unstable
compound that decomposes to give carbon dioxide and releases a large amount of energy. This
energy is absorbed by the fluorophore and it gets excited. The excited dye emits a photon of light
and decays back to the ground level.
This reaction is favored if the conditions are slightly basic as the intermediate produced is a
peroxyacid ester and thus the reaction between the Diphenyl Oxalate and Hydrogen Peroxide
gets accelerated. Mostly a weak base is used such as Sodium Salicylate. This reaction is a
temperature-dependent reaction.
The reaction can be represented as follows:
C14H10O4 (Diphenyl Oxalate) + H2O2 → C6H5OH (Phenol) + C2O4 (1, 2-Dioxetanedione)
C2O4 + Fluorophore → CO2 + [Fluorophore]*
[Fluorophore]* → [Fluorophore] + Light
Structure of Diphenyl Oxalate:
Structure of 1, 2-Dioxetanedione:
Reaction can be represented as:

30. Figure 16: Overall Glowstick reaction
This is the general reaction for a Glowstick Chemiluminescence. For an effective reaction, the
Diphenyl Oxalate that is mostly used is bis-(2, 4, 6-trichlorophenyl) oxalate or TCPO. All
Diphenyl Oxalates are trademarked as Cyalume.
TCPO is synthesized from an Esterification reaction between Phenol and Oxalic acid. Mostly an
Oxalyl Chloride is used, which is a symmetric acid chloride that is highly electrophilic and
reactive because of the Chloride leaving group present in the molecule. Since a diester is
required, two molecules of trichloro-Phenol are taken which reacts with one molecule of Oxalyl
Chloride to form bis-(2, 4, 6-trichlorophenyl) oxalate or TCPO. A basic conditions is used to
absorb the 2 molecules of HCl that is produced during the formation of diester. The base that is
used is Triethylamine.
Figure 17: Synthesis of TCPO
The TCPO formed is a solid which is easier to handle. Plus, the trichlorophenolate group that is
present on the molecule is a better leaving group and helps in accelerating the reaction. Mostly it
is dissolved in a solvent such as diethyl phthalate or ethyl acetate that helps in binding the
compound to the oxidant.

31. Reaction with TCPO as the reactant:
Figure 18: Reaction with TCPO as reactant
Hydrogen Peroxide exchanges oxygen with TCPO. One trichloro-Phenol is released while the
two oxygen molecules of the Hydrogen Peroxide joins the oxalate molecule. This intermediate
spontaneously transforms into 1, 2-dioxetanedione and gives out a molecule of trichloro-Phenol.
The square diester formed experiences a lot of ring-strain which makes it unstable. It quickly
decomposes to give two molecules of carbon dioxide and releases a lot of energy in the process.
The fluorophore picks up this energy, gets excited and decomposes back to the ground level by
emitting a photon of light.
The color of light emitted depends on the fluorophore used which in most case is an anthracene
derivative that exhibits chromogenic property.

32. KINETICS OF THE REACTION
As the Dioxetanedione fragments, it releases energy that “excites” the colorizer or the
fluorophore. The colorizer gets excited from its ground state level to a higher energy level. When
it relaxes back to the ground state level, it releases a photon of light. In case the energy gap
between the two levels ΔE lies in the visible region of the electromagnetic spectrum, visible
photon of a distinct color is released. This is responsible for the bright color of the Glowstick.
The reagent TCPO is not a fluorophore. It is not responsible for the bright glow of the stick. It
acts an energy supplier that excites the fluorophore. Therefore, it is necessary that a fluorophore
is present when the Glowstick reaction takes possible.
Figure 19: Energy levels of the fluorophore
Some key features of the excitation/relaxation process:
1) The energy gap of HOMO (Highest occupied molecular orbital) and the LUMO (Lowest
unoccupied molecular orbital) determines the photon frequency and color of the photon
released.
2) For most organic compounds, the HOMO-LUMO gap doesn’t lie in the visible frequency
(7.5X1014
Hz- 3.75X1014
Hz).
3) To have the HOMO-LUMO gap in the visible spectrum, the molecule must show
extensive conjugation.
4) The fluorophore is required in a catalytic amount. The excitation/relaxation is a cyclic
process that regenerates the original molecule in the ground state which is ready to repeat
the process.

33. FACTORS AFFECTING THE REACTION:-
1. TEMPERATURE: Heat has a direct impact on the rate of the reaction. As the
temperature is increased, the reaction proceeds faster. Heating the Glowstick increases
the rate of reaction and the Glowstick starts glowing brightly for a brief period. Cooling
the Glowstick reduces the rate of the reaction. Thus, the Glowstick will glow dimmer but
it will last for a longer period of time.
2. BASIC CONDITIONS AND LEAVING GROUPS: A slightly basic reaction condition
favors the reaction as the reaction between Diphenyl oxalate and Hydrogen Peroxide
produces a peroxyacid ester. The basic condition accelerates the reaction by neutralizing
the acid-ester. TCPO is favored for commercial Glowsticks because of the
trichlorophenolate leaving group present on the molecule. One of the trichlorophenolate
leaving group easily detaches itself from the molecule. This accelerates the reaction and
the Glowstick glows brighter as compared to a normal phenol ester.
3. FLUOROPHORE WAVELENGTH [18]
: The light emitted by the Glowstick depends
upon the fluorophore. A fluorophore absorbs light radiation of a specific wavelength and
re-emits a light radiation having a longer wavelength. The structure of the fluorophore
influences which wavelength the fluorophore absorbs, the efficiency of energy transfer
and the time before which the fluorophore re-emits the radiation of longer wavelength.
The chemical environment (acidic or basic) also influences the properties of the
fluorophore as the excited molecule of the fluorophore reacts with surrounding molecules
present in the reacting medium. As the emitted radiation has a longer wavelength, it has
lower energy. In most cases, the emission spectrum of fluorophores lies in the visible to
infrared region. Fluorophores also show fluorescence under ultraviolet radiation.
WHY THE REACTION PROGRESSES SLOWLY?
The forward reaction proceeds slowly and releases light radiation rather than heat is because the
forward activation energy of this reaction is sufficiently high. The reverse reaction is the 2+2
photocyclic addition of the 1, 2-dioxetandione. This reaction is a forbidden reaction, i.e., the
probability of the reverse reaction occurring is low. The activation energy of the reverse reaction
is extremely high which is why the reaction never occurs. Plus the reaction violates the
Woodward-Hoffmann rules in organic chemistry which states that a peri-cyclic reaction is
allowed if the energy barrier is low whereas the reaction is a forbidden reaction if the energy
barrier is high.

34. VARIATIONS AVAILABLE IN THE MARKET:-
At times, the manufacturers vary the ratio of concentrations of the chemicals used in the
Glowstick for certain purposes. They alter the brightness and the time-duration of the Glowstick
using this method. In some situations, the Glowstick produces a bright light for a short period of
time (for example, recreation purposes) whereas in some cases, the Glowstick produces a dim
light for a long period of time (for example, for use in the armed forces).
By changing the ratio of concentration of the chemicals used, the manufacturers essentially
negate the effect of temperature on the rate of the reaction. This way they are able to produce
Glowsticks that show a temperature-independent reaction and can be used satisfactorily in either
hot or cold climates.
Some Glowsticks are available in the market which contain a fluorescent plastic tube. The plastic
tube is coated with a fluorophore which fluorescence under black light.
The following picture shows a disassembled orange Glowstick which has a fluorescent plastic
tube.
The first shot shows the original, intact Glowstick. Second shot shows the disassembled
Glowstick: Hydrogen Peroxide mixture is in the measuring cylinder and the fluorophore is in the
glass vial. Third shot shows all three of them in black light- As it can be seen, the plastic tube is
also showing fluorescence under black light. Fourth shot shows the fluorophore mixed with
Hydrogen Peroxide mixture giving a greenish-yellow glow. Fifth shot shows the light given off
by the Glowstick when the glowing activated mixture of Fluorophore and Hydrogen Peroxide is
added to the plastic tube-Note: The color given off is now orangish which is the intermediate
color between the reddish-orange glow given off by the plastic tube under black light and yellow
glow produced by the Fluorophore alone under black light.
Figure 20: Disassembled orange Glowstick
The following graph shows the light radiation expressed in terms of radiative irradiance versus
wavelength emitted. The green line represent the mixture of Hydrogen Peroxide and fluorophore
(4th
shot in the picture). The yellow line represent the fluorescence of the fluorophore alone (3rd
shot in the picture). The orangish red line represent the actual glow of the stick when the H2O2-
Fluorophore mix is added back to the plastic tube (5th
shot in the picture). The red line represent
the fluorescence of the empty plastic tube under black light (3rd
shot in the picture).

35. Figure 21: Graph showing the emission spectra of the orange Glowstick
Note the change in the wavelength emitted when the H2O2-Fluorophore mixture was added to the
plastic container. The final color emitted by the Glowstick lies between the color emitted by the
Fluorophore (yellow line) and that emitted by the empty plastic tube under black light (red line).
EMISSION PROPERTIES OF THE GLOWSTICK
For most Glowsticks, the emission spectrum shifts towards the reddish region of the
electromagnetic spectrum. After Hydrogen Peroxide is added to the mixture and the chemicals
get activated, the light intensity is the brightest. As seen with Luminol reaction, the light
intensity in this reaction as well decreases exponentially as the reaction proceeds.
FURTHER DISCUSSION ABOUT THE GLOWSTICK [19]
:-
A particular study conducted by University of California, Davis proposed the alternative
pathways the Glowstick reaction can undergo. The reaction shown by the Glowsticks is also
called Peroxyoxalate Chemiluminescence or Sensitized Chemiluminescence as the energy
produced in the initial reaction between H2O2 and TCPO gets transferred to the fluorophore
which excites itself to a higher energy state.

36. Apart from the 1,2- Dioxetanedione produced in the reaction, scientists predict that other
intermediates like hydroperoxyoxalate can also be produced. The Glowstick reaction can be
summarized in 3 steps:
1) Initial oxidation of TCPO by H2O2 to produce the high energy intermediate.
2) The reaction between the intermediate and the Fluorophore.
3) Reaction of excited fluorophore to produce light.
The alternative pathways that the scientists are suggesting can lead to:
1) TCPO getting hydrolyzed instead of oxidation.
2) The intermediate reacting with a quencher and getting oxidized; intermediate
decomposing due to presence of residual H2O2; no excited fluorophore produced when
the intermediate reacts with the fluorophore.
3) The excited fluorophore decaying by producing heat instead of light.
These alternative routes are controlled by a number of factors: solvent and buffers used; pH of
the reacting medium; catalyst used; type of fuel- variety of phenyl oxalates used; oxidant and
fluorophore properties. Change in any of these parameters can cause the reaction to follow an
alternative pathway. Using this information, scientists are able to measure analytically the
concentration of H2O2 or the fluorophore content present in the system by changing one or more
parameters of the reaction.
The experiment conducted by the university aimed at studying the wavelength emission of the
Peroxyoxalate Chemiluminescence system. They took a mixture of TCPO and 9, 10-
diphenylanthracene (DPA) (fluorophore) both of concentration 1X10-3
M. The fluorophore emits
a blue light of wavelength (λ) 425nm (blue region of the visible spectrum). The solvent used for
this reaction was tetrahydrofuran (THF) and a dilute solution of H2O2 (0.3%) was taken. The
reaction was carried out at a temperature of 25⁰C. They used a fluorescence spectrometer in
Chemiluminescence mode to observe the emission spectrum. The emission spectrum was
scanned at the rate of 1200nm/min immediately after the components were mixed. The graph
obtained showed that the Chemiluminescent reaction had an emission that was around 425nm
which is also the normal fluorescent emission wavelength for diphenylanthracene.

37. DEMONSTRATION EXPERIMENT [20]
:-
PRECAUTION: The following experiment must be performed only by or under direct
supervision of an experienced chemist. Chemicals used in this experiment are toxic in
nature and must be handled with utmost caution. Synthesis must be performed in a fume-
hood or a fume-cupboard.
STEP 1: SYNTHESIS OF TCPO [bis-(2, 4, 6- trichlorophenyl) oxalate]
CHEMICALS REQUIRED: 2, 4, 6- trichlorophenol, dry toluene, ice bath, Triethylamine or
similar suitable organic base, oxalyl chloride, methanol or ethanol.
APPARATUS REQUIRED: Molecular sieves, suction or vacuum pump, syringes.
PROCEDURE:
1. Dissolve 2, 4, 6-trichlorophenol in dry toluene. (15-20 ml of toluene per gram of TCP).
Azeotropic distillation is used to obtain a dry solution.
2. Chill the above solution in an ice bath to 0⁰C.
3. Add one molar equivalent of Triethylamine. A dried base, usually over a molecular sieve,
is preferred.
4. The solution needs to be stirred in the chilled state and 0.5 mol equivalents of oxalyl
chloride is added drop wise to the mixture. This is an exothermic reaction with extensive
evolution of heat. A thick off-white precipitate is formed.
5. After the addition of oxalyl chloride, the mixture is allowed to warm to room
temperature. Stirring the mixture overnight increases the purity of the product obtained.
6. Suction filter the mixture on a fritted funnel. Discard the filtrate. TCPO is the fine, white
powder which is mixed with triethylammonium chloride at this stage.
7. The mixture is washed with methanol or ethanol to remove triethylammonium chloride
and then thoroughly dried, preferably under vacuum.
We have now obtained TCPO which is further used to carry out the Glowstick reaction.
STEP 2: PERFORMING THE GLOWSTICK REACTION
CHEMICALS REQUIRED: a) 3mg of 9,10-bis(phenyethynyl) anthracene; b) 300mg of sodium
acetate or sodium salicylate; c)10 ml of ethyl acetate or diethyl phthalate; d) TCPO; e) 3ml
Hydrogen peroxide (H2O2).
APPARATUS REQUIRED: a small beaker or a flask with a cap, a test tube for the H2O2
PROCEDURE:
1. Take diethyl phthalate or ethyl acetate in the flask. Add the fluorophore i.e., 9,10-
bis(phenyethynyl) anthracene to this. It has an orange color in the solid state but it
dissolves in diethyl phthalate to give a green color. Shake well so that the fluorophore
dissolves properly
2. Add TCPO to this mixture followed by the addition of sodium acetate. Shake well.

38. 3. Turn the lights off and add H2O2 to this mixture. As soon as H2O2 is added to the mixture,
it starts emitting a green light.
We now have our Glowstick. Usually, H2O2 is kept in a glass ampoule in the commercially
available Glowstick. The Glowstick is bend to crack this glass ampoule and allow H2O2 to mix
with the fluorophore and TCPO.
Instead of sodium acetate, sodium bicarbonate and sodium salicylate can also be used.
Instead of TCPO, DNPO [bis-(2, 4-dinitrophenyl) oxalate] and CPPO [bis-(2, 4, 5-trichloro-6-
(pentyloxycarbonyl) phenyl) oxalate] can be used.
Various fluorophores emit various colors. Some of the common ones are:
9, 10-bis (phenyethynyl) anthracene emits green light at 486nm.
Rubrene (C42H28) has a bright red color in solid state. It dissolves to give an orange color. It
emits orange-yellow light at 550nm.
9, 10-diphenylanthracene (DPA) is off-white in color in solid state. It dissolves to give a clear
solution. It emits blue light at 425nm.
Rhodamine-B (C28H31ClN2O3) is green in color in solid state. It dissolves to give a bright red
color. It emits red light at 610nm. It decays quickly therefore it is not really used in Glowsticks.
It also breakdowns in contact with CPPO which shortens its shelf life.
Ethyl acetate ensures that the glow lasts for several minutes in the dark whereas diethyl phthalate
allows the glow to last for several hours.
Figure 4: Glowsticks

39. ACKNOWLEDGEMENT:-
I would like to thank Mr.Marazban Kotwal, our Chemistry teacher and our honors program
teacher for helping us with the project. I approached him with many doubts and every time he
was gracious enough to solve them and explain them to me. I would also like to thank my friend,
Ms.Poornima Ramesh, a student at IISER, Kolkata, who is pursuing a degree in biology, for
helping with the concepts of ELISA blot, Western Blot and Pyrosequencing of DNA.
I would also like to thank Dr. Wolfgang Kaim, professor at Stuttgart University, Germany, who
gave me some vital information regarding the probable causes of Chemiluminescence like
blocking the non-radiative pathways and enhancing the Chemiluminescent pathway and the
mechanism in which a bond breaks, thereby influencing light emission.

40. REFERENCES:-
1. Information about Phosphorescence, Chemiluminescence, Fluorescence and
Triboluminescence: http://www.rocksinmyheadtoo.com/Fluor.html
2. Radioluminescence: http://en.wikipedia.org/wiki/Radioluminescence
3. Chemiluminescence: http://en.wikipedia.org/wiki/Chemiluminescence ; Lumigen Inc.,
Chemiluminescence explained; http://www.webexhibits.org/causesofcolor/4AD.html;
http://www.scienceclarified.com/everyday/Real-Life-Chemistry-Vol-
5/Luminescence.html#b;
4. Nitric Oxide detection using Ozone: Research Article: Homogenous Chemiluminescent
measurement of Nitric Oxide with Ozone. Implications for continuous selective
monitoring of gaseous air pollutants (Authors: Arthur Fontijn, Alberto J. Sabadell and
Richard J. Ronco.; http://www.innovateus.net/science/what-
chemiluminescence#What+causes+luminescence%3F
5. Luminol Reaction: Research Article: The Preparation of 3-Aminophthalhydrazide for
Use in the Demonstration of Chemiluminescence (Authors: Ernest Huntress, Lester
Stanley, Almon Parker- Journal of the American Chemical Society) ; Chemiluminescence
of Luminol: The Chemical Reaction (Author: Emil H. White, John H.M. Hill, Oliver
Zafiriou, Heinz H. Kagi-Journal of the American Chemical Society);
http://en.wikipedia.org/wiki/Luminol; http://science.howstuffworks.com/luminol.html.
6. Luminol Chemiluminescence: UC Davis
http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Electronic_Spectroscopy/
Chemiluminescence#Introduction
7. Elemental Phosphorus Oxidizing in air:
http://www.webexhibits.org/causesofcolor/4AD.html;
8. Luciferin: Firefly reaction: Aldo Roda- Chemiluminescence and Bioluminescence: Past,
Present and Future, Royal Society of Chemistry, 2010;
http://en.wikipedia.org/wiki/Firefly_luciferin;
http://www.scientificamerican.com/article/what-is-chemiluminescence/
9. Enhanced Chemiluminescence: http://en.wikipedia.org/wiki/Chemiluminescence
10. Causes/Mechanisms of Chemiluminescence: Lumingen Inc., Chemiluminescence
Explained.
11. Applications of Chemiluminescence: Journal of Chemiluminescence and
Bioluminescence
12. ELISA and Western Blot technique: Journal of Chemiluminescence and
Bioluminescence- Volume 1: Issue 1 and Issue 2; http://en.wikipedia.org/wiki/ELISA;
http://www.bio.davidson.edu/genomics/method/Westernblot.html;
http://en.wikipedia.org/wiki/Western_blot
13. Pyrosequencing of DNA: http://en.wikipedia.org/wiki/Pyrosequencing
14. Forensic Studies: http://en.wikipedia.org/wiki/Luminol; Stuart H. James and William G.
Eckert, Interpretation of Bloodstain Evidence at Crime Scenes, 2nd edition;
http://science.howstuffworks.com/luminol.html.

41. 15. Cancer Research: http://en.wikipedia.org/wiki/Chemiluminescence- (Biological
Applications).
16. Combustion Analysis: Evaluation of Chemiluminescence as a Combustion Diagnostic
under Varying Operating Conditions (Authors- Venkata Nori and Jerry Seitzman);
http://scholar.lib.vt.edu/theses/available/etd-03142001-
144036/unrestricted/05Chapter_1.pdf.
17. Chemistry Behind a Glowstick: Chem365 Labbook-2013 (Author- Craig Jasperse);
http://chemistry.about.com/od/howthingsworkfaqs/a/howlightsticks.htm;
http://science.howstuffworks.com/innovation/everyday-innovations/light-stick.htm;
“What’s that Stuff? Light Sticks”-Chemical and Engineering News( Author-Elizabeth
Wilson); http://en.wikipedia.org/wiki/Glow_stick; http://ocw.mit.edu/high-
school/chemistry/demonstrations/videos/anatomy-of-a-glowstick/glowstick.pdf;
http://en.wikipedia.org/wiki/Diphenyl_oxalate; http://en.wikipedia.org/wiki/TCPO;
18. Fluorescence: http://en.wikipedia.org/wiki/Fluorophore;
19. Peroxyoxalate Chemiluminescence:
http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Electronic_Spectroscopy/
Chemiluminescence#Introduction
20. Demonstration Experiment: http://www.wikihow.com/Make-a-Glowstick;
www.nurdrage.com; http://science.wonderhowto.com/how-to/make-your-own-
homemade-glow-sticks-0146580/; www.youtube.com/nurdrage.
FIGURES:
1. Luminol: http://en.wikipedia.org/wiki/File:Luminol.svg.
2. Luminol Synthesis: http://en.wikipedia.org/wiki/File:Luminol_synthesis.png.
3. Luminol reaction with H2O2:
http://en.wikipedia.org/wiki/File:Luminol_chemiluminescence_molecular_representation
.svg.
4. Overall Luminol reaction with H2O2: www.photochemistryportal.net
5. Wavelength emission and decay rate of emission:
http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Electronic_Spectroscopy/
Chemiluminescence#Introduction
6. Bioluminescent pathway for Luciferin reaction in fireflies:
http://en.wikipedia.org/wiki/File:Luciferase_Mechanism.gif;
http://en.wikipedia.org/wiki/File:Luciferasepathways.gif
7. Glowing lower body of a male firefly: www.firefly.org
8. Testing wells for ELISA blot: http://en.wikipedia.org/wiki/ELISA
9. Different types of ELISA: www.abnova.com
10. Illustration of Western Blot: www.leinco.com
11. Western Blot: www.comparative-hepatology.com
12. Pyrosequencing of DNA: www.clinchem.org
13. Pyrosequencing: www.bio.unipd.it
14. Blue glow of Luminol at a crime scene: www.unitednuclear.com
15. Using a Glowstick: jeanbont.pbworks.com
16. Overall Glowstick reaction: http://en.wikipedia.org/wiki/File:Cyalume-reactions.svg
17. Synthesis of TCPO: Chem-365 Labbook-2013 (Author: Craig Jasperse)

42. 18. Reaction with TCPO as reactant: Chem-365 Labbook-2013 (Author: Craig Jasperse)
19. Energy levels of the Fluorophore: Chem-365 Labbook-2013 (Author: Craig Jasperse)
20. Disassembled orange Glowstick:
http://en.wikipedia.org/wiki/File:Lightstick_disassembly.jpg
21. Graph showing the emission spectra of the orange Glowstick:
http://en.wikipedia.org/wiki/File:Chemoluminescent_lightstick_spectral_curves.png
22. TCPO/DPA Chemiluminescence:
http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Electronic_Spectroscopy/
Chemiluminescence#Introduction
23. Glowsticks: www.geekosystem.com