Chemiluminescence is the emission of light from a chemical reaction. It occurs through various processes in living organisms, electrical reactions, mechanical actions, and other means. Some key examples of chemiluminescence discussed in the document include fireflies, luminol forensic applications, and glow sticks. Chemiluminescence has many applications including DNA hybridization detection, immunoassays, forensic investigations, food analysis, and western blotting.

1. LUMINESCENCE AND CHEMILUMINISCENCE
Department of Pharmacology & toxicology
National Institute of Pharmaceutical Education and Research, S.A.S Nagar, 160062
Punjab, INDIA
Presentation by :-
Mohammad Adeel Zafar
M.S. Research Scholar

2. Flow of presentation
Introduction
Sources and process
Luminescence and incandescence
Early investigation
Types of luminescence
Chemiluminescence
Applications of chemiluminescence
2

3. LUMINESCENCE
• Luminescence is emission of light by certain materials when they are relatively cool. It is in
contrast to light emitted from incandescent bodies, such as burning wood or coal, molten
iron, and wire heated by an electric current
• Luminescence may be seen in neon and fluorescent lamps; television, radar, and X-ray
fluoroscope screens; organic substances such as luminol or the luciferins in fireflies and
glow worms; certain pigments used in outdoor advertising;
and also natural electrical phenomena such as lightning
and the aurura borealis
3

4. • In all these phenomena, light emission does not result from the material
being above room temperature, and so luminescence is often called cold
light. The practical value of luminescent materials lies in their capacity to
transform invisible forms of energy into visible light
4

5. • Luminescence emission occurs after an appropriate material has absorbed energy from a
source such as ultraviolet or X-ray radiation, electron beams, chemical reactions, and so on
• The energy lifts the atoms of the material into an excited state, and then, because excited
states are unstable, the material undergoes another transition, back to its unexcited ground
state, and the absorbed energy is liberated in the form of either light or heat or both (all
discrete energy states, including the ground state, of an atom are defined as quantum
states)
SOURCES AND PROCESS 5

6. • The excitation involves only the outermost electrons orbiting around the nuclei of the
atoms
• Luminescence efficiency depends on the degree of transformation of excitation energy
into light, and there are relatively few materials that have sufficient luminescence
efficiency to be of practical value
6

7. • Luminescence is characterized by electrons undergoing transitions from excited quantum
states
• The excitation of the luminescent electrons is not connected with appreciable agitations of
the atoms that the electrons belong to
• When hot materials become luminous and radiate light, a process called incandescence, the
atoms of the material are in a high state of agitation
• Of course, the atoms of every material are vibrating at room temperature already, but this
vibration is just sufficient to produce temperature radiation in the far infrared region of the
spectrum.
LUMINESCENCE AND INCANDESCENCE 7

8. • With increasing temperature this radiation shifts into the visible region. On the other
hand, at very high temperatures, such as are generated in shock tubes, the collisions of
atoms can be so violent that electrons dissociate from the atoms and recombine with
them, emitting light: in this case luminescence and incandescence become
indistinguishable
8

9. • Although lightning, the aurora borealis, and the dim light of glow worms and of fungi have
always been known to mankind, the first investigations (1603) of luminescence began with
a synthetic material
• When Vincenzo Cascariolo, an alchemist and cobbler in Bologna, Italy, heated a mixture
of barium sulphate (in the form of barite, heavy spar) and coal the powder obtained after
cooling exhibited a bluish glow at night
• And Cascariolo observed that this glow could be restored by exposure of the powder to
sunlight
EARLY INVESTIGATIONS 9

10. • The name lapis solaris, or “sunstone,” was given to the material because alchemists at
first hoped it would transform baser metals into gold, the symbol for gold being the Sun
• The pronounced afterglow aroused the interest of many learned men of that period, who
gave the material other names, including phosphorus, meaning “light bearer,” which
thereafter was applied to any material that glowed in the dark
10

11. 11

12. Types of Luminescence
• Biomolecules (i.e. The light emiting living organism )
• The light generating molecules become excited by a chemical reaction; bioluminescence is thus related
to chemiluminescence
• Some biological entities like bugs, e.g. fireflies, denizens of the deep like anglerfish, some mushrooms and even
bacteria can control this reaction
• They produce the chemicals luciferin (a pigment) and luciferase (an enzyme). The luciferin reacts with oxygen to
create light. The luciferase acts as a catalyst to speed up the reaction
12

13. Bioglow Fire flies
13

14. The following are types of luminescence :
• Chemiluminescence :- the emission of light as a result of a chemical reaction
• Bioluminescence :- a result of biochemical reactions in a living organism
• Electrochemiluminescence :- a result of an electrochemical reaction
• Lyoluminescence :- a result of dissolving a solid (usually heavily irradiated) in a liquid
solvent
• Candoluminescence :- It is light emitted by certain certain materials at elevated
temperatures, which differs from the blackbody emission expected at the temperature in
question
14

15. • Crystalloluminescence :- produced during crystalization
• Electroluminescence :- a result of an electric current passed through a substance
• Cathodoluminescence :- a result of a luminescent material being struck by electrons
• Mechanoluminescence :- a result of a mechanical action on a solid
• Triboluminescence :- generated when bonds in a material are broken when that material
is scratched, crushed, or rubbed
15

16. • Fractoluminescence :- generated when bonds in certain crystals are broken by
fractures
• Piezoluminescence :- produced by the action of pressure on certain solids
• Sonoluminescence :- a result of imploding bubbles in a liquid when excited by sound
• Photoluminescence :- a result of absorption of photons
• Fluorescence :- photoluminescence as a result of singlet–singlet electronic relaxation
(typical lifetime : nanoseconds)
16

17. • Phosphorescence :- photoluminescence as a result of triplet–singlet electronic
relaxation (typical lifetime: milliseconds to hours)
• Raman emission :- photoluminescence as a result of inelastic light scattering,
(lifetime: nanoseconds)
• Radioluminescence :- a result of bombardment by ionizing radiation
• Thermoluminescence :- the re-emission of absorbed energy when a substance is
heated
• Cryoluminescence :- the emission of light when an object is cooled (an example of
this is wulfenite)
17

18. Chemiluminescence
• Chemiluminescence is the production of light by a chemical reaction
• Two chemicals react to form an excited intermediate, which breaks down releasing
some of its energy as photons to reach its ground state
18

19.  Chemiluminescence is the emission of light and heat as the result of a chemical reaction. It is a form of
electromagnetic radiation which just happens to fall in the visible spectrum, making it easy to see and identify
 The best-known example of chemiluminescence is probably the chemical reaction used to make glow sticks light
up, which is the result of cracking a small vial of one chemical into a chemical it will react with
 When chemiluminescence occurs in animals, it is known as bioluminescence Several animal species use this
process as a signal, or to lure prey; fireflies are notable producers of chemiluminescent reactions, as are some algae
 When subjecting chemicals to an electrical current creates a luminescent reaction, it is termed
electrochemiluminescence
19

20. There are all sorts of interesting applications for chemiluminescence. For example, Luminol,
a chemical widely used in the forensics community, reacts with iron to generate a luminescent
reaction
When forensics teams want to determine whether or not traces of blood are at a crime scene,
they can spray the site with Luminol to generate a reaction
It can also be used to check for traces of other reactants at a site, assuming that a chemical
which generates a chemiluminescent reaction can be obtained
Some gas phase reactions also exhibit this property, so chemiluminescence can be used to
check for traces of certain impurities in the air.
20

21. 
21

22. Luminol (a glow-in-the-dark chemical)
• The release of a photon of light from a molecule of luminol is a fairly complex, multi-
stage process
• In a basic (alkaline) solution, luminol exists in equilibrium with its anion, which bears a
charge of -2. The anion can exist in two forms (or tautomers), with the two negative
charges delocalised on either the oxygens (the enol-form) or on the nitrogens (the ketol-
form)
• Molecular oxygen (O2) combines with the enol-form of the luminol anion, oxidising it
to a cyclic peroxide
22

23. The required oxygen is produced in a redox reaction (i.e. one in which both reduction
and oxidation occur) involving hydrogen peroxide (H2O2), potassium hydroxide and
(for example) potassium hexacyanoferrate(III) (K3[Fe(CN)6], also known as potassium
ferricyanide)
The hexacyanoferrate(III) ion ([Fe(CN)6]3-) is reduced to the hexacyanoferrate(II) ion
([Fe(CN)6]4-, giving potassium ferrocyanide, K4[Fe(CN)6]), while the two oxygen
atoms from the hydrogen peroxide are oxidised from oxidation state -1 to 0
23

24. Chemistry of glow sticks:
When diphenyl oxalate reacts with hydrogen peroxide (H2O2), it is oxidized to give phenol
and a cyclic peroxide. The peroxide reacts with a molecule of dye to give two molecules of
carbon dioxide (CO2) and in the process, an electron in the dye molecule is promoted to an
excited state. When the excited (high-energy) dye molecule returns to its ground state, a
photon of light is released. The reaction is pH-dependent. When the solution is slightly
alkaline, the reaction produces a brighter light
24

25. Safety note
Phenol is toxic, so if your glow stick leaks, take care not to get the liquid on your hands; if you
do, wash them with soapy water straight away
25

26. What makes glow sticks different colours ?
• The dyes used in glow sticks are conjugated aromatic compounds (arenes)
• The degree of conjugation is reflected in the different colour of the light emitted
when an electron drops down from the excited state to the ground state
26

27.  DNA hybridization detection
 chemiluminescence immunoassay
 forensic science
 food analysis
 western blotting
APPLICATIONS OF CHEMILUMINESCENCE 27

28.  SOUTHERN BLOTTING involves DNA seperation, transfer and hybridization
 HYBRIDIZATION :- Process of formation of double stranded DNA molecule
between a single stranded DNA probe and a single stranded target DNA
molecule
DNA hybridization detection
28

29. • It provides a sensitive, high throughput alternative to conventional colorimetric
methodologies
• Principle: -same as ELISA
-uses chemiluminescent substrate,
-hydrogen peroxide, enhancers
-stopping reagent is not required
-Incubation period is small
Chemiluminescence Immunoassay
29

30. Forensic science
• Chemiluminescence is used by criminalists to detect traces of blood at crime scene
• Solution: luminol powder (C8H7O3N3), hydrogen peroxide, and a hydroxide (eg.
KOH) sprayed where blood might found
• Tiny amount of iron from Hb in blood serves as catalyst for the chemiluminescence
reaction, luminol to glow
30

31. Food analysis
• Organophosphorous are most popular pesticide
• Most commonly used organophosphorous: QUINALPHOS (O,O diethyl-o-quinoxalinly
phosphorothioate)
• Reaction involved
Quinalphos +H2O2 peroxophosphonate
Peroxophosphonate+ luminol 3aminophthalate anion*
3-aminophthalate* 3-aminophthalate +observed emission
31

32. Western blotting
Western blotting (or protein immunoblotting) is a technique widely used to detect specific
proteins in samples of tissue homogenate, cell lysates, cell culture supernatants or body
fluids.
32