CHM 151
Lab 15: The Chemiluminescence of Luminol
Last Updated Dec. 2012
Introduction
Chemiluminescence is the emanation of light from a chemical reaction. The glow associated with
fireflies, for example, comes from a series of reactions involving the oxidation of the pigment luciferin
(often known as bioluminescence when it’s done by living organisms). In a laboratory setting,
chemiluminescence can be demonstrated by the oxidation of 3-aminophthalhydrazide, commonly known as
luminol.
The name luminol may sound familiar to fans of crime dramas or Court TV, as this reagent is commonly
used to identify trace samples of blood at crime scenes. There, the oxidizing agent is the iron found in
hemoglobin. However, one drawback to using luminol in this way (a point often raised by defense
attorneys) is that common household chemicals, such as bleaches and detergents, can also act as oxidizing
agents.
In this lab luminol will be prepared from the reaction of 3-nitrophthalic acid and hydrazine, followed by the
reduction of –NO2 to –NH2. The reaction solvent for the initial reaction will be triethylene glycol (bp =
285C). Once the luminol is prepared you will oxidize it with a basic solution of hydrogen peroxide
(H2O2). The peroxide solution also contains potassium ferricyanide, K 3Fe(CN)6, which acts a catalyst.
NO2
O
C
NO2

 C
OH
+
C
N2 H 4
OH
O
3-nitrophthalic acid
hydrazine
O
NH2
C
N
H
N
H
Na 2S2O4
O
C
N
H
N
H
- H2 O
C
C
O
O
luminol
Procedure
Part A: Synthesis of luminol
1.
2.
3.
4.
5.
6.
7.
Combine 1 g of 3-nitrophthalic acid and 3 mL of 8% aqueous hydrazine in a 50 mL Erlenmeyer flask.
Heat the flask until the solid dissolves.
Add 10 mL of triethylene glycol and a single boiling chip. Heat the solution to 150 C and continue
heating at this temperature for 15 minutes. The color of the solution should darken to a dark orange or
brown color.
[Warnings: make sure your thermometer reads to at least 200 ºC, in case you overheat your solution
(odds are, you will). Also, don’t just leave your (much taller) thermometer in the flask leaning against
the (much smaller) flask, as that greatly increases the chance of a spill. Use a clamp.]
Allow the solution to cool below 100 C. While the solution is cooling heat 15 mL of water to boiling.
After the reaction solution has sufficiently cooled add the boiling water, then cool the solution further
with an ice bath. A light yellow precipitate should form after several minutes. Collected the solid by
vacuum filtration. After the precipitate has collected rinse with 2-5 mL water.
Transfer the solid to a 50 or 100mL beaker and a add 5 mL 10% NaOH. Stir the solution with a glass
stir rod until the solution turns a deep red brown.
Slowly add 3 g sodium hydrosulfite (Na2S2O4), and then heat the solution to boiling. Allow the
solution to gently boil for 5 minutes. Do not allow too much water to boil off. If this starts to occur
add a small amount of water to keep the solution volume at approximately 5-8mL.
Remove the beaker from the hot plate. Add 2 mL acetic acid to the solution and cool in an ice bath.
Collect the resulting yellow precipitate by vacuum filtration.
Part B: The Chemiluminescence of Luminol
1.
2.
3.
4.
5.
Dissolve the luminol in 30 mL of 10% NaOH and 70 mL water.
Transfer 25 mL of this solution to a 250 mL Erlenmeyer flask and add 25 mL of water. Set the
remainder of the solution from step 1 aside.
Prepare a second solution in a 250mL beaker using 20mL 3% K 3Fe(CN)6, 20mL 3% H2O2 and 10mL
water.
When you have the solutions from steps 2 and 3 prepared, your instructor will take you to a dark room
where the solutions can be combined.
If time allows you may repeat steps 2-4 using the remainder of you solution from step 1.
Waste Disposal
Used triethylene glycol should be disposed of using the bottle for organic waste (not water soluble) bottle.
All other waste should go into the bottle for organic waste (water soluble). Used boiling chips can go in the
trash. Used filter paper should go in the solid waste container.
Name: _____________________________
Section: ________
Post-lab Questions
1. What color is emitted during the oxidation of luminol?
2. The purpose of the boiling chip is to prevent the solvent from heating unevenly and splattering - an
effect known as “bumping” (this, by the way, is usually what happens when soups or liquids “explode” as
you heat them in the microwave).
a) Is the reaction solvent really boiling during step A2? Explain.
b) Based on your observations, how does a boiling chip prevent the solvent from bumping?
3. If triethylene glycol was not available for this lab, could the reaction be done in an aqueous solution
instead? Why or why not?
Name: _____________________________
Section: ________
Pre-lab Questions
1. Define the following
a) Oxidation
b) Reduction
2. Is Na2S2O4 a reducing agent or oxidizing agent in today’s reaction? What type of agent is hydrogen
peroxide?
3. Using Bohr’s model of the atom, calculate the energy required to move an electron from a ground state
of n = 2 to an excited state of n = 3. Express your answer in both J/photon and kJ/mol.
4. If you were using Bohr’s model to estimate the wavelength of light emitted during the oxidation of
luminal, what shortcomings of the model will affect the accuracy of your answer?