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JoVE Lab Manual
Lab: Chemistry

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Synthesis of Luminol

Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. The difference in energy between reactants and products is known as the enthalpy of the reaction. This is found by calculating the difference in energies between the products and the reactants, or ΔH. If ΔH is positive, the reaction is endothermic; if it is negative, the reaction is exothermic. While chemical reactions are traditionally thought of as a conversion of reactants into products, many reactions take place in multiple steps, forming intermediates. In chemiluminescent reactions, these intermediates transition from a high energy state to the ground state, releasing a photon of light that may be visible as the reaction progresses.

Atomic Energy Levels

Niels Bohr, a Danish physicist, proposed the theory that an electron orbiting the nucleus of the atom can occupy only certain orbits or energy levels. An atom with all of its electrons in the lowest possible energy level is said to be at its ground state. When an electron occupies an energy level higher than the ground state, the atom is in its excited state.

A key assumption in Bohr’s theory is that an electron remains in the ground state until it is disturbed. Thus, an electron is only elevated to an excited state by absorbing energy. When the electron relaxes back down to the ground state, it releases that energy. Often, this is in the form of a photon of light, whose wavelength is directly related to the energy difference between the excited and ground states.

When an electron is excited to a higher energy level by absorbing light of a certain energy or wavelength, the phenomenon is called fluorescence. This effect can be seen if you expose a laundered white T-shirt to a black light or UV-light. The characteristic glow is caused by the fluorescence of the detergent and bleaching compounds in the shirt, which make it look white under regular light.


In a chemiluminescent reaction, an electron is excited by absorbing heat that is released during a reaction. Then, light is released when the electron relaxes back down to the ground state. The major difference between chemiluminescence and fluorescence is that the energy that excites the electrons in chemiluminescence comes directly from the reaction.

One practical application of chemiluminescence is in commercial glow sticks. A glow stick contains two separate solutions: one containing peroxide and the other containing diphenyl oxalate and a color dye. When the two solutions are mixed, which occurs upon activating the glow stick, the resulting reaction between the peroxide and diphenyl oxalate produces energy that excites the dye to a higher energy state. As the reactants are exhausted, the dye returns to the ground state and releases light. This is what gives glow sticks their characteristic color.


Luminol is a chemical that exhibits chemiluminescent properties and is utilized in a wide range of applications, most notably in forensics. Luminol (C8H7N3O2) emits blue when it is mixed with an oxidizing agent. In the case of forensics, luminol reacts with the iron in hemoglobin, enabling forensic scientists to identify very small traces of blood.

Luminol is synthesized by the dehydration reaction of 3-nitrophthalic acid with hydrazine. The reaction is heated to remove water, and triethylene glycol is added to further increase the temperature. The nitro group of the 3-nitrophthalhydrazide is then reduced using sodium dithionite to form an amino group at high pH. In basic conditions, the 3-nitrophthalhydrazide is soluble. The addition of glacial acetic acid precipitates the luminol.

With potassium hydroxide, luminol forms a dianion. The hydroxide anions deprotonate the two hydrogens that are attached to the nitrogens in the luminol. Oxygen gas oxidizes luminol to its excited state. As it relaxes back to the ground state, it releases a blue-white light.


  1. Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2015). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.

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