What is Chemiluminescence?
Chemiluminescence is the production of light as a result of a chemical reaction. It occurs when two chemicals react to form an excited, or high-energy, intermediate which breaks down and releases some of its energy as photons of light in order to reach its ground state—the lowest allowed energy state, or most stable configuration of an atom, molecule, or ion (Welsh, 2011). In some instances, a limited amount of heat may also be emitted. The general equation of a chemical reaction causing chemiluminescence is represented by:
A + B —–> AB* —–> Products + Light
[Note: AB* refers to the excited/high-energy intermediate formed by the reaction of chemicals A and B]
The Luminol Test: A Method of Tracking Evidence in Forensic Science

Criminal investigators mix the Luminol powder with a liquid substance containing hydrogen peroxide (H2O2), a hydroxide (OH–), and other chemicals, and would pour this mixture into a spray bottle. The hydrogen peroxide and Luminol are actually the main reagents in this chemical reaction but in order for the reaction to emit an intense glow, a catalyst is needed to accelerate the process. The iron in the blood’s hemoglobin serves as the catalyst in this reaction. In performing the Luminol test, criminalists must spray the mixture evenly on the area where they think the blood may be present.

As seen in Figure 2 above, the iron in the hemoglobin accelerates a reaction between the hydrogen peroxide and the luminol once the hemoglobin in the blood and the luminol mixture come into contact. Harris (2002) explains, “In this oxidation reaction, the luminol loses nitrogen and hydrogen atoms and gains oxygen atoms, resulting in a compound called 3-aminophthalate. The reaction leaves the 3-aminophthalate in its energized state—the electrons in the oxygen atoms are moved up to higher orbitals. The electrons quickly fall back to a lower energy level, emitting the extra energy as a light photon. With iron accelerating the process, the light is bright enough to see in a dark room.”
Luminol test is widely-used by forensic investigators because of their efficiency in yielding results, low cost, easy availability of reagents, and the simplicity of preparing the procedure. The luminescent areas are marked for their detection once the light emission is beginning to fade, and the light obtained can be photographed or filmed (Khan, et al., 2014).
Factors Affecting the Reaction Rate in the Chemiluminescence of Luminol
- Presence of a Catalyst
- The iron present in the hemoglobin, an oxygen-carrying protein in the blood, serves as a catalyst in the reaction. Iron is only required in trace amounts wherein a tiny amount of blood, or hidden blood stains, are capable of producing a positive result (Welsh, 2011).
- pH Level
- pH level has an important role in the chemiluminescence of the Luminol within the system (Khan, et al., 2014). It was observed that in low pH, the intensity of light emitted reduces drastically. On the other hand, in higher pH, a significant increase in light intensity was noticed. As seen from Figure 3 below, the chemiluminescent intensity begins to increase at an alkaline pH range of pH 7.0 to pH 8.0, while the maximum intensity was reached at around pH 10.0.
- Concentration
- Agustini, et al. (2012) noted that “the intensity of the light depends on the nature and concentration of the catalyst used, may present and this effect can be photographed and lasting approximately one minute.”
- Surface Area
- Another variable to the efficacy of the Luminol test, at least in criminalistics, relies on the surface area in which the reaction will take place. Surfaces made of absorbent materials would usually enable a positive result, even after a clean-up of the crime or a long period of time from the crime’s occurrence (Agustini, et al., 2012).
Other Real-World Applications
The chemiluminescent property of luminol is used by biologists in cellular assays to detect the presence of copper, iron, cyanide, as well as specific proteins via western blotting. Moreover, this property is also beneficial within sanitary practices in medical protocols where the application of luminol for the detection of blood among medical pieces of equipment helps to prevent infection, especially before performing surgical procedures.
Works Cited:
- Agustini, B. C., da Silva, A. L. L., da Silva, R. R., & Frigeri, H. R. (2012). Luminol in Forensic Science. Journal of Biotechnology and Biodiversity, 3(4), 172–177. doi: 10.20873/jbb.uft.cemaf.v3n4.rogiskisilva
- Boudreaux, K. (n.d.). Luminol: A Glow-in-the-Dark Reaction. Retrieved May 3, 2020, from https://www.angelo.edu/faculty/kboudrea/demos/luminol/luminol.htm
- Harris, T. (2002). How Luminol Works. Retrieved May 3, 2020, from https://science.howstuffworks.com/luminol3.htm
- Khan, P., Idrees, D., Moxley, M. A., Corbett, J. A., Ahmad, F., Figura, G. V., … Hassan, M. I. (2014). Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical Application and Future Uses. Applied Biochemistry and Biotechnology, 173(2), 333–355. doi: 10.1007/s12010-014-0850-1
- Minnesota Department of Public Safety. (n.d.). Luminol (Blood). Retrieved May 3, 2020, from https://dps.mn.gov/divisions/bca/bca-divisions/forensic-science/Pages/forensic-programs-crime-scene-luminol.aspx
- Welsh, E. (2011). What is Chemiluminescence? Retrieved May 3, 2020, from https://www.scienceinschool.org/2011/issue19/chemiluminescence
This post was initially submitted in partial fulfillment of the requirements for obtaining a credit under Grade 12 Chemistry (Ontario Curriculum). All photo credits belong to the rightful owners of the images used in this post.

