Luminol Chemiluminescence Test for Blood

The luminol chemiluminescence reaction is responsible for the glow of lightsticks. The reaction is used by criminalists to detect traces of blood at crime scenes. In this test, luminol powder (C₈H₇O₃N₃) is mixed with hydrogen peroxide (H₂O_s) and a hydroxide (e.g., KOH) in a spray bottle. The luminol solution is sprayed where blood might be found. The iron from the hemoglobin in the blood serves as a catalyst for the chemiluminescence reaction that causes luminol to glow, so a blue glow is produced when the solution is sprayed where there is blood. Only a tiny amount of iron is required to catalyze the reaction. The blue glow lasts for about 30 seconds before it fades, which is enough time to take photographs of the areas so they can be investigated more thoroughly. Here's how you can detect blood yourself or demonstrate how to do it:

Luminol Materials

• luminol stock solution (2 g luminol + 15 g potassium hydroxide + 250 mL water)

• 3% hydrogen peroxide in water (common over-the-counter concentration)

• potassium ferricyanide or a sterile blood lancet and sterile alcohol pad

Performing the Test or Demonstration

• In a clear test tube or cup, mix 10 ml of the luminol solution and 10 ml of the peroxide solution.

• You can activate the glow either by adding ~0.1 g of potassium ferricyanide to the solution or with a drop of blood. The blood must be on the alcohol pad. The forensic test is for dried or latent blood, so the reaction between the alcohol and fresh blood is necessary.

Notes About the Luminol Test

• In addition to iron and iron compounds, other substances can catalyze the luminol reaction. Copper and its compounds, horseradish, and bleach also cause the solution to glow. So, you could substitute any of these materials for the drop of blood or potassium ferricyanide in the demonstration. Similarly, the presence of these chemicals at a crime scene affects testing for blood. If a crime scene was washed in bleach, for example, the whole area would glow when sprayed with luminol, making it necessary to use a different test to find traces of blood.

• If you are doing the reaction as a chemiluminescence demonstration, you can kick the display up a notch by dissolving the potassium ferricyanide in the peroxide solution and using a fractionating column or glass spiral to react the solutions rather than a test tube. You could pour a small amount of fluorescein in the bottom of a flask, pour the potassium ferricyanide solution through the spiral into the flask, and (in a darkened room) finish by adding the luminol solution. The spiral will glow blue as it passes through the column, but the glow will change to bright green once the luminol touches the fluorescein in the flask.

• Don't drink the luminol solution. Don't get it on your skin or in your eyes. If you prepare a spray bottle of luminol solution to check for traces of blood, keep in mind the solution might be damaging to some surfaces. That's not a big factor at a crime scene, but it's something to keep in mind at home or in class. Don't spray upholstery or clothing or people.

• I am told you can use much less luminol (~50 mg) and still achieve enough luminescence for a demonstration or for crime work.

How the Test Works

The iron in the hemoglobin found in blood catalyzes an oxidation reaction in which the luminol gains oxygen atoms while losing nitrogen and hydrogen. This produces a compound called 3-aminophthalate. The electrons in the 3-aminophthalate are in an excited state. Blue light is emitted as energy is released when the electrons return to the ground state.