Luminol: How Crime Scene Investigators Find Blood

The lights go off in a room that looks perfectly clean. An investigator sprays a fine mist across the floor, and a faint blue glow appears: a smear along the skirting board, drips leading to the door, the outline of a wiped-up stain. The glow lasts only about half a minute, but it’s long enough to photograph. The chemical responsible is luminol, and its story is a neat example of redox chemistry, catalysis and light.

What is luminol?

Luminol is a pale yellow organic solid with the formula C₈H₇N₃O₂. It was first made in the 1800s, and its ability to glow was reported in 1928. Its use for detecting blood was developed in the late 1930s.

For crime scene use, luminol is dissolved in an alkaline solution (for example with sodium carbonate or sodium hydroxide) together with an oxidising agent, usually hydrogen peroxide. The mixture is sprayed on to surfaces.

Chemiluminescence: light from a reaction

Most chemical reactions release energy as heat. A few release it as light. This is called chemiluminescence.

Here is the idea, without the full mechanism:

1. In alkaline solution, luminol loses protons to form a negatively charged ion. See what is a base.
2. The ion is oxidised (by oxygen from hydrogen peroxide). Nitrogen gas is released and a molecule called 3-aminophthalate forms.
3. Crucially, the 3-aminophthalate is formed in an excited state: some of its electrons are in higher energy levels than normal.
4. As those electrons drop back to the ground state, the energy is released as a photon of blue light, with a wavelength of about 425 nm.

It’s the same basic physics as the colours in a flame test (electrons dropping from higher to lower energy levels), except that the energy comes from a chemical reaction instead of a flame.

Fireflies and glow sticks work in similar ways: fireflies use an enzyme-driven reaction (bioluminescence), and glow sticks use the oxidation of an ester by hydrogen peroxide, with a dye that emits the colour.

Why blood makes it glow

Luminol and hydrogen peroxide react only very slowly by themselves. They need a catalyst to speed up the reaction. Blood provides one.

Red blood cells are packed with haemoglobin, the protein that carries oxygen. Each haemoglobin molecule contains four haem groups, and each haem group holds an iron ion at its centre. This iron catalyses the breakdown of hydrogen peroxide and the oxidation of luminol. See reaction rates and catalysts and transition metals for why iron is good at this: it can switch easily between oxidation states.

Because a catalyst isn’t used up, a tiny amount of blood catalyses a lot of reaction. Luminol can detect blood diluted thousands of times or more, and it works on stains that are old, dried or have been cleaned. Attempts to wash blood away often leave enough behind to glow.

Why it’s only a presumptive test

Luminol is extremely sensitive but not specific. Anything that catalyses the same reaction can make it glow, including:

• Bleach (sodium hypochlorite), a strong oxidising agent. Ironically, a scene cleaned with bleach can glow all over. However, bleach glows brightly and briefly, and its effect fades as the bleach decomposes over time.
• Copper and iron compounds, such as in some metal surfaces and rust.
• Plant peroxidases in some vegetables (such as horseradish and turnip).

Luminol can also react with animal blood as well as human blood.

For this reason, a luminol glow tells investigators where to look, not what they’ve found. Any glowing area is sampled and sent for confirmatory testing. See forensic chemistry for the difference between presumptive and confirmatory tests.

Using luminol at a scene

• Darkness is essential: the glow is faint, so the room must be as dark as possible.
• Photography: long-exposure photographs record the glow, usually with a second exposure in dim light to show the room layout.
• Timing: the glow lasts about 30 seconds in one area; spraying again renews it, but repeated spraying dilutes and spreads the stain.
• Order of tests: luminol can dilute a sample, so visible stains are usually sampled for DNA before spraying.

Modern luminol formulations are designed to be compatible with later DNA analysis.

Other presumptive blood tests

Several colour tests use the same principle: haem catalyses the oxidation of a colourless compound by hydrogen peroxide, producing a coloured product.

Test	Positive result
Kastle–Meyer (phenolphthalin)	pink colour
Leucomalachite green	green-blue colour
Tetramethylbenzidine (TMB)	blue-green colour
Luminol	blue glow in the dark
Fluorescein	yellow-green fluorescence under a light source

In the Kastle–Meyer test, a swab of the suspected stain is treated with phenolphthalin (a reduced, colourless form of phenolphthalein) and then hydrogen peroxide. If haem is present, it catalyses the oxidation of phenolphthalin back to phenolphthalein, which is pink in the alkaline reagent. It’s a lovely link between forensic science and a titration indicator.

Colour tests are used on visible stains to check whether they might be blood; luminol is used to find stains that aren’t visible at all.

After the glow: confirmatory tests

Once a possible blood stain has been found, the laboratory can confirm:

• Is it blood? Tests for specific blood proteins, or microscopic examination for blood cells.
• Is it human? Tests using antibodies that bind only to human proteins.
• Whose is it? DNA profiling.

Bloodstain pattern analysis also uses the shapes and distribution of stains revealed by luminol to reconstruct events, such as the direction of movement.

Luminol in the classroom

Luminol is a popular demonstration because the glow is so striking. A typical teacher demonstration mixes an alkaline luminol solution with a dilute hydrogen peroxide solution containing a catalyst such as potassium hexacyanoferrate(III) or a little copper(II) sulfate, then pours them together in a darkened room. The blue glow appears instantly and fades as the reagents are used up.

The demonstration shows several ideas at once:

• Energy changes don’t always appear as heat; here some of the energy leaves as light. See exothermic vs endothermic reactions.
• Catalysts change the rate dramatically: without one, the glow is almost invisible.
• Rate and concentration: a more concentrated mixture glows more brightly but for a shorter time.

Luminol solutions and hydrogen peroxide are irritants, so eye protection and gloves are needed, and the demonstration should follow a proper risk assessment.

Limits of the glow

Luminol doesn’t reveal the age of a stain, the amount of blood with any precision, or whose blood it is. Very heavily diluted or chemically degraded stains may give no glow at all, and a large area of glow from bleach can hide a real pattern. Investigators treat the glow as one piece of a larger picture that includes the scene layout, witness accounts and laboratory results.

Key takeaways

• Luminol glows blue when it’s oxidised in alkaline solution: an example of chemiluminescence.
• The light comes from an excited product molecule releasing energy as a photon.
• Iron in haemoglobin catalyses the reaction, so even tiny, cleaned-up traces of blood glow.
• Bleach, some metals and plant enzymes also cause a glow, so luminol is only a presumptive test.
• Confirmatory tests and DNA profiling follow up any area that glows.