How luminol works, the science behind blue stains at crime scenes

You don’t have to be a Crime series fan to know it: the luminola chemical compound used by the Scientific Police, stands out and is remembered for its spectacularity, much loved by TV criminologists. Just turn off the lights, spray a mysterious solution on a spotless floor and luminescent spots appear to reveal the crime. The luminescence we see is caused by a oxidation-reduction reaction between hydrogen peroxide and luminol. But then what does blood have to do with it? The iron present in hemoglobin catalyzes (i.e. speeds up) this reaction: the bluish color appears much more vividly and quickly in the presence of iron, which is why it is used for the detection of bloodstains. However, the luminescence is not as long-lasting as we see it on TV, and furthermore, it did not arise only from bloodbut also from urine, feces and some plants, so it is only a preliminary test.

What is luminol: history of the luminescence most loved by criminologists

If you were to buy luminol (C₈H₇OR₃No₃), you would find yourself faced with one yellowish crystalline powder, whose chemical name is decidedly more complicated than the common one: 5-amino-2,3.dihydro-1,4-phthalazindione. It seems to have been discovered already around 1853, synthesized in 1902 by AJ Schmitz, but it is Albrecht the first to describe, in 1928, the intense luminescence it emits in particular conditions.

The name “luminol” appears for the first time in reports from 1934, while it was in 1937 that Specht, a great enthusiast and scholar of luminol, proposed using it for medico-legal investigations. Specht also discovered that dried blood stains produced much more vivid chemiluminescence than fresh ones: according to him, the older the bloodstains were, the more intense the reaction would be.

Today, in addition to investigations, luminol is also used in the detection of other biological molecules, as a biosensor and in the pharmaceutical industry for cell localization.

How the reaction occurs to see the traces of blood

There chemiluminescence it is an emission of light following a chemical reaction and, in our case, it occurs thanks to the oxidation-reduction reaction between luminol and water oxygenated (H₂OR₂). Iron (Fe²⁺) present in the heme group of hemoglobin catalyzes the reaction, that is, it speeds it up: the reaction would still occur, but very slowly. In fact, the luminescence we see ci reveals the presence of the catalystin our case the iron, just as if it were telling us: “Hey, there’s some iron here!”

The complete reaction involves a series of intermediate steps, with shifts of charges and electrons, but the bottom line is that the bond between the two atoms of nitrogen present in the molecule; are replaced by two oxygen atoms and a is formed intermediate structure full of energy, 3-aminophthalate. This intermediate cannot sustain all this energy and must vent it somehow: the aminophthalate in the excited state releases this energy in the form of light (hν) at a wavelength of approximately 450 nm and returns to its fundamental energetic state.

Since it is in the visible light spectrum, we can see it with the naked eyebut it is necessary to turn off the light, because an external light source would cover the light emitted by the reaction.

How luminol is prepared

When we think of luminol as we see it on TV, we imagine a single substance that “ignites” traces of blood. In reality, what we see sprayed is a solution composed of different substances. First luminol, then an oxidizing agent, usually hydrogen peroxide (H2O2) and finally an agent that makes the environment basic, i.e. with a pH greater than 7, usually sodium hydroxide (NaOH).

This basic environment is necessary because at neutral pH, the luminol molecule forms one zwitterionthat is, it has both a positive and negative charge within the same molecule. The reaction between hydrogen peroxide and luminol also occurs when the pH is acidic, but is weaker and the luminescence is seen less. The formulation has changed over time, but the most used one published in 1966 by Weber involves preparing three initial solutions with:

• 8 g of sodium hydroxide (NaOH) in 500 ml of water
• 10 ml of 30% hydrogen peroxide in 490 ml of water
• 0.354 g of luminol in 62.5 ml of 0.4 N sodium hydroxide diluted in 500 ml of water.

Then, you take 10 ml of each of these initial solutions and add 70 ml of water. Now you’re ready to go to the crime scene with Grissom from CSI.

Lights and shadows, in every sense

Luminol is able to detect blood stains not visible to the naked eye in any crevice, even if it is washed or diluted: think that it detects dilutions of up to 1μl of blood in 1 L of water or other solution! That’s why, no matter how well the killer cleans the scene, luminol leaves him no escape. Its use in investigations is in fact based on the concept that nothing vanishes without leaving a trace and that blood can also resist on different surfaces decades! However, there is immediately a first myth to dispel. The luminescence is much shorter than what we see on TV: in fact it lasts, about 30 secondsand for this reason it is essential to be able to take a good photo of the crime scene.

The upside is that it can be used to analyze large surfaces, just like we see our favorite forensic technicians doing. One of the main problems with using luminol is that it is not strictly specific for blood and can give gods false positives. Luminescence is in fact also triggered by other ions that act as catalysts, such as cobalt, manganese, nickel or copper. It can be activated by enzymes, such as peroxidase present in horseradish or feces, and by oxidizing molecules such as those present in bleach. That’s why it is considered a preliminary test and not definitive. Imagine: if you use bleach to wash the floors, the luminol reacts anywayit will then be up to the detectives to understand if you were hiding the evidence of a heinous crime or if you are simply a lover of cleanliness.

Another problem is represented by the type of surfaces: if sprayed on non-absorbent materials such as linoleum, glass or vinyl, the solution can “slip” diluting the blood and effectively ruining the test. This is why technicians use the minimum quantity necessary and they hurry to photograph the crime scene. On carpets and absorbent materials, however, stains can also be left to dry and sprayed with luminol even several timesrepeating the analysis.