The Chemist on the Case – Chemiluminescence Activated by Blood
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Investigation
Many substances exhibit chemiluminescence, including luciferin with luciferase, white phosphorus, singlet oxygen, lophine, or the pyrogallol-formaldehyde system. The strongest luminescence is produced by luminol oxidized in an alkaline environment. The luminescence of oxidized luminol can be triggered by the presence of blood, a phenomenon commonly used for its detection.
Take a look at the piece of filter paper shown below:
The paper appears clean; in today's experiment, we will determine whether any traces of blood were left on it.
What Do We Need?
We need to gather the substances listed below.
- Hydrogen peroxide H2O2 30% (perhydrol),
- Sodium hydroxide NaOH,
- 3-Aminophthalic hydrazide (luminol) C8H7N3O2.
Warning: This experiment involves NaOH, a strong base with highly corrosive properties. Perhydrol is also corrosive and can cause necrotic damage upon skin contact. Luminol is an allergen and is suspected of being carcinogenic! Avoid any contact with the eyes or skin! The use of blood of unknown origin may pose an infection risk for certain diseases, so extreme caution is required! Protective gloves and safety goggles are mandatory! The author assumes no responsibility for any damage that may result. You perform this experiment at your own risk!
3-Aminophthalic hydrazide, more commonly known as luminol, is a relatively expensive reagent compared to more commonly used laboratory chemicals like hydrogen peroxide or sodium hydroxide. Fortunately, only very small amounts are needed, so even 0.5 g (0.018 oz) of this substance is enough for many fascinating reactions. Its structural formula:
It is also possible to synthesize luminol independently, although this requires some effort. However, for those who are determined, nothing is too difficult! For those interested in synthesizing this substance on their own, I have provided a description of my luminol synthesis. Below is an image of a sample obtained using this method, which was also used in this experiment.
Once we have gathered all the necessary components, we can proceed with the analysis.
Experiment
We need to prepare the appropriate solution. Dissolve 1 g (0.035 oz) of NaOH in 50 cm3 (1.69 fl oz) of distilled water, then add a few milligrams (a fraction of an ounce) of luminol (this small amount is sufficient; there is no need to use more). Luminol dissolves rather slowly in water, so continuous stirring is required until it fully dissolves. A magnetic stirrer can be very useful for this purpose. The prepared solution is stable and can be stored for future use.
Next, cut the material to be analyzed into small fragments:
Just before analysis, add 1.5 cm3 (0.05 fl oz) of perhydrol to the solution. Pour about 5 cm3 (0.17 fl oz) of the prepared solution into a test tube, then drop in one fragment of the material and stir.
As a result of these steps, the solution begins to glow with a faint but clearly visible blue light.
The luminescence confirms that blood was indeed present on the paper.
Explanation
We have detected blood on the paper. But how did it get there? In this case, there was no crime involved—rather, it demonstrates that science sometimes requires small sacrifices. I accidentally injured myself while preparing for another experiment, and after dressing the wound and stopping the bleeding, I wiped my skin with a sterile filter paper. There was no visible trace of blood, but as it turns out, an extremely small amount must have transferred onto the paper. This method is capable of detecting even completely invisible or aged blood traces. It is, in fact, widely used in forensic science.
The mechanism of the reaction is based on the oxidation of luminol in an alkaline environment using H2O2 in the presence of a catalyst. A detailed explanation of this process can be found here. The reaction can be catalyzed by complex compounds of copper or iron. In other experiments, potassium ferricyanide K3[Fe(CN)6] is used as a trigger, but for obvious reasons, it is not present in blood. So, how does blood catalyze the oxidation of luminol and cause its glow?
The key factor here is the red blood pigment—hemoglobin. Hemoglobin consists of a protein component, globin, and a prosthetic non-protein element, heme. The organic part of heme has a structure analogous to porphyrin. In the heme molecule, a specific porphyrin binds an iron ion (Fe2+ or Fe3+) through four iron-nitrogen bonds. Formally, two of these are covalent bonds and two are coordinate bonds, although in practice, they are equivalent. The structural formula of heme:
Due to its iron in a complexed form, heme exhibits catalytic properties that promote the oxidation of luminol by H2O2. This process results in the emission of characteristic blue chemiluminescence, which can assist in blood detection.
It is important to note that similar reactions can be triggered by specific plant enzymes, particularly peroxidases. To rule out false positives caused by these enzymes, the test sample should be heated for a few minutes at a minimum of 100°C (212°F). High temperatures deactivate enzymes, ensuring that any remaining luminescence can be attributed solely to the presence of blood.
Marek Ples