Is It Blood? Exploring Forensic Investigations
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The following article was originally published in the journal for educators Biologia w Szkole (eng. Biology in School) (2/2022):
Let's take a moment to stop at our handy library. Whether crime novels are our favorite literary genre or not, Agatha Christie's books are – in my humble opinion – beyond reproach [1]. The unforgettable Hercule Poirot in one of them states that:
A case of murder is a case of murder whether it happened yesterday or sixteen years ago
This is indisputable. But how can we know if a murder really took place, or if it was just an unfortunate accident? That’s when the investigation begins…
One of the most important tasks is determining the presence of blood at the crime scene, on objects that could be the instruments of the crime, or on items belonging to the suspect. However, today’s investigator has more effective methods at their disposal than the hero of an Agatha Christie novel could have ever imagined. One of them is a technique for detecting even very small amounts of blood based on the chemiluminescent properties of specific substances.
Experiment
Chemiluminescence can be defined as a phenomenon during which electromagnetic radiation is emitted – mainly from the visible light spectrum, but many definitions also allow ultraviolet and infrared radiation – as a result of specific chemical reactions. The products formed in this process exist in a thermodynamically unstable excited state, which then transitions to a lower-energy ground state. The energy difference is released into the environment (directly or via other molecules) as light [2].
Many substances exhibit chemiluminescence, including luciferin with luciferase (used by, for example, fireflies from the Lampyridae family), white phosphorus, singlet oxygen, lofina, the pyrogallol-formaldehyde system, polyphenols from green tea, Wöhler’s siloxane – an interesting organosilicon compound, and many others.
A highly effective chemiluminophore is luminol C8H7N3O2, which is the hydrazide of 3-amino-phthalic acid (Fig. 1). Under normal conditions, luminol is usually in the form of a fine crystalline powder, ranging from cream to yellowish to light brown in color [3].
Interestingly, under appropriate conditions, luminol can help detect even very small amounts of blood, or more precisely, the red respiratory pigment contained in erythrocytes – hemoglobin.
To prepare the reagent for detecting blood, we only need potassium hydroxide KOH (or sodium NaOH) and hydrogen peroxide H2O2 with a concentration of 3%, which is the typical hydrogen peroxide available in pharmacies.
To start the experiment, we need to dissolve a few pellets of potassium hydroxide in about 20 cm3 (0.68 fl oz) of distilled water – the exact amount isn’t critical. Then, to the alkalized solution, we add a small amount of luminol – think of the size of a matchstick tip as a guide to how small an amount of this substance is needed (Fig. 1).
It’s worth noting that luminol dissolves very poorly in pure water, but much better in a basic solution. Next, we add 5 cm3 (0.17 fl oz) of hydrogen peroxide (3%) to the solution. The resulting reagent is almost colorless, with just a very faint yellowish tint (Fig. 2).
This reagent is unfortunately unstable and should be used quickly – ideally within a few hours. The alkaline luminol solution can be stored longer in the dark and in a refrigerator, but in such cases, hydrogen peroxide should be added directly before use.
To test the reagent, we encounter a problem, as blood is needed. I would like to caution against using even small blood samples of unknown origin, as they may be infectious. A good and safe method is to use dried animal blood – for example, from pigs – available commercially and often used by fishermen for bait. Photo 3 shows the isolated and dried bovine hemoglobin used in my experiments.
Pour a small amount of the reagent into a small beaker and add a drop of blood or a pinch of dried blood (or hemoglobin isolate). As seen in Fig. 4A, the isolate floats on the surface of the liquid – we can only notice slight foaming of the solution due to the oxygen released from the decomposition of hydrogen peroxide.
In the dark, however, we will easily notice that the solution, when in contact with a substance containing hemoglobin, starts to glow brightly in a beautiful blue color (Fig. 4B). If we stir the liquid, the effect becomes even more spectacular, as the entire volume of the solution glows with bright light (Fig. 5).
The described reaction is actually used as a preliminary test for the presence of blood at a crime scene or on specific objects. This is done by spraying the reagent onto the area or object in question. The observation is not made with the naked eye, as the emitted light signals may be too weak – photographs with extended exposure times are used to help with this.
However, this method has many limitations. To understand one of them, and how to overcome it, we need to prepare a simple simulation of a crime scene situation. For this, we smear some fresh or dried blood, or hemoglobin isolate, at a specific spot on a clean sheet of paper, and next to it, we place, for example, some juice squeezed from parsley root Petroselinum crispum or horseradish Armoracia rusticana. The paper should then be dried at room temperature, preferably away from direct sunlight. Both spots on the paper should be marked with pencil-drawn circles, as shown in Fig. 6.
As seen, after spraying the reagent in the dark, we can observe that the smear of blood (hemoglobin) starts to glow. Interestingly, it turns out that chemical substances in the parsley root extract also have the same property of catalyzing luminol chemiluminescence. Unfortunately, chemiluminescence caused by components of blood cannot be easily distinguished from the glow caused by contact with many plant extracts, which for various reasons can be found almost everywhere.
Let’s now check the effect of increased temperature on the experiment. The second sheet of paper, prepared in the same way, with smears of the substances of interest, should be dried at an elevated temperature (at least 140°F/60°C), for example by pressing it with an iron, and then sprayed with the reagent as before (Fig. 7).
Note that after treating with elevated temperature, the plant extract smear lost the ability to make the reagent glow, while the blood smear retained this ability. As we see, heating the samples allows the elimination of false positives caused by plant extracts.
Additionally, we must remember that the luminol reagent test is destructive, meaning that samples on which it has been performed cannot be used for further analysis. Due to its limitations, this test should be treated as a preliminary or supportive method.
Explanation
The mechanism of the reaction involves the oxidation of luminol in an alkaline environment in the presence of a catalyst. In an alkaline solution, luminol dissociates to form a doubly charged anion. Due to keto-enol tautomerization, two different forms of the compound are produced, differing in the structure and distribution of negative charge: the ketonic form, where the negative charge is located on nitrogen atoms, and the enol form, with the charge concentrated on oxygen atoms. The enol form undergoes further reaction. It is oxidized in an alkaline environment by hydrogen peroxide, producing a cyclic peroxide. Because of the peroxide bridge in its structure, this chemical species is highly unstable, leading to its rapid spontaneous decomposition, which produces nitrogen molecules and 3-amino-phthalan. Importantly, the latter compound is formed in an excited state, which then transitions to the ground state. According to the principle of energy conservation, the excess energy is emitted into the environment as light, here blue light.
The catalyst for this reaction can be various complex compounds, such as iron. In many experiments with luminol as a catalyst, potassium ferricyanide K3[Fe(CN)6] is used, although it is, of course, absent from blood.
In the case of blood, the catalyst for the described reaction is hemoglobin. It consists of a protein part – globin – and a prosthetic heme group with a structure similar to porphyrins (Fig. 2) [4]. In the heme molecule, the appropriate porphyrin contains a ferric cation, immobilized by four iron-nitrogen bonds. Formally, two of these bonds are covalent, and two are coordinative, although in reality, they are equivalent.
It should be noted that similar reactions are exhibited by various plant enzymes, such as peroxidases found in horseradish or parsley roots. These are, of course, proteins, so subjecting them to high temperatures causes denaturation and loss of catalytic ability. Heme, being a non-protein component, is more resistant to elevated temperatures, which is why we can perform a differentiating test.
References:
- [1] Hart A., Herkules Poirot – życie i czasy (biografia według Agathy Christie), Prószyński i S-ka, 1998 back
- [2] Ples M., Fiolet świeci - chemiluminescencja powszechnie dostępnego związku manganu (eng. Glowing Purple – Chemiluminescence of a Common Manganese Compound), Chemia w Szkole (Chemistry in School), 6 (2018), Agencja AS Józef Szewczyk, 16-19 back
- [3] Huntress E., Stanley L., Parker A., The Preparation of 3-Aminophthalhydrazide for Use in the Demonstration of Chemiluminescence, Journal of the American Chemical Society, 56(1), 1934, str. 241–242 back
- [4] Solomon E. P., Berg L. R, Martin D. W., Villee C. A., Biologia, Multico Oficyna Wydawnicza, Warszawa, 1996, str. 68 back
All photographs and illustrations were created by the author.
Marek Ples