What the Eye Can’t See – Tracking Blood with Chemiluminescence

Polish version is here

The following article was originally published in the journal for educators Chemia w Szkole (eng. Chemistry in School) (1/2015):

Ples M., Na tropie - fluorescencyjne wykrywanie śladów krwi (eng. What the Eye Can’t See – Tracking Blood with Chemiluminescence), Chemia w Szkole (eng. Chemistry in School), 1 (2015), Agencja AS Józef Szewczyk, pp. 25-26

Introduction

What role does chemistry play in criminal investigations? Surprisingly, quite a lot. Even Sherlock Holmes, the iconic fictional detective created by Arthur Conan Doyle, understood the power of chemistry in solving crimes. One of the most common dilemmas a forensic investigator faces is determining whether a crime has actually occurred at the scene. One crucial clue is the potential presence of blood traces.

In this article, I’ll walk you through a simple yet highly sensitive method of detecting blood using a derivative of the fluorescent dye fluorescein (Fig. 1).

Fig. 1 – Structural formula of fluorescein

In an alkaline solution, fluorescein exhibits intense fluorescence under UV light — the emitted glow appears yellow-green (Photo. 1).

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Photo. 1 – Fluorescein in aqueous sodium hydroxide solution, showing distinct yellow-green fluorescence

Video: supplementary material

Preparing the Dye Solution

To carry out a blood detection test, we first need to prepare a solution of a pale yellow leuco compound — the reduced form of the dye. For this, gather the following chemicals:

fluorescein C₂₀H₁₂O₅
sodium hydroxide NaOH
zinc Zn

The zinc used in this experiment should be in powdered form or as finely granulated as possible.

Dissolve 0.1 g (0.0035 oz) of fluorescein and 1 g (0.035 oz) of sodium hydroxide in 25 cm³ (0.85 fl oz) of distilled water. Then add 2 g (0.07 oz) of powdered zinc. The resulting mixture turns a dark red, almost black color (Photo. 2).

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Photo. 2 – Dark red mixture

The zinc suspension in the fluorescein and sodium hydroxide solution must be stirred vigorously for about an hour — a magnetic stirrer is ideal for this. During this time, the color changes to a pale yellow (Photo. 3). If this transformation does not occur at room temperature, gently heat the mixture to around 60°C (140°F) and continue stirring.

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Photo. 3 – Mixture after the reaction

This color change is caused by the reduction of fluorescein by zinc, producing leucofluorescein, also known as fluorescin. The resulting solution can be stored for some time, but it is sensitive to oxidation. Therefore, it should be kept in a tightly sealed container. One useful trick is to leave the excess zinc powder in the solution — any oxidized dye will be reduced again by the metal. For this reason, the solution should be filtered only just before use.

Blood Detection

To demonstrate the forensic utility of leucofluorescein, two solutions must be prepared:

Solution A – Dissolve 2 cm³ (0.068 fl oz) of filtered leucofluorescein solution in 48 cm³ (1.62 fl oz) of distilled water
Solution B – An aqueous 3% hydrogen peroxide H₂O₂ solution

Solution A is unstable and should be used within a few hours. For ease of application, both solutions can be transferred into small spray bottles.

For the purpose of the experiment, a small blood sample was smeared onto filter paper (Photo. 4). The sample was left in a dry place for several days, during which the blood dried and its color changed from red to brown.

Note: Never handle unknown blood samples without proper safety precautions. They may pose a biological hazard!

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Photo. 4 – Blood sample used in the experiment

In this example, the bloodstain is clearly visible to the naked eye. However, real crime scenes often present a more complex scenario — the presence of blood may be concealed by various organic and inorganic contaminants, dirt, dust, and other debris.

Illuminating the stain with UV light alone does not significantly change its appearance (Photo. 5A).

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Photo. 5 – Blood sample exposed to UV light; A – before treatment with leucofluorescein and hydrogen peroxide; B – after treatment

To confirm the presence of blood, spray the sample with Solution A, followed by Solution B, and observe it under UV light. Within moments, a distinct yellow-green fluorescence appears around the stain (Photo. 5B). This strongly indicates the presence of human or animal blood. The fluorescence is bright, clearly visible to the naked eye, and easily photographed — an important advantage when documenting crime scenes.

Explanation

The phenomenon behind this reaction is relatively straightforward. Unlike fluorescein, leucofluorescein does not fluoresce under ultraviolet light.

However, it can be reoxidized to fluorescein in the presence of a suitable oxidizing agent — in this case, hydrogen peroxide H₂O₂. This reaction normally proceeds slowly but is significantly accelerated by the presence of heme, the prosthetic group found in hemoglobin. Heme acts as a catalyst, accelerating the oxidation of leucofluorescein to fluorescein, resulting in bright fluorescence under UV light.

It's important to note that excessively high concentrations of heme can quench fluorescence — this can be observed in Photo. 5B, where the center of the stain glows less brightly than its edges. Therefore, this method is most effective for detecting small or trace amounts of blood. Due to its convenience and sensitivity, it is widely used in forensic applications.

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