RESEARCH STARTER

Reagents in crime scene investigation

Reagents play a crucial role in crime scene investigation by facilitating the detection of hidden evidence that may not be visible to the naked eye. These substances can induce chemical reactions with specific items, revealing important forensic information such as the presence of fingerprints, bodily fluids, and illegal substances. For example, reagents like luminol can produce a glowing reaction in the presence of blood, whereas ninhydrin can highlight fingerprints by reacting with proteins left behind.

In addition to biological evidence, reagents are also used to identify drugs and poisons through distinct color changes during chemical reactions. However, the selection of a reagent must consider the type of surface involved as well as the environment—some reagents may work better on porous materials while others are suited for laboratory settings.

It's essential for forensic scientists to handle reagents with care due to potential hazards, such as carcinogenic or explosive properties, ensuring strict adherence to safety protocols. Overall, the strategic use of reagents in forensic science enhances the ability to gather critical evidence, aiding in the pursuit of justice.

Full Article

DEFINITION: Substances that bring about chemical reactions when added to particular other substances.

SIGNIFICANCE: Forensic scientists use reagents at crime scenes to discover “hidden” evidence, such as the presence of fingerprints that cannot be seen with the naked eye, and to conduct preemptive tests to determine whether substances present may be blood or other bodily fluids. Reagents are also useful for determining the presence of illegal substances, such as drugs.

Reagents are used to test questioned substances at crime scenes and in forensic laboratories. In the presence of the substances with which they are designed to react, reagents produce chemical reactions that usually involve color changes or other changes that can be seen, such as causing a substance to glow under a black light. If the substance with which a reagent is known to react is not present, the chemical reaction does not occur. For example, if a reagent known to react with blood does not react on a stain, that stain is not blood. If that reagent does react on a stain, the stain may be blood, but because other substances could cause the same reaction, the scientist cannot conclude that the stain is blood until further testing is done.

Chemical Reactions

Reagents react chemically with substances, usually either through synthesis or through replacement. In synthesis, the reagent and the substance form a new substance. In replacement, the reagent acts on the substance by replacing some of the substance’s elements with its own elements.

Forensic scientists must take care when preparing and using reagents. A reagent must be pure enough to react properly with the substance it is intended to detect, so it must be prepared according to strict procedures Because chemical reagents may be carcinogenic, caustic, or explosive, they must be handled with caution. The use of protective clothing and equipment is essential. Strict safety protocols should be followed, including the use of personal protective equipment (PPE), such as lab coats, gloves, and goggles, as well as fume hoods for volatile substances.

Uses in Biological and Chemical Labs

Many different reagents can be used to identify blood or other bodily fluids on different surfaces. The choice of reagent depends on the surface on which the bodily fluid is thought to be and whether the reagent is being used at the crime site or in the laboratory. For example, some reagents work well on porous materials but are poor choices for nonporous materials. Other reagents may cause violent chemical reactions and must be used only in laboratory settings.

Some of the reagents that identify blood are amido black, leuco crystal violet (LCV), luminol, and Takayama reagent. Amido black reacts with the protein in blood to produce a blue-black color, whereas leuco crystal violet, along with hydrogen peroxide, reacts with the hemoglobin or its derivatives in blood to produce a deep violet color.

Luminol reacts with the oxidizing activity of hemoglobin derivatives in blood to produce a blue-white chemiluminescent reaction (that is, it glows in the dark), so luminol tests must be conducted in darkness. The reaction produced by luminol does not last long (30 seconds to three minutes), and photographs of the tested area need to be taken quickly to preserve the results. Luminol can produce false positives from metal ions, bleach, urine, saliva, or plant enzymes, and false negatives, so forensic scientists use preliminary tests like phenolphthalein (also known as the Kastle Meyer Test) to confirm heme in blood; it also cannot distinguish human from animal blood. Takayama reagent (a mixture of glucose, sodium hydroxide, pyridine, and deionized water) reacts with the hemoglobin in blood when heated to form pink needle- or rhomboid-shaped crystals that can be seen under a microscope.

Other reagents can identify other bodily fluids, such as semen.  The Christmas Tree stain confirmatory test for sperm uses picroindigocarmine to color the neck and tail green/blue, followed by Nuclear fast red (Kernechtrot) to stain heads red and tips pink for positive visual identification. Reagents can also identify illegal or poisonous substances. For example, copper sulfate reacts with ephedrine, pseudoephedrine, or norephedrine to produce a purple color. The Scott test, developed by L.J. Scott Jr. in 1973, turns blue when cobalt thiocyanate reacts with cocaine or phencyclidine (PCP). A reagent made from solutions with sodium phosphate, chloramine-T, and a color reagent reacts with cyanide to produce a red color. The reagent used in the Dille-Koppanyi test reacts with barbiturates to produce a violet color, and the Duquenois-Levine (D-L) reagent, along with hydrochloric acid and chloroform, reacts with marijuana resins to produce a purple, then pink, color. Kits containing these reagents may be used by law enforcement to make a preliminary determination of what a substance might be.

Although the D-L field test for marijuana is non-specific, prone to false positives, and inadequate for prosecution, law enforcement’s false claims of accuracy have resulted in wrongful convictions and denial of due process.

Recent developments in forensic reagents include carbon quantum dots (CQDs) for enhanced chemical sensing, trace evidence imaging, and crime scene reconstruction due to their tunable optical properties, alongside green chemistry approaches like solvent-free vacuum techniques and safer, degradable reagents to minimize hazards in fingerprint development and toxicology analyses.

Uses with Impression Evidence

Powder or chemical reagents can be used to bring out fingerprints where none can be seen. For example, ninhydrin is a reagent that reacts with some of the protein left behind by fingerprints. When ninhydrin is placed on a porous surface that has fingerprints, a deep purple (Ruhemann’s purple) color highlights the prints. Gentian violet or crystal violet is another substance used to develop latent fingerprints. These types of reagents must be used with caution, however, because they destroy any DNA (deoxyribonucleic acid) evidence that may have been left behind with fingerprints.

Reagents can also be used with shoe prints to determine the type of soil in which the person wearing the shoe walked. For example, if the soil contains iron, ammonium/potassium thiocyanate will react with the iron to produce a reddish-brown color. Hydroxyquinoline reacts with calcium, magnesium, iron, aluminum, and other metals that may be present in small amounts in shoe or tire impressions to produce fluorescence that can be seen under an ultraviolet light. Iodine reacts with small amounts of waxy or oily substances on footprints or tire tracks and may be used in enhancing wet impressions.



Bibliography

American Chemical Society Committee on Analytical Reagents. Reagent Chemicals: Specifications and Procedures. American Chemical Society, 2005.

Bell, Suzanne. Forensic Chemistry. Pearson Prentice Hall, 2006.

Beshahwored, Siyum Shewakena. “Carbon Quantum Dots (CQDs) in Forensic Investigations: A Review of Current Applications and Future Perspectives.” RSC Advances, 22 Sept. 2025, doi:10.1039/D5RA05791D.

“Blood & Bodily Fluids.” Forensic Resources, forensicresources.org/forensic-disciplines/blood-bodily-fluids/. Accessed 20 Jan. 2026.

Cobb, Cathy, et al. Crime Scene Chemistry for the Armchair Sleuth. Prometheus Books, 2007.

Connon, C. C. “Forensic DNA Analysis: An Overview of the Laboratory Process.” Forensic DNA Analysis, edited by C. Cupples Connon, vol. 2685, Humana, 2023, pp. 3–15, doi:10.1007/978-1-0716-3295-6_1.

“Forensic Science.” Encyclopædia Britannica, www.britannica.com/science/forensic-science. Accessed 20 Jan. 2026.

Johll, Matthew. Investigating Chemistry: A Forensic Science Perspective. Freeman, 2007.

Kelly, John F., et al. “The Non-Specificity of the Duquenois-Levine Field Test for Marijuana.” The Open Forensic Science Journal, vol. 5, 2012, pp. 4–8, benthamopen.com/contents/pdf/TOFORSJ/TOFORSJ-5-4.pdf. Accessed 20 Jan. 2026.

Lundblad, Roger L. Chemical Reagents for Protein Modification. CRC Press, 2004.

“Performance of the Previous Examination in the Necropsy Room with the Scott Test (Ammonium Thiocyanate and Cobalt Chloride – in Acid Medium) to Identify Cocaine: Valid Conduct or Waste of Time?” Forensic Research and Criminology International Journal, MedCrave Online Publishing, medcraveonline.com/FRCIJ/performance-of-the-previous-examination-in-the-necropsy-room-with-the-scott-test-ammonium-thiocyanate-and-cobalt-chloride-ndash-in-acid-medium-to-identify-cocaine-valid-conduct-or-waste-of-time.html. Accessed 20 Jan. 2026.

Polacco, Sumiko, et al. “Quantifying Chemiluminescence of the Forensic Luminol Test for Ovine Blood in a Dilution and Time Series.” Forensic Science International, vol. 290, 2018, doi:10.1016/j.forsciint.2018.06.026.

Singh, Rajvinder. “Narration and Legacy of Important Chemical Spot Tests in Forensic Investigation.” Critical Reviews in Analytical Chemistry, vol. 52, no. 1, 2022, doi:10.1080/10408347.2020.1785837. Accessed 20 Jan. 2026.

Ünal, B. K., et al. “Greener Approaches/Materials for Forensic Sciences.” Essential Chem, vol. 2, no. 1, 2025, pp. 1–21, doi:10.1080/28378083.2024.2434238.

Wang, Zerong. Comprehensive Organic Name Reactions and Reagents. Wiley-Interscience, 2008.

Full Article

DEFINITION: Substances that bring about chemical reactions when added to particular other substances.

SIGNIFICANCE: Forensic scientists use reagents at crime scenes to discover “hidden” evidence, such as the presence of fingerprints that cannot be seen with the naked eye, and to conduct preemptive tests to determine whether substances present may be blood or other bodily fluids. Reagents are also useful for determining the presence of illegal substances, such as drugs.

Reagents are used to test questioned substances at crime scenes and in forensic laboratories. In the presence of the substances with which they are designed to react, reagents produce chemical reactions that usually involve color changes or other changes that can be seen, such as causing a substance to glow under a black light. If the substance with which a reagent is known to react is not present, the chemical reaction does not occur. For example, if a reagent known to react with blood does not react on a stain, that stain is not blood. If that reagent does react on a stain, the stain may be blood, but because other substances could cause the same reaction, the scientist cannot conclude that the stain is blood until further testing is done.

Chemical Reactions

Reagents react chemically with substances, usually either through synthesis or through replacement. In synthesis, the reagent and the substance form a new substance. In replacement, the reagent acts on the substance by replacing some of the substance’s elements with its own elements.

Forensic scientists must take care when preparing and using reagents. A reagent must be pure enough to react properly with the substance it is intended to detect, so it must be prepared according to strict procedures Because chemical reagents may be carcinogenic, caustic, or explosive, they must be handled with caution. The use of protective clothing and equipment is essential. Strict safety protocols should be followed, including the use of personal protective equipment (PPE), such as lab coats, gloves, and goggles, as well as fume hoods for volatile substances.

Uses in Biological and Chemical Labs

Many different reagents can be used to identify blood or other bodily fluids on different surfaces. The choice of reagent depends on the surface on which the bodily fluid is thought to be and whether the reagent is being used at the crime site or in the laboratory. For example, some reagents work well on porous materials but are poor choices for nonporous materials. Other reagents may cause violent chemical reactions and must be used only in laboratory settings.

Some of the reagents that identify blood are amido black, leuco crystal violet (LCV), luminol, and Takayama reagent. Amido black reacts with the protein in blood to produce a blue-black color, whereas leuco crystal violet, along with hydrogen peroxide, reacts with the hemoglobin or its derivatives in blood to produce a deep violet color.

Luminol reacts with the oxidizing activity of hemoglobin derivatives in blood to produce a blue-white chemiluminescent reaction (that is, it glows in the dark), so luminol tests must be conducted in darkness. The reaction produced by luminol does not last long (30 seconds to three minutes), and photographs of the tested area need to be taken quickly to preserve the results. Luminol can produce false positives from metal ions, bleach, urine, saliva, or plant enzymes, and false negatives, so forensic scientists use preliminary tests like phenolphthalein (also known as the Kastle Meyer Test) to confirm heme in blood; it also cannot distinguish human from animal blood. Takayama reagent (a mixture of glucose, sodium hydroxide, pyridine, and deionized water) reacts with the hemoglobin in blood when heated to form pink needle- or rhomboid-shaped crystals that can be seen under a microscope.

Other reagents can identify other bodily fluids, such as semen.  The Christmas Tree stain confirmatory test for sperm uses picroindigocarmine to color the neck and tail green/blue, followed by Nuclear fast red (Kernechtrot) to stain heads red and tips pink for positive visual identification. Reagents can also identify illegal or poisonous substances. For example, copper sulfate reacts with ephedrine, pseudoephedrine, or norephedrine to produce a purple color. The Scott test, developed by L.J. Scott Jr. in 1973, turns blue when cobalt thiocyanate reacts with cocaine or phencyclidine (PCP). A reagent made from solutions with sodium phosphate, chloramine-T, and a color reagent reacts with cyanide to produce a red color. The reagent used in the Dille-Koppanyi test reacts with barbiturates to produce a violet color, and the Duquenois-Levine (D-L) reagent, along with hydrochloric acid and chloroform, reacts with marijuana resins to produce a purple, then pink, color. Kits containing these reagents may be used by law enforcement to make a preliminary determination of what a substance might be.

Although the D-L field test for marijuana is non-specific, prone to false positives, and inadequate for prosecution, law enforcement’s false claims of accuracy have resulted in wrongful convictions and denial of due process.

Recent developments in forensic reagents include carbon quantum dots (CQDs) for enhanced chemical sensing, trace evidence imaging, and crime scene reconstruction due to their tunable optical properties, alongside green chemistry approaches like solvent-free vacuum techniques and safer, degradable reagents to minimize hazards in fingerprint development and toxicology analyses.

Uses with Impression Evidence

Powder or chemical reagents can be used to bring out fingerprints where none can be seen. For example, ninhydrin is a reagent that reacts with some of the protein left behind by fingerprints. When ninhydrin is placed on a porous surface that has fingerprints, a deep purple (Ruhemann’s purple) color highlights the prints. Gentian violet or crystal violet is another substance used to develop latent fingerprints. These types of reagents must be used with caution, however, because they destroy any DNA (deoxyribonucleic acid) evidence that may have been left behind with fingerprints.

Reagents can also be used with shoe prints to determine the type of soil in which the person wearing the shoe walked. For example, if the soil contains iron, ammonium/potassium thiocyanate will react with the iron to produce a reddish-brown color. Hydroxyquinoline reacts with calcium, magnesium, iron, aluminum, and other metals that may be present in small amounts in shoe or tire impressions to produce fluorescence that can be seen under an ultraviolet light. Iodine reacts with small amounts of waxy or oily substances on footprints or tire tracks and may be used in enhancing wet impressions.



Bibliography

American Chemical Society Committee on Analytical Reagents. Reagent Chemicals: Specifications and Procedures. American Chemical Society, 2005.

Bell, Suzanne. Forensic Chemistry. Pearson Prentice Hall, 2006.

Beshahwored, Siyum Shewakena. “Carbon Quantum Dots (CQDs) in Forensic Investigations: A Review of Current Applications and Future Perspectives.” RSC Advances, 22 Sept. 2025, doi:10.1039/D5RA05791D.

“Blood & Bodily Fluids.” Forensic Resources, forensicresources.org/forensic-disciplines/blood-bodily-fluids/. Accessed 20 Jan. 2026.

Cobb, Cathy, et al. Crime Scene Chemistry for the Armchair Sleuth. Prometheus Books, 2007.

Connon, C. C. “Forensic DNA Analysis: An Overview of the Laboratory Process.” Forensic DNA Analysis, edited by C. Cupples Connon, vol. 2685, Humana, 2023, pp. 3–15, doi:10.1007/978-1-0716-3295-6_1.

“Forensic Science.” Encyclopædia Britannica, www.britannica.com/science/forensic-science. Accessed 20 Jan. 2026.

Johll, Matthew. Investigating Chemistry: A Forensic Science Perspective. Freeman, 2007.

Kelly, John F., et al. “The Non-Specificity of the Duquenois-Levine Field Test for Marijuana.” The Open Forensic Science Journal, vol. 5, 2012, pp. 4–8, benthamopen.com/contents/pdf/TOFORSJ/TOFORSJ-5-4.pdf. Accessed 20 Jan. 2026.

Lundblad, Roger L. Chemical Reagents for Protein Modification. CRC Press, 2004.

“Performance of the Previous Examination in the Necropsy Room with the Scott Test (Ammonium Thiocyanate and Cobalt Chloride – in Acid Medium) to Identify Cocaine: Valid Conduct or Waste of Time?” Forensic Research and Criminology International Journal, MedCrave Online Publishing, medcraveonline.com/FRCIJ/performance-of-the-previous-examination-in-the-necropsy-room-with-the-scott-test-ammonium-thiocyanate-and-cobalt-chloride-ndash-in-acid-medium-to-identify-cocaine-valid-conduct-or-waste-of-time.html. Accessed 20 Jan. 2026.

Polacco, Sumiko, et al. “Quantifying Chemiluminescence of the Forensic Luminol Test for Ovine Blood in a Dilution and Time Series.” Forensic Science International, vol. 290, 2018, doi:10.1016/j.forsciint.2018.06.026.

Singh, Rajvinder. “Narration and Legacy of Important Chemical Spot Tests in Forensic Investigation.” Critical Reviews in Analytical Chemistry, vol. 52, no. 1, 2022, doi:10.1080/10408347.2020.1785837. Accessed 20 Jan. 2026.

Ünal, B. K., et al. “Greener Approaches/Materials for Forensic Sciences.” Essential Chem, vol. 2, no. 1, 2025, pp. 1–21, doi:10.1080/28378083.2024.2434238.

Wang, Zerong. Comprehensive Organic Name Reactions and Reagents. Wiley-Interscience, 2008.

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