13.5: Forensic Chemistry

Forensic chemistry is the unsung hero of the world of forensics. Forensic chemistry deals with the identification of substances by their composition; the exploration of their chemical properties and the way they interact, couple, and transform; and the manner in which they form new substances; and to apply all of this to civil and criminal courtroom proceedings. The important role of forensic chemistry is twofold: identification and enhancement. Identification comes in the form of presumptive and confirmatory testing of substances using chemicals and atomic analysis. Enhancement is the process of using chemicals to either improve the visibility of a substance, or to make a substance previously not visible to the human eye visible. We will therefore look at both functions of forensic chemistry separately.

Presumptive and Confirmatory Testing

In forensic chemistry, presumptive testing is the preliminary testing of a substance to establish sufficient cause for either an arrest or to determine the need to proceed with confirmatory testing, which will produce a much higher degree of certainty. Although inexpensive and quick, presumptive testing may not be accurate owing to interpretation by the human eye or cross-contamination of the substance that might prove a false-positive or false-negative result. Confirmatory analysis is often conducted on the microscopic or molecular level and therefore can rule out false conclusions. Presumptive testing is available for patrol officers and forensic technicians alike for the preliminary identification of substances such as controlled substances and narcotics, gunshot residue, blood, semen, urine, and the presence of lead and copper from the impact of bullets. We will start with the presumptive testing of controlled substances and narcotics.

Microcrystalline or color tests are chemical tests resulting in the formation of unique micro-crystals for a given substance when combined with a specific reagent. These are the tests generally used by the patrol officer or investigator in the field to determine if a substance in the possession of a suspect is suspected to be a drug or another product that is legal to possess. They are easy to use and are generally accepted for their ability to detect the characteristics of a family of drugs. Interestingly, most crystalline and color tests are largely empirical in nature. This means that even scientists do not fully understand why there is a chemical reaction between the substance and the chemicals, but the result is predictable and consistent.

Marquis Reagent is composed of 2% formaldehyde in a sulfuric acid base. It is the test that provides results for more than one substance and therefore is often the first test used when the substance being tested is unknown. When combined with a suspected opioid, such as heroin or morphine, the reaction will be a purple coloration with wispy fingers emanating from the substance. When combined with amphetamines, the color reaction is orange to brown. Lysergic acid diethylamide (LSD) will turn a dull olive-black color, whereas mescaline and fentanyl will produce strong orange coloration.

Figure \(\PageIndex{1}\): Color reaction of black tar heroin with the Marquis Reagent. Author's collection

Mecke Reagent is a mixture of 95% selenious acid and 1 gram of concentrated sulfuric acid. When combined with opiates, the color change will be a bluish green. Lysergic acid will produce a greenish black color change. Your author prefers to use the Mecke Reagent for opiates that are in the pill form so as not to have a false reaction to the coloring dye used to give the pill its distinct pharmaceutical appearance.

Scott’s Reagent is a proprietary color test for the presence of cocaine hydrochloride. It was developed by L. J. Scott, a forensic chemist and former Drug enforcement Administration special agent. The three-part test contains cobalt (II) thiocyanate, acetic acid and glycerin, plus concentrated hydrochloric acid and chloroform. Operator error can produce false-positive results for the presence of powder cocaine owing to the need to observe a blue “speckling” reaction as opposed to just a blue color change.

Simon’s Reagent is a two-part color test method for the presence of amphetamines and methamphetamines. Part one is a mixture of 2% sodium nitroprusside and 2% acetaldehyde in water. Part two is a solution of 2% sodium carbonate in water. The reaction with secondary amines will produce a bright blue coloration.

Duquenois-Levine is a three-part color test for the presence of delta-9 tetrahydrocannabinol (Delta-9 THC) in marijuana. The first step of the test is 2% vanillin with 2% acetaldehyde in ethyl alcohol. A small portion of the marijuana leaf is saturated with this chemical mixture. The addition of hydrochloric acid will produce a vibrant purple color change. The addition of chloroform will produce a pink bubble or “eyeball” at the bottom of the test tube. This last part of the test supports the presence of Delta-9 THC). Of interest, the criminalistic laboratory will confirm the substance as marijuana by examining the underside of the leaf for cystolithic hairs, a non-glandular trichome, using a Scanning Electron Microscope, which are unique to the cannabis plant as well a nettles and figs. Of course, since nettles and the fig leaf lack Delta-9 THC they will not react to the Duquenois-Levine test.

Figure \(\PageIndex{2}\): Cystolithic hairs on the underside of a marijuana leaf. Author's collection

Figure 13.5 - Cystolithic hairs on the underside of a marijuana leaf

Confirmatory testing is conducted by the forensic laboratory technician primarily using the gas chromatograph mass spectrometer (GCMS). The GCMS has two separate functions. Gas chromatography separates mixtures based on their distribution between a stationary liquid phase and a moving gas phase (carrier gas). The results are plotted on a graph reflecting the recorder response (vertical axis) versus time (horizontal axis). The laboratory technician cannot identify the substance based solely on the graph produced by the gas chromatograph. The mass spectrometer allows the material to exit from the gas chromatograph into a high-vacuum chamber where a beam of high-energy electrons is aimed at the sample molecules. The electrons collide and cause them to lose one electron and gain one positive electron (ion). The ions then form unstable smaller fragments that pass through an electronic or magnetic field, where they are separated according to their masses. The fragment patterns are then compared against the known molecular structures of the questioned substance. Under laboratory-controlled conditions, no two substances will produce the same fragmentation pattern.

The chemical composition of gunshot residue, or GSR, is lead, antimony, and barium. Gunshot residue can be found on the hands or sleeves of the person who fired a gun, or is standing in proximity to a gun that is being fired, on garments that are near the muzzle of the gun or are being worn by the person who fired the gun, and on the defect caused by the bullet after it left the barrel of the gun. Magnetic “stubs” are the preferred method of sampling GSR from the hands of the person suspected of firing a gun. The stubs are examined for the presence of lead using the Scanning Electron Microscope.

The Scanning Electron Microscope, or SEM, is a microscope that uses an electron beam to produce a high-resolution image of the surface of a substance in order to identify its chemical composition. The SEM is often paired with energy-dispersive X-ray spectroscopy, or EDS, for precise chemical identification, allowing for the definitive confirmation of particles based on their unique chemical signature. A suitable presumptive test for the presence of GSR sampled from a firearm or clothing is the Sodium Rhodizonate, or NaRho, color test method. This test is composed of sodium nitrate in aqueous solution followed by the application of a buffer solution of sodium bitartrate and tartaric acid in distilled or deionized water and an application of hydrochloric acid mixed with distilled or deionized water. The reaction with lead will produce a bright purple coloration. If the suspected GSR is on dark clothing, it is recommended to perform the Bashinski Transfer Technique, which involves places laboratory filter paper over the test area and spraying it with a 15% solution of glacial acetic acid in distilled or deionized water. Apply pressure to transfer the suspected GSR onto the filter paper. Place the filter paper on clean butcher’s paper and place several layers of dry filter paper on top of the sample filter. Apply heat from a dry iron until the filter is dry. You may then perform the color test. Once dry, the filter paper with the test results can be analyzed using the SEM. Sodium rhodizonate can also be used to detect the presence of lead in and around bullet defects in walls, metal, or clothing. Dithiooxamide, or DTO, is a chemical color test for the presence of copper transferred from jacketed bullets upon impact with a target.

There are several good presumptive color test products for the presence of suspected blood; however, your author will only present two: phenolphthalein, or the Kastle-Meyer test, and leucomalachite green. Phenolphthalein is a three-part color test which reacts with the hemoglobin of blood as a catalyst. A sample of suspected blood is collected on a cotton-tipped swab using distilled water or alcohol. One or two drops of reduced phenolphthalein is then dropped onto the swab, followed by a drop or two of hydrogen peroxide, producing a bright pink reaction. The reaction should be immediate. Any reaction after 15 seconds may be the result of oxidation, which would be considered a false-positive. An interesting sidenote is that phenolphthalein has been an ingredient in laxative medication for many years. Leucomalachite green, or LMG, is a two-step test using the leucomalachite green solution followed by the application of hydrogen peroxide. This test also reacts with the hemoglobin in blood as a catalyst agent that oxidizes the color-less leucomalachite solution and turns it into a blue-green colored result. Although the test is highly sensitive, it is prone to false-positive results. It is important to understand that these tests are presumptive in nature, and the presence of blood should be conducted using confirmatory testing. Contrary to common belief, DNA typing is not a confirmatory test for the presence of blood. It is a confirmatory test for the presence of DNA. Confirmatory tests for blood include the Teichmann and Takayama tests as well as the Rapid Stain Identification-Blood test, which detects a specific red blood cell antigen, glycophorin A.

Figure \(\PageIndex{3}\): The three-step Kastle-Meyer test. – Shop Evident

The OBTI Bluestar Immunochromatographic Rapid Test, developed by Bluestar Forensics, is a presumptive and confirmatory test for the presence of blood that is human in origin. It is highly sensitive and easy to use. A sample of suspected blood is collected on a sterile cotton swab, which is placed in the solution for 30 seconds. The solution is then poured onto a test cassette that records a positive or negative response.

The Acid Phosphate Test, also known as the Walker Test or Brentamine spot test, is a presumptive test for semen. The male prostrate gland secretes a high quantity of the enzyme acid phosphate, or AP. The test involves the application of a reagent chemical, such as naphthyl phosphate or sodium alpha-naphthyl phosphate, and a dye, such as Fast Blue B, to a sample of a suspected stain. If semen is present, the enzyme acid phosphatase hydrolyzes the substrate, thus separating the phosphate from the alpha-naphthyl compound. The color change will depend on the dye used in the mixture; however, the reaction with Fast Blue B will be a deep purple color change. Confirmatory testing for semen is conducted using either the Christmas Tree Stain process, which is a dye stain that allows for the microscopic visualization of sperm, or the Rapid Stain Identification-Semen test, which identifies the presence of the seminal vesicle-specific antigen, or semonogelin, which is unique to human semen.

The Phadebas Forensic Press Test is used to detect the enzymatic activity of the alpha-amylase enzyme, which is found in saliva. It is a color test method that uses a laboratory filter paper impregnated with the proprietary Phadebas solution, which is a synthetic biochemical assessment product for the detection of the α-amylase enzyme. A blue “spot” on the filter paper indicates a positive result for suspected saliva. The darker the blue coloration, the higher the presence of saliva. As there is the potential for false-positive results, a second test using the Rapid Stain Identification-Saliva test, which detects the alpha-amylase molecule itself, and specifically, the alpha-amylase from human saliva should be performed. When combined, both tests provide confirmatory results for the presence of human saliva.

Para-dimethylaminocinnamaldehyde, or DMAC, is a simple, fast, and safe test for the detection of urine. However, DMAC reacts with urea rather than creatinine, ammonia or uric acid, and therefore can have inaccurate results. It is essential that confirmatory testing be conducted

Chemical Enhancement

As mentioned in previous chapters, laboratory technicians use various chemicals to enhance, or make visible, evidence that is either latent or degraded to the point where ordinary, non-chemical efforts to visualize the evidence are ineffective. Your author has provided some information regarding the use of chemicals in latent fingerprint development and bloodstain visualization; however, in this chapter we will examine additional chemicals that were not mentioned in that chapter, especially those that fluoresce or have chemiluminescent qualities.

1,2-Indanedione-Zinc chloride, or IND/Zn, is an amino-acid reactive reagent that is an effective process for visualizing latent fingerprints on paper and is considered the best method for developing fingerprints on paper that is old. Fingerprints have been successfully recovered from documents that were 80-years old. Developed fingerprints are visualized using ALS light at 515nm to 570nm green light using a red barrier filter. The application of liquid nitrogen as an end process may increase the quality of the developed prints.

Oil-Red-O is a lysochrome, or lipid-soluble dye that adheres to the fatty components of fingerprint residue, such as those from sebum. It is used on wet or damp paper or cardboard products. The process is simple and inexpensive and involves dipping the item in a bath of the solution for approximately 60 to 90 minutes, followed by a rinse to lower the pH, and then drying the item for a few hours. The developed fingerprints will be dark red in color.

Sudan Black is likely the best development method for latent fingerprints deposited on greasy or waxy non-porous surfaces such as milk cartons, candy wrappers, candles, and the inner surface of latex gloves. It is a lipophilic dye, which means that it will bind with the fatty acids in sebaceous oils. The item is immersed in a bath of the solution until the fingerprint is visualized as a dark blue or black color. It is important to photograph the reaction as quickly as possible because it will degrade in time.

M. B. D., or 7-(p-Methoxybenzylamino)-4-Nitrobenz-2-oxa-1,3-Diazole, is a fluorescent dye stain used on multi-colored non-porous items that have been previously treated with cyanoacrylate ester fuming development medium. M.B.D. will adhere to the polymerized fingerprint ridges developed with cyanoacrylate fuming. The item can be sprayed with M.B.D. or dipped and gently rinsed. The resulting stains will fluoresce brightly in a yellow-green color using an ALS light between 415nm to 535nm with an orange or red barrier filter.

Leuco-Crystal Violet, or LCV, has been mentioned in a previous chapter, but it is well worth mentioning again. LCV is a very effective protein stain used to develop or enhance fingerprints or footwear impressions that were deposited in blood. Leuco-crystal compounds have been used in the dyeing process of fabrics for thousands of years. They are colorless dyes that can be treated to produce oxidation, resulting in a vibrant color change. Heme, the iron-containing compound in hemoglobin, acts as a catalyst that speeds up the oxidation process. The reaction converts the colorless LVC into a bright violet oxidized form called crystal violet. It is easy to apply by pouring or as a fine misting spray, works best on horizontal surfaces, does not require a destaining rinse, and most manufacturers provide a blood fixative. Your author thinks LCV is, well, brilliant.

Luminol and Bluestar are chemicals commonly used for the detection of blood either in crime scenes or on evidence collected from the crime scene or during the course of an investigation. The reaction between these chemicals and even the smallest traces of blood will produce a chemiluminescent glow that will be bright blue and consistent in color. Luminol, or 3-Aminophthalhydrazide, was originally synthesized in the 1850’s in Germany for the detection of copper in mining. It was further developed in the early-1920’s, and in 1937, German chemist Walter Specht suggested that it could be used to detect blood in crime scenes when he noticed that luminol produced a reaction when it was catalyzed by the lead in hemoglobin. In 1966, the formula was improved with the addition of sodium hydroxide and hydrogen peroxide. Although it improved the detection quality, luminol needed to be refrigerated until mixed, mixed at the scene, and could only be viewed in total darkness, which presented a problem for photographic documentation. The technician had to pre-focus the camera in manual mode and only had one chance to get the photograph correct. One of the biggest drawbacks of Luminol is that it will significantly degrade DNA, especially with repeated applications. In 2000, Jean-Marc Lefebvre-Despeaux, the president of BLUESTAR™ Forensics, commissioned Loïc Blum, biochemistry professor at Claude Bernard University in Lyon, France, to develop a luminol-based that was more stable, easier to uses, and would not degrade DNA. The result is a highly sensitive blood-visualization reagent that can be used with background lighting that makes photographic documentation easier. It is also easier to detect false positives with Bluestar™ as a reaction with blood is a steady and consistent blue glow, whereas a reaction with copper will be dull and of a short duration, and the reaction with bleach is violent and of a much shorter duration like a super-nova. To highlight the significance of Bluestar™, your author will tell yet another story.

In a city near where your author works as an investigator and Senior Forensic Specialist, in the early morning hours, a patrol officer observed a half-naked, beaten and bruised man staggering down the middle of a street. The story the man gave before being whisked away to the hospital was that he had been kidnapped, beaten repeatedly, and threatened with death by three men at a nearby house, but had managed to escape. The police contacted two of the men at the house and took them into custody, but for some reason no effort was made to locate and process the crime scene. A few days later, a jail deputy was listening to a telephone call placed by one of the men in custody to his mother. During the call, the man instructed his mother to, “Take some bleach out to the garage and do a very good job cleaning everything,” and, “Wash the blanket he was wrapped in with bleach.” The deputy contacted his supervisor who in turn called the District Attorney’s Office. A search warrant was authorized for the house, and the services of your author were requested as mutual-aid. Although no blood was detected in the house, three small blood drops were immediately detected with white light near an expansion joint on the floor of the garage in proximity to the garage door. The lights were turned out and the window covered. An application of Bluestar™ was misted onto the horizontal surfaces revealing the bright, super-nova reaction with bleach. It looked like the mother had done her job well, except for one thing: Mom had forgotten…the doorknob.

Figure \(\PageIndex{4}\): Bluestar reaction with blood on a doorknob. Author's collection

The doorknobs on both sides of the door leading from the garage to the house bore evidence of blood transfer. In the laboratory, your author later detected the presence of blood on the blanket, in spite of having been washed with bleach in a washing machine, and on a pair of tennis shoes worn by one of the suspects. The blood was matched to the victim by DNA. The use of Bluestar™ provided a visual representation of the blood-letting event and the efforts of the mother to destroy the evidence of the crime that was significant to telling the story during the subsequent trials, of which there were three; they caught the third suspect a few months after the crime. Oh, and the victim, it was a case of mistaken identity. They thought he was someone else.