William Brooke OShaughnessy A Pioneer in Pharmacology

Sir William Brooke O'Shaughnessy (1809-89) was a British forensic chemist who enjoyed a wide-ranging career. He took on a project in 1830 at the request of the editor of the prestigious medical journal The Lancet related to a rash of poisonings seemingly traceable to candy. Ever the methodical chemist, he developed a systematic method of testing for the presence of organic and inorganic poisons that found their way into candy by accident or design. Mindful of the tendency of children to suck on the candy wrappers, he considered those as a possible mode of ingestion. He eventually identified a number of contaminants and adulterants, including compounds containing lead, antimony, mercury, copper, and the dye Prussian blue. His findings led to further investigation of adulterated products, with offenders having their names published in The Lancet.

O'Shaughnessy joined the East India Company as an assistant surgeon and went to Calcutta, India, in 1 833. He had wanted to return to his practice of medicine, but his forensic chemical skills kept interfering with this plan. In India, he was given the post of chemical examiner at Calcutta Medical College. This assignment was in addition to his medical duties. It was there that he demonstrated the Marsh test and began to use it. He also was among the first to point out one of its shortcomings, such as the potential for antimony to react similarly to arsenic, leading to a false positive result.

O'Shaughnessy's contributions to forensic chemistry were among many other accomplishments. As a doctor, he championed the use of cannabis (marijuana) to English medical practice as a treatment for tetanus, cholera, and convulsive problems. Having worked with many cholera victims early in his career, he became adept at looking at stomach contents and judging if a death was due to a disease or arsenic poisoning. In India he became an assayer at the Calcutta Mint and was instrumental in bringing telegraphic service to the country. He wrote a book on forensic chemistry in the form of a medical manual for students in Calcutta and was knighted in 1856.

24 drugs, poisons, and chemistry the final color, much like with indigo. The process required thousands of purple snails to make any appreciable amount of dye, and it is not surprising the species nearly became extinct. The dye was one of the most valuable commodities in the ancient world. Alizarin was another ancient dye, red in color.

The dye industry was central to manufacturing and trade from the ancient times until well into the 19th century. The nature of the manufacturing process was labor- and material-intensive, making dyes too expensive for most people to afford. In the 1700s interest turned to finding ways to synthesize dyes from cheaper and more abundant resources. One of the first to try was Peter Woulfe, who began with indigo in 1771. He treated it with nitric acid to make picric acid, a lovely yellow compound. Its dye applications were short-lived. When dry, picric acid is unstable and detonates easily, a generally undesirable feature for clothing. Regardless, the recognition that dyes could be converted to other dyes with chemical treatments was an important step forward, even if picric acid (thankfully) never made a significant impact on the dye industry.

In 1858 Johann Peter Griess (1829-88), a German chemist, described a type of reaction called diazotization in which an amine compound (one containing an -NH2 group) reacts with nitrite ion (NO2-) under acidic conditions to yield diazonium salts that are often highly colored. The reaction can continue further to coupling reactions that produce highly colored products. Uncovering the reaction led to production of azo dyes by 1861. The coupling reactions, often considered a nuisance in salt production, have been utilized extensively in forensic chemistry as color-based presumptive tests for drugs and in other forensic applications.

For much of the last century, the Griess test was one of the principal screening tests used for detection of gunshot residue (GSR). In this test, the analyte of interest is not the amine but the nitrite, which is a byproduct of combustion of gunpowder. An acidic solution of the starting amine (naphthylamine) is added to the sample suspected of containing gunshot residue. If nitrite is present, the dye formation proceeds to yield a red dye. A modified version detects nitrates (NO3-) in water. The source of this material is usually runoff water containing fertilizers, which are high in nitrates. Ammonium nitrate (NH4NO3) is one of the main culprits and is used as an ingredient in explosives. The bomb used in Oklahoma City explosion in 1995 was made of ANFO, a combination of ammonium nitrate and fuel oil.

Griess did not actually make the first synthetic dye. That honor goes to William Henry Perkin (1838-1907), mentioned briefly above. He was an 18-year-old chemist who was working to improve treatments for malaria. He was interested in making quinine, which at the time (1856) was the only known effective treatment for the disease. Quinine was made from extracts of coal tar in a complex process that Perkins hoped to sidestep by starting from simpler ingredients. He did not succeed in that quest, but he did manage to create an intense purple-blue solution that not only dyed cloth but also did not weather, fade, or wash out. He quickly abandoned the quest for quinine, patented the dye, called mau-veine (mauve), and started a company in London called Perkin & Son. The name was a gesture of gratitude to his father, who helped fund his work. Perkins contribution to dye chemistry was to show that it was possible to start from compounds found in coal tar. This and other related materials were and remain relatively cheap and abundant. This freed the dye chemists from scarce and costly natural materials such as plants, mollusks, and other living precursors.

By 1868 Carl Liebermann and others had created colorants consisting of metals and dyes. They understood that the key structural feature of dyes responsible for color was a series of alternating double bonds, such as those found in mauve. Knowing this, Liebermann was able to focus his efforts. During the 1870s and 1880s, he developed color tests based on dye formation and remained a central figure in early colorant and dye chemistry. The Liebermann test is still used occasionally to detect the presence of phenol groups (benzene rings with -OH attached, as in aspirin). A positive reaction produces a colored dye product. The Ehrlich reagent (1901) consists of p-dimethylaminobenzaldehyde, which reacts with indoles such as LSD and mescaline to form colored dyes, purple in the case of LSD.

Paul Ehrlich (1854-1915) was one of the more productive and colorful of the color chemists. He often carried a box of cigars under one arm, of which he smoked about 25 a day. He was also reported to eat little and to frequent beer halls, where he got into spirited discussions and debates. Despite—or because of—this eccentricity, he won a Nobel Prize in medicine in 1908 for work related to both medicine and dyes. He worked with stains used to color tissues and microorganisms. His work in the late 1880s was the foundation of the Gram staining procedure still used today to differentiate bacterial types. In the 1890s Ehrlich turned his attention to immunological work and later to medicine and pharmacology. He screened hundreds of compounds to treat the spiro-chete that was known to cause syphilis. Prior to his work, mercury was the only viable treatment for the disease, resulting in many fatalities. After screening more than 600 compounds, Ehrlich identified one that contained arsenic and became known as salvarsan, discussed previously. Another German dye-making company, Hoechst Dye Works, marketed the drug starting in 1910. In a replay of the Baeyer aspirin story, the success of the drug allowed the company to move more aggressively into pharmaceuticals.

Perhaps the most versatile color test used in forensic drug analysis is the Marquis test published in 1896. The ingredients are simple— formaldehyde and sulfuric acid. The Marquis reagent reacts with many alkaloid drugs to form a variety of colors. Amphetamine and metham-phetamine create orange dyes, while the opiates react with the reagent to form purple ones. Ninhydrin, a common reagent used for visualizing fingerprints, was introduced in the early 1900s and reacts with the amino acids to form a purple-colored dye. As a result of the rapid advances in the late 1800s, forensic chemists entered the 1900s with a rich assortment of extraction and detection methods, all based on classical wet chemistry.

Chemists now know that the results of color tests are not absolute and there is always more than one compound that can produce what appears to be a positive result. If a chemist tests a substance with a color test reagent and sees a characteristic color change, the best way to interpret the result is "more likely than not." The sample more likely than not contains the substance of interest, but it is not certain. The analyst must always be mindful of false positives, in which a substance that is not the target produces the color change. Similarly, if a test reagent should cause a color change and does not, this is called a false negative. All presumptive tests are conducted with these possibilities in mind, and it is why all preliminary findings are confirmed with other techniques.

FLOW OF FORENSIC ANALYSIS

When a chemist receives a powdered or other solid sample for analysis, the first thing he or she usually does is take a few milligrams of the material and test it with several reagents. The color change observed, if there is one, provides the analyst with a reasonable idea of the sample's contents. The analyst will also have a good idea of what is not present, but the results are not conclusive. If the reagent used to test for cocaine (cobalt thiocyanate) gives a positive color change (turning from pink to blue), this means that the sample more likely than not contains cocaine or a compound related to it. Further tests will prove or disprove this hypothesis. In the field police officers used color tests to determine if a suspicious powder might contain an illegal or dangerous substance. The modern forensic chemist has an arsenal of color tests to choose from, as shown in the table on page 28.

THIN-LAYER CHROMATOGRAPHY

The original spot tests were the basis of another technique that is used in forensic chemistry, thin-layer chromatography (TLC). This technique was developed in the 1800s and led to many advances in sample preparation and, eventually, in instrumentation. Chromatography means "color writing," and the word is thought to relate to early experiments in which plant pigments were separated this way. All chromatography, including TLC, is based on the idea of selective partitioning between two phases. Although it sounds exotic, partitioning is something everyone is familiar with, even if they do not realize it. Vinaigrette salad dressing is a mixture of oil and vinegar, two liquid phases that are not soluble in each other. Vinegar is a solution of dilute acetic acid in water. Even when shaken, the oil and water will eventually separate into two distinct layers.

Now consider table salt, which is sodium chloride (NaCl). Salt dissolves easily in water but not in oil. If salt is added to the salad dressing and the dressing is shaken, the salt will end up in the watery vinegar solution and not in the oil layer, since salt dissolves in water but not significantly in oil. This is an example of partitioning. The salt has greater affinity for the water and will end up in the water phase. There may be traces of salt left in the oil, but the concentration of salt in oil would be very small. The same would happen if sugar were added to the oil-vinegar mix; sugar is soluble in water and not in oil, so the sugar would end up in the water phase.

Which compounds will dissolve in a given solvent depends on the chemical structure of compounds involved. For example, table salt

presumptive tests (color tests)

used in forensic chemistry

Drug, evidence,

Color test

Color change

or analyte

(positive)

amphetamine/

Mandelin's/Marquis

green/orange-brown

methamphetamine

barbiturates

Dilli-Kopanyi

red

Zwikker's

purple

benzodiazepines

Zimmerman

purplish-pink

cocaine

cobalt thiocyanate

silvery blue

Liebermann's

yellow

heroin

Froehdes

purple

Liebermann's

black

Marquis

purple

LSD

Erlich's

purple

UV light

fluorescence

marijuana

Duquenois-Levine

purple

and related

Froehdes

green or yellow

mescaline

Mandelin's

green

Marquis

orange

morphine

Marquis

purple

Liebermann's

black

mescaline (peyote)

Marquis

orange

Froehdes

green

psilocin/psilocybin

Ehrlich's

gray or purple

Marquis

orange or yellow

Water molecules have what is called a permanent dipole because the electron cloud surrounding it is unevenly distributed. Water is classified as a polar molecule because of it. It is often drawn as a boomerang shape to show how the charges are oriented on the molecule. Dipoles are something like Earth's North and South Poles or the positive and negative terminals on a battery. Because of this permanent separation of charges, water dissolves many ionic compounds, as well as other polar compounds. This is an example of the principle of "like dissolves like."

is an ionic compound, meaning that it consists of positively charged sodium ions (Na+) and negatively charged chloride ions (Cl-). Water is a polar molecule that has partial charges. Because of this, water can dissolve many ionic compounds. Oil contains long chains of hydrocarbon molecules that repel water; therefore, oil and water are not miscible (they will not mix or dissolve in each other). The basis of this type of partitioning is a principle informally referred to as "like dissolves like."

The way partitioning works in TLC is more complex than the salt-oil-water example. The first phase is the solvent mixture (a mobile liquid), and the second is the paper, which is stationary. When the solvent reaches the sample spot on a piece of paper, anything that can dissolve will dissolve and move with the solvent. As the solvent front moves forward, the dissolved compounds can do one of two things: stay in the paper matrix or move with the solvent. The more affinity they have for the solvent, the faster they will move up the paper. The more affinity they have for the paper, the slower they will move, and the closer they will remain to the original spot or origin. Similar principles were exploited in the spot tests described earlier. The end result is separation of the h2o

Oxygen

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Hydrogen Hydrogen d~

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Initial

Three inkspots on TLC plate

During "run"

Three inkspots on TLC plate

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Completed run

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Thin-layer chromatography (TLC). The sample drop is placed near the bottom of the paper (the origin) and allowed to dry. The paper is placed upright in a shallow solvent mixture. Like water absorbed into a paper towel, the solvent is drawn up the paper and encounters the spot. The components in the spot that dissolve in the solvent move up the paper with it and separate.

components of the mixture if the solvent and paper have the proper chemical structures to effect that separation.

Modern TLC is performed on plates of glass or plastic coated with a powdery solid. These solid phases are designed to have affinities for different materials. By selecting different TLC plates, a chemist can separate a larger variety of compounds than could be done with paper alone. An instrument called a densitometer can be used to measure the density of the spots on the plate when the analysis is complete. The denser a spot, the more material is present. Such an observation provides what is called semi-quantitative information. Hundreds of solvent combinations have been tried over the years, and specialized developing sprays are used to develop spots that might otherwise be invisible.

As useful as TLC is, by the middle of the 20th century chemists needed more powerful techniques to separate and identify compounds. This need, along with advances in technology around the time of World War II, spurred the invention of instrumental methods of analysis that have become the heart of forensic chemistry. Nonetheless, the one instrument that many forensic scientists consider to be their most valuable tool was invented nearly 400 years ago.

DEVELOPMENT OF INSTRUMENTAL TECHNIQUES

Many people think of large, expensive, and exotic room-filling machines when they think of chemical instruments, yet this is an uncommon sight in modern forensic labs. The first police laboratory, which was established by Edmund Locard (1877-1966) in 1910 in Lyon, France, reportedly had two types of instruments: microscopes and spectrophotometers.

Microscopes are not usually considered instruments in the modern sense, but they were one of the first pieces of equipment available to chemists and forensic chemists. Without microscopes there would be no forensic science. Interestingly, a trip to a modern forensic chemistry lab reveals an abundance of two types of devices—microscopes and spectrophotometers—and combinations of the two. These two devices are related in other ways as well. Both use energy (light) to examine and probe the structure of matter. In many ways the history of modern chemical instruments begins with the microscope.

MICROSCOPES: MOVING LIGHT

The microscope was invented by Antonie van Leeuwenhoek (16321723). The instrument is composed of a series of lenses that are used to magnify images. A lens is a curved piece of glass that focuses light rays and magnifies images in a microscope. The simplest lens used in forensic chemistry is a magnifying glass. This lens creates a false image on the retina in the eye that appears anywhere from four to 10 times as large as the real sample (4X-10X). This false image is also called a virtual image. Unlike the image produced from a movie projector, a virtual image does not actually exist in a place in space. When the magnifying glass is moved away from the sample, the image disappears; it only exists when one is looking directly through the lens.

Aside from virtual images, there are lenses that create real images such as those projected on a movie screen. When light from the projector passes through the front lens, a larger real image is projected into the theater and focuses on the distant screen. Unlike the virtual image, a real image exists no matter where the viewer is, and it does not depend on the person looking through a special lens. Both real and virtual images are used in a microscope.

A microscope is an optical system that focuses an intense light through a tiny sample and through a series of lenses to create a highly magnified image of the sample in the viewer's eye. Light originates from an intense bulb and is focused onto a mirror that directs it into the condenser, which compresses the rays into a bright cone of light directed through the sample. This design is called transmitted light because the light moves through the sample. Another form of microscopy relies on reflected light, which is used when samples are too thick to allow light through.

Once the light has passed through the sample, it is collected by the objective lens (the one nearest to the sample), which adds the first magnification. Most microscopes have a series of objective lenses ranging from 10X to 100X. The image is magnified again by the ocular, or eyepiece, lens, which is usually 4X or 10X. If the eyepiece is 4X and

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A simple lens. As light passes an object that is far away from a lens and enters the lens, the light rays are bent. They converge or focus at the point called the principal focus. The imaginary line running down the center of the lens is called the optic axis.

A simple lens. As light passes an object that is far away from a lens and enters the lens, the light rays are bent. They converge or focus at the point called the principal focus. The imaginary line running down the center of the lens is called the optic axis.

Fertilizer Used Make Drugs
(25 mm long)

Virtual image of fiber (100 mm)

Virtual image of fiber (100 mm)

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How a simple magnifying lens works. Light passes by the specimen through the lens and creates a magnified image inside the eye that appears much larger than the real sample. This larger image, which is not real, is called the virtual image.

the objective lens in place 100X, total magnification of the sample is 400X.

Forensic chemists use microscopes to study crystals, examine samples, and perform microchemistry experiments and measurements. Many simple qualitative and quantitative tests that are performed using large equipment can be done with the microscope. This is often

34 drugs, poisons, and chemistry

Ocular lens Eyepiece

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Ocular lens Eyepiece

Tube

Objective

Objective lens

Object plane

Sub-stage condenser

Light source

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A microscope. A modern biological or compound microscope contains a series of lenses. Light passing upward through the sample is called transmitted light. The drawing shows where the lenses create virtual and real images.

important because forensic scientists must conserve as much of a sample as possible. Fibers, hairs, and soils are also studied microscopically. One of the most useful developments in the past 15 years has been the marriage of microscopes with other chemical instruments.

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