Walter McCrone and Microchemistry

Negative head and torso views of the Shroud of Turin, dated May 18, 2006 (© P Deliss/Godong/Corbis)

At first it might seem as if history, forensic chemistry, and microscopes have little in common. Nothing could be further from the truth. Many chemical tests used as part of forensic chemistry and toxicology can be performed on a small scale, so small that the results require the use of a microscope to observe. Specialized tests and analyses using microscopes have also been developed and used. The man most responsible for the modern marriage of chemistry and microscopy is Dr. Walter McCrone (1916-2002).

McCrone was well known for his microscopic detective work in famous cases such as the Shroud of Turin. The shroud was a sacred relic of the Catholic Church believed by many to be the burial cloth of Jesus. The image of a man, arms crossed and eyes closed, looks much as one would expect the body to look after crucifixion. In 1 978 a scientific team that included McCrone was given access to the shroud to perform tests. McCrone used tape to collect samples from the linen cloth for microscopic examination. He studied the particles adhering to the tape and discovered particles of pigments and collagen, a material used as the binding media in many paint formulas. McCrone provided strong evidence the Shroud of Turin is a painting created with very dilute paints, much like modern watercolors. The finding was controversial but was supported by later scientific analyses that used carbon dating techniques to date the linen used in the shroud to the early 1 300s. He worked on many other art forgery cases, but none brought the same attention as did the Shroud of Turin.

Negative head and torso views of the Shroud of Turin, dated May 18, 2006 (© P Deliss/Godong/Corbis)

HYPHENATED INSTRUMENTS: SEPARATION AND DETECTION

Aside from microscopes, forensic chemists rely on analytical instruments. Development of modern chemical instruments began in the late 1800s and accelerated dramatically around 1940. The most important classes of instruments in forensic chemistry are spectrophotometers and those that use "hyphenated" techniques. A hyphenated instrument has two modules that are linked together in one unit. The first module is a separation module, while the second is a detector. These two aspects were mentioned earlier as part of spot tests and TLC. The difference here is that the process now takes place inside a modular instrumental system. A sample that contains more than one component can be introduced into the first module, where it will be separated into individual components, much as in TLC. In an instrument, however, flow continues to move, pushing components one at a time into the detector. This separation is essential because most detectors cannot distinguish between different compounds.

The most commonly used hyphenated instrument in forensic chemistry is a gas chromatograph-mass spectrometer (GC-MS). A GC works similarly to TLC except hot gases are the solvent (the mobile phase) and the solid material (solid phase) is coated on the inside of a GC column. The column is long and thin with a diameter smaller than a piece of thin spaghetti. The solid material is coated in a layer that is too thin to see, and most of the inside of the column is empty space, like a tiny pipe with a thin layer of paint on the inside. The sample molecules can stay in the gas and move quickly through the pipe, or they may stick in the solid phase (the paint) and move more slowly. The molecules that tend to stick to the solid coating will come out of the column later than materials that prefer the fast-moving gas. Because GC is an effective means of separation, complex mixtures can be introduced into the instrument. This means that sample preparation does not have to be long and involved, which is a significant advantage.

Another type of hyphenated instrument is the gas chromatographflame ionization detector (GC-FID). In FID the detector is sensitive to the compounds found in gasoline, kerosene, and many other substances. The GC-FID is used for the analysis of arson cases. Finally, some labs use a high-pressure liquid chromatograph (HPLC) as a separation module

Gas chromatograph-mass spectrometer (GC-MS). This instrument has two modules. The GC component employs partitioning to separate a complex mixture into the individual components A and B. A comes out of the GC first and enters the MS, with B emerging a few seconds or minutes later. The mass spectrum is used to identify the compound and, in some cases, to perform quantitative analysis. The detector (the MS) sees each compound alone, so it is much easier to identify.

coupled to spectroscopic detectors or to mass spectrometers. Even DNA analysis uses hyphenated chemical instruments to separate the DNA pieces and then detect the pieces using a specialized detector. Like so many forensic instruments, this detector is based on the principles of spectrophotometry.

SPECTROPHOTOMETRY:

INSTRUMENTS AND ELECTROMAGNETIC ENERGY

Spectrophotometry (also called spectroscopy) is the study of the interaction of electromagnetic energy with matter. Electromagnetic energy can be described as a wave. Think of a rock dropped into water. Waves emanate from the rock and propagate outward through the water. The waves are not new matter added from the rock, only ripples in the water caused by the rock.

The energy of waves is related to their wavelength and frequency. The wavelength is the distance from one wave crest to the next. In the visible range of the spectrum (energy that humans detect as colors), the wavelength is between 400 and 700 nanometers (1 nanometer [nm] equals 1 one-billionth of a meter). The frequency of energy is the number of waves that pass a fixed point per second. In the rock and water example a person standing on the shore could count how many waves hit the shoreline per second, which would be the frequency of the waves. Radios are calibrated in frequency, so a radio station picked up at the setting 95.5 would be broadcasting energy waves with a frequency of 95.5 kHz, or 95.5 million waves arriving per second. The hertz (Hz) is a measure of frequency, with 1 Hz equaling one wave per second. If in the rock in the pond example five waves hit the shore each second, the frequency of the waves would be 5 Hz. Wavelength and frequency are related by the equation c = \u, which states that the speed of light (3 x 108 m/sec.) equals the frequency of a given wave times its wavelength.

There are spectrophotometers that work in all ranges of the electromagnetic spectrum. The first developed was based on colorimetry, in which the visible portion of electromagnetic energy is used. A colorimeter is useful for analyzing color, which might seem unnecessary, but color can be an important characteristic of forensic evidence, such as the results of color tests or the color of paints, hair, ink, or fibers. People perceive color in different ways, but an instrument only measures characteristics. An instrument can also often detect subtle differences in color that the human eye cannot. Variations of colorimetry are used forensically in the analysis of drugs, paint, and fibers.

Wavelength (crest to crest)

Wavelength (crest to crest)

High energy Short wavelength

Low energy Long wavelength

Gamma rays

High energy Short wavelength

Gamma rays

X-rays

Ultraviolet

1

Infrared

Electromagnetic radiation

Low energy Long wavelength

Electromagnetic radiation

Visible light

Violet Indigo Blue Green Yellow Orange Red 400 nm 700 nm

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The electromagnetic spectrum. The highest energy waves are X-rays and cosmic rays, which have short wavelengths and high frequency. At the opposite end of the spectrum are television waves, with long wavelengths and low frequency. The visible portion is the only part humans can sense directly, and it incorporates the colors of the rainbow. When combined, these appear white.

All spectrometers consist of an energy (light) source, a mechanism or device to filter the source energy and select the wavelength(s) of interest, a device or method to hold the sample, and a detector system, which converts electromagnetic energy (light) to a measurable electrical current. The way in which energy is absorbed reveals information about the chemistry of the materials that are absorbing it. Spectrophotometers that work in the X-ray range of the electromagnetic spectrum, for example, are used to identify metals such as lead and antimony that are part of gunshot residues. On the other hand, the ultraviolet-visible (UV-Vis) range is useful for inks, paints, and drugs.

The most versatile form of spectrophotometry used in forensic chemistry is infrared (IR) spectroscopy. IR spectroscopy is used in drug analysis, toxicology, and in the analysis of inks, paints, fibers, papers, tapes, and many other types of physical evidence. Humans can sense

Source of white light

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Colorimetry. In this style of spectrophotometry the eye is the detector, the energy is sunlight, and the matter is a test tube with some blue food coloring. Visible light can be broken into the colors of the rainbow: red, orange, yellow, blue, indigo, and violet. When the sun or a lightbulb shines through the test tube, the molecules that make up the food coloring absorb most of the red, yellow, and orange light. This leaves the blue light free to travel through the solution to the eye, where nerve cells fire and are interpreted by the brain as "blue."

IR indirectly as heat because it is related to the motion of molecules. The more molecules move, the more energy they have and the more energy they can donate in a collision. This is a complicated way of saying "do not put your hand on a hot stove." The stove feels hot because the molecules in the burner are moving very quickly. Temperature is an expression of the relative speed and motion of molecules. Motion can be translational (going from one place to another), rotational, or vibrational (shaking and wiggling). IR spectroscopy is based on vibra-tional motion.

When molecules absorb IR energy, it causes them to vibrate. To visualize this, atoms within a molecule can be thought of as tiny steel marbles and the chemical bonds connecting them as springs. Absorption of the IR radiation causes the spring to flex and bend, but the energy is not sufficient to break the spring. Since the molecular motion is three dimensional, there are many different types of motion that can occur, as shown in the figure. Because each bond between two different atoms has many different possible motions and because molecules are composed

of many such atoms and bonds, the IR absorption pattern for any one molecule is unique.

To produce an IR spectrum of a sample, it is placed in the IR spectrophotometer such that it is exposed to different wavelengths of IR energy. Some of these wavelengths will be absorbed, causing the water molecule to vibrate as just described. Other wavelengths will pass through the water without being absorbed. A detector records the results, and a plot is made of the degree of absorbance at each wavelength. The resulting plot is called the IR spectrum of that sample. Most IR spectra are complex, but that complexity allows for identification. IR spectrophotom-etry is an important technique for forensic chemists who work in drug analysis.

Many spectrophotometric techniques are based not on the absorption of energy but on the emission. Inductively coupled plasma atomic

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