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Atom Bond Atom

Atom Bond Atom

Symmetric stretch

Symmetric stretch

Asymmetric stretch

Scissoring

Rocking in-plane

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Asymmetric stretch

Scissoring

Rocking in-plane

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Infrared (IR) motion. When molecules absorb IR energy, they can move in different ways. This motion is what humans measure as temperature and call "heat"—the more motion, the hotter the temperature.

42 drugs, poisons, and chemistry emission spectroscopy (ICP-AES) relies on the emission of characteristic wavelengths of light for elemental analysis. Fluorescence methods exploit the absorption of energy, typically in the ultraviolet range, to produce emissions that reveal information about the element or compound.

IR radiation

Ai K

Molecule such as H2O

Xi K

Not absorbed

Absorbed; causes rocking

Partially absorbed; causes wagging

Partially absorbed; causes twisting

Partially absorbed; ^ causes stretching

Not absorbed

x Absorbed

X1 Xo X3 X4 X5

Absorbance

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Recording a spectrum. The IR energy directed at the sample is absorbed in different ways that are characteristic of the molecule. A plot of the amount of energy absorbed at each IR wavelength is called the IR spectrum.

(Opposite) Sample IR spectra. The wavelength of IR energy is changing across the X axis, and the deeper the "valley" in the spectrum, the more strongly the IR energy is absorbed. The spectra look complex, but this complexity allows forensic chemists to distinguish the three molecules.

Monchromator

Detector

Hollow cathode lamp Metal atoms (light source)

Monchromator

Detector

Hollow cathode lamp Metal atoms (light source)

Dissolved sample

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Dissolved sample

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Atomic absorption spectroscopy (AAS). The sample is dissolved in an acid and drawn into a flame, which frees the metal atoms, such as lead, from the solution. The atoms absorb energy from the lamp, and the more atoms of a given metal that are present, the more energy absorbed. Each element requires a different lamp.

Examples include X-ray fluorescence (XRF) and spectrofluorometry. Spectrophotometers can also be used as the detector module in hyphenated instruments, but these are not as common in forensic chemistry as are GC-MS instruments. Finally, an instrument called an atomic absorption spectrophotometer (AAS) is used in forensic science to detect and give quantitative results for metals such as lead, mercury, arsenic, and antimony. Many of these metals are poisonous, so the instrument is particularly useful in toxicology.

MICROSPECTROPHOTOMETRY

Forensic chemists have recently added a powerful technique to their toolboxes, one that combines a microscope with a spectrophotometer to create a microspectrophotometer (MSP). This is a natural marriage because a microscope is designed to focus light through a sample, and spectrometry studies the interaction of energy with matter. The

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Spectrum no. 1

Spectrum no. 2

Spectrum no. 3

Spectrum no. 4

Microspectrophotometer

Microspectrophotometer

Layered paint chip

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Layered paint chip

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Depiction of surface mapping. A layered paint chip is mounted on a slide and placed on the stage of a microspectrophotometer (MSP). A spectrum is obtained from the first layer and then the stage is moved to the left for the next spectrum. Because each paint layer is chemically different, each spectrum in the map will show these differences.

most difficult aspect of designing MSPs is making sure that enough electromagnetic energy flows through the sample to get a meaningful interaction and a useful spectrum. The breakthrough came with the development of detectors so sensitive that they have to be cooled in liquid nitrogen to make sure that normal electrical and background noise does not drown out the tiny signal produced by the MSP. An MSP instrument consists of a microscope and lenses, as described above, but the lenses and other materials must consist of material that will not interfere with the measurements. For example, glass is a good absorber of IR energy. Because of this, the lenses in an MSP that works with IR energy cannot be made of glass. If they were all the IR energy would be absorbed by the lenses and never detected. It would be like building a microscope for looking at small specimens and making the lenses out of wood.

MSPs can be used in the X-ray, UV-Vis, and IR portions of the electromagnetic spectrum. Forensic chemists use them to study tiny paint chips, tapes, fibers, paper, coatings, plastic, and many other types of material. Combining the instrument with a motorized stage allows for surface mapping. To create a surface map of a fiber or other evidence the instrument focuses on a tiny portion of the sample, takes a spectrum, and then moves to the next point. This method is very useful for studying samples that are not the same throughout, such as a layered paint chip.

IMMUNOLOGICAL METHODS IN TOXICOLOGY

In addition to the techniques and instruments already described, toxi-cologists have another procedure useful for testing blood, urine, and other bodily fluid samples. This technique is called immunoassay, and there are several variations. Immunoassay relies on an antigen-antibody reaction between the drug being tested and an antibody specific for it. This is the same general kind of reaction that occurs when someone catches a cold; the body manufactures antibodies that attack the cold virus (the antigen). For immunoassay the antibody is attached to a solid surface, such as the bottom of a plastic or glass well. A complex that consists of the drug coupled to a label is then added to the container. The label is something that can be detected; for example, a radioactive tag would be detectable using a radiation detector, while a fluorescent label would be detected by light that it emits.

When this complex of drug and label is added to the container, a reaction occurs. As a result the labeled drug is bound to the antibody. A sample that may contain the drug, such as urine, is next added to the plastic well. If there is no or little drug present, the labeled drug-antibody complex will remain undisturbed. If there is a large concentration of the drug, this will displace the labeled drug from the antibodies, releasing the labeled drug into solution. The higher the drug concentration in

Drug ' Label Antibody

Sample added with no or low drug concentration

VVV

Sample added with high drug concentration

Detection

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Detection

A simplified depiction of an immunoassay

ELISA

EMIT

-Label is an enzyme that catalyzes this reaction

-Label is an enzyme that catalyzes this reaction

Label is an enzyme that catalyzes this reaction

- Label is radioactive, detect with a counter

- Label is radioactive, detect with a counter

FPIA

Label is fluorescent

Label is fluorescent

Drug

Label

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How the different methods of immunoassay work the urine sample, the greater the displacement of the labeled drug. The amount of the displaced labeled drug is then measured.

The type of label used determines the type of immunoassay technique. In an enzyme-linked immunoassay (ELISA), a chemical compound called a substrate is added to the solution in the well. The label is an enzyme that catalyzes a reaction in which the substrate is changed, forming a colored solution. The deeper the color, the greater the concentration of the enzyme label present, which implies a greater concentration of drug in the original sample. The enzyme-multiplied immunoassay technique (EMIT) is similar; the label catalyzes a common biological reaction, the conversion of nicotinamide adenine dinucleotide (NAD) to a reduced form of the same molecule called NADH. The more NADH detected, the greater the concentration of drug in the sample. A radioimmunoassay (RIA) uses a radioactive label that can be detected by counting equipment. Finally, a fluorescent polarization immunoassay (FPIA) uses a fluorescent label and the directions in which the fluorescence is emitted.

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