Chromatography Notes and Labs

Background: Organic analysis of unknown substances includes analytical techniques such as Chromatography, Spectophotometry and Mass Spectrometry. Chromatography is a means of separating and identifying organic components. It is especially useful to separate mixtures i.e. many illicit drugs contain many different materials to dilute the drug of interest. Gas Chromatography separates molecules using high temperature system to vaporize all the components and subsequently separated on a column.

Theory of chromatography uses the different polarities of the stationary phase, mobile phase and the liquid mixture of different compound to be separated. The compounds that have the most similar polarity to the mobile phase i.e. solvent or eluant will move the fastest and be closer to the solvent line than the other types of molecules which have less similar polarity to the solvent. The compounds more attracted to the stationary phase i.e. paper or thin layer plate will also move more slowly. Thus, molecules can be separated from each other based on their different polarities.

Gas Chromatography (GC) separates mixtures based on their distribution between a stationary liquid phase and a mobile gas phase. This is a more accurate technique. Paint chip s, fibers and plastics can be tested in a GC. A chromatogram with a specific pattern is produced and can be used to identify materials found at a crime scene as belonging to a suspect.

HPLC (high performance liquid chromatography) uses a stationary phase that is a thin film with a mobile gas phase. Again a pattern specific to particular molecules is produced and can be matched to standard references or to a suspect.

TLC (thin layer chromatography) can be an inexpensive screening test which is often used prior to more expensive analytical tests. This chromatography is done on thin layer plates of glass or plastic coated with silica (SiO2) or alumina(Al2O3). These compounds are run in an appropriate solvent and visualized with ultraviolet light.

Toxicology Student Notes

These notes are based on the textbook, "Criminalistics: An Introduction to Forensic Science" by Saperstein pp. 277-287 and includes information from different websites

Toxicology is the study of toxins i.e. poisons. Toxins can be natural or synthetic poisons. For example, in 1982 seven people died in the Chicago area after taking over the counter Tylenol capsules containing cyanide. The killer was never discovered. Johnson and Johnson still offer a $100,000 reward for the prosecution and conviction of the perpetrator (http://www.personal.psu.edu/users/w/x/wxk116/tylenol/). This case resulted in the current types of protective packaging of products in order to prevent product tampering. A naturally occurring toxin is Tetrodotoxin found in Puffer fish which can be fatal (http://www.cfsan.fda.gov/cgi-bin/bbbglos?Tetrodotoxin).

Drugs can cause physiological or psychological effects. These substances can affect the structure and/or function of living tissue via chemical reactions. Drugs can be legal or illegal, addictive (e.g. narcotics), or not addictive. Drugs covered by law are known as "controlled substances". Taken in excess, these drugs can possibly cause illness or death. Seventy five percent of the evidence to be analyzed in forensic labs is drug-related. Pharmaceutical companies send new drugs to the FBI Crime lab to be categorized for future forensic analysis and comparison. Some drugs standardized in forensic labs include: Marijuana, Cocaine, Methamphetamine, Ecstasy, Ritalin, Heroin, and Lysergic Acid Diethylamide (LSD).

Forensic analyses include organic and inorganic analytical techniques. Color Tests, Chromatography and Immunoassay tests are used in forensic labs. Some of these techniques are based on the solubility of substances in different solvents e.g. Chromatography (a means of separating and identifying organic components). This technique is especially useful in order to distinguish mixtures of illicit drugs, which can contain different substances used to dilute the drug. One of the most precise analytical techniques is Gas Chromatography combined with the mass spectrophotometer, GCMS. The GCMS gives a conclusive individual image (much like a fingerprint) for thousands of different chemical substances, no two of which are alike.

Drugs must be carefully collected, labeled and preserved as forensic evidence for future laboratory and legal analysis, maintaining the "Chain of Custody". (Saperstein, Ch. 9 p. 228)

Drug recognition Expert: This expert initially categorizes drugs using a microscope. The drugs then undergo further testing. Different types of microscopes can be used to analyze forensic evidence including drugs. These include the compound microscope, comparison microscope, stereoscopic microscope, polarizing microscope, microspectrophotometer, and the Scanning electron microscope (SEM). (Saperstein, pp. 164-174)

Forensic Toxicology includes the analysis of alcohol and drugs. Techniques used in Toxicology include setting up an analytical scheme or series of tests to detect, isolate and identify a specific substance. (p.278, Saperstein)

1. Drugs can be acidic (barbiturates and aspirin) or basic (phencyclidine, methadone, amphetamines and cocaine) in character. Acidic drugs are extracted with acidic water, basic drugs are extracted with organic solvents e.g. chloroform.
2. Color Screening Tests:
   
   1. Marquis
   2. Dillie-Koppanyi
   3. Duquenois-Levine
   4. Van Urk
   5. Scott Test

   A quick color-screening test using TLC (thin layer chromatography) can indicate the presence of a drug. If positive, then more sensitive and expensive analyses can confirm the actual identity of suspected drugs. Microcrystalline tests can also indicate the presence or absence of a specific drug, but confirmation tests with the GCMS are required to identify the drug.

3. Confirmation tests for drugs can include spectrophotometry using ultraviolet (UV) and infrared (IR) light http://www.cem.msu.edu/~parrill/movies/ir.html). The infrared spectrum is unique for each compound and can be considered analogous to a "fingerprint" of that compound. The most powerful analytical tool is the GC/MS, gas chromatography/mass spectrometer, that combines the purification of chromatography with the specificity of a mass spectrometer (See Fig. 10-12 and 10-13 pp. 280-281, Saperstein). In a GCMS, the drug sample passes through the chromatography column which separates all compounds in the sample which are then sent to the mass spectrometer which fragments the compound(s) giving a specific "fingerprint" pattern for each substance. Different compounds can be seen on a print out-as peaks from the GC and fragments from the MS (Saperstein, see fig 5- 9 for heroine and cocaine on p. 254). This GCMS technology allows for detection of very low levels of drugs in the blood or urine.
4. Alcohol tests can determine the concentration of alcohol in the blood or urine is a commonly required analysis. Alcohol concentrations can be determined from the breath or the urine. A Breathalyzer measures a color change produced from the reaction of potassium dichromate with alcohol (p. 268 Saperstein). More accurate blood alcohol levels can be determined by Gas chromatography (see p. 273 Saperstein, fig. 10-9).

Drug use can result in dependence (i.e. addiction), which can be physiological and/or psychological. Early laws focused on "habit-forming" drugs like opium and cocaine. These laws classified drugs as Narcotic, Hallucinogenic, Depressant, Stimulant, or Anabolic Steroids.

1. Narcotics: Morphine, Heroin, Methadone, and Codeine. These are analgesics and relieve pain by depressing the central nervous system.
2. Depressants: Barbiturates (can be short-acting or long-acting), Alcohol, Methaqualone (Quaalude), Meprobamate (Miltown, equanil), Diazepam (Valium), and Chlordiazepoxide (Librium). These drugs depress the central nervous system.
3. Stimulants: amphetamines, cocaine (originally used as an anesthetic and painkiller), caffeine, and nicotine. These drugs can result in a high euphoric feeling.
4. Hallucinogens: Marijuana, Lysergic Acid Diethylamide (LSD), Phencyclidine (PCP), Ecstasy (MDMA, a modified amphetamine), psilocybin, and mescaline. Hallucinogens cause visual, auditory and mood alterations from the norm.
5. Anabolic Steroids: related to testosterone. These steroids can cause liver cancer, masculinize females and diminish sex drive in males.

Drug-Control Laws categorize drugs according to their addictive nature and whether or not they have medicinal value. Criminal penalties are present at each level from 1-20 years in prison and up to a million dollars in fines. (Saperstein p. 247 for table of Schedule I-V drugs).

References and Resources: Saperstein, Richard, "Criminalistics: An Introduction to Forensic Science", Prentice Hall, Inc., Upper Saddle, NJ 07458, 2001. ISBN 0-13-013827-4 (textbook with lots of background information and figures)

Toxin Websites: http://www.personal.psu.edu/users/w/x/wxk116/tylenol/ http://www.cfsan.fda.gov/cgi-bin/bbbglos?Tetrodotoxin CHEMISTRY ANALYSIS QUALITATIVE AND QUANTITATIVE websites: GCMS: http://www.shsu.edu/%7Echm_tgc/sounds/pushmovies/GC.gif Gas chromatography originally from http://www.shsu.edu/~chm_tgc/sounds/sound.html from Sam Houston State University Infrared Analysis: fair site: http://www.cem.msu.edu/~parrill/movies/ir.html Univ. of Arizona Infrared Spectroscopy tutorial: http://www.organicworldwide.net/infrared.html HPLC Chromatography tutorial: http://www.ionsource.com/tutorial/chromatography/rphplc.htm Arizona

Spectroscopy Notes

A branch of analytical chemistry devoted to identification of elements and elucidation of atomic and molecular structure by measurement of the radiant energy absorbed or emitted by a substance in any of the wavelengths of the electromagnetic spectrum in response to excitation by an external energy source (instrumental analysis).

Spectroscopy includes all the major spectroscopic techniques: mass spectrometry, nuclear magnetic resonance, magnetic resonance imaging, x-ray imaging, and infrared spectroscopy.

Spectroscopy is a branch of analytical chemistry devoted to the identification of elements and the elucidation of atomic and molecular structure by measurement of the radiant energy absorbed or emitted by a substance in any of the wavelengths of the electromagnetic spectrum in response to excitation by an external energy source. The types of absorption and emission spectroscopy are usually identified by the wavelength involved. For example: gamma ray, X ray, UV, visible, infrared, microwave and radiofrequency all cause electron excitation and can be used to analyze substances. Spectroscopic analysis was originated by Fraunhofer in 1814 when discovering certain dark (D) lines in the solar spectrum, later identified as characterizing the element, sodium. In 1861 Kirchhoff and Bunsen produced emission spectra and showed their relationship to Fraunhofer lines. X-ray spectroscopy was used by Moseley (1912) to determine the precise location of elements in the periodic table. Since then, a number of highly specialized techniques have been developed including Raman spectroscopy, NMR (Nuclear Magnetic Resonance), nuclear quadrupole resonance, dynamic reflectance spectroscopy, laser, microwave and gamma ray spectroscopy.

Forensics: Prior to Spectroscopic analysis, less expensive tests are performed: white powder analysis and/or Thin Layer Chromatography. Subsequently, the samples are sent for analysis by IR, NMR or GC mass spectroscopy.

Hair and Fiber Notes

Background: Hair and fiber evidence is often used to identify victims and suspects from a crime. These crimes can include murder, sexual assault, hit and run accidents and burglary. Not only can it be used to identify people, it can show the entrance or exit route of the perpetrator. Hair and fiber can be used to identify clothes or shoes belonging to the suspect.

Hair structure: Hair grows from follicles in the skin and covers the surface of mammals. A small muscle that helps the hair stands up surrounds each follicle. A nerve connects the hair follicle to the brain with a sebaceous gland next to the follicle producing sebum, an essential oil. The hair is embedded in the skin follicle at the root and extends the length of the hair shaft. A cross section of a hair includes the cuticle on the outside next to the cortex and finally the inner core is called the medulla. Most of the hair is cortex which contain the color pigments. Common characteristics of hair include color continuous or fragmented medulla, width of hair, artificial coloring.

Forensics scientists first determine if the hair is human or animal, then compare to suspect's hair or known pets. A comparison microscope works best for this analysis.

References and Resources: Notes are from "Crime Scene Investigations - Real Life Science Labs for Grades 6-12" by Pam Walker and Elaine Wood and "Criminalistics" Lab Manual, seventh edition by Clifton E. Meloan, Richard E. James and Richard Saperstein, Prentice Hall 2001.
Carolina Biological supply Company kit# 69-9871 "His Hair, Her Hair, Whose Hair"
R. P. Cargille Laboratories, 55 Commerce Road, Cedar Grove, New Jersey 57009-1289, 973-239-6633, fax- 973-239-6096

Hair http://www.natural-hair.com/structure.html (nice figure of hair structure)
http://www.salonweb.com/gold/tri.htm (discusses hydrogen bonds and beta structure of the hair)

Glass and Soil Student Notes

Often, forensic scientists need to determine the characteristics of items found at a crime scene and compare these items to ones found on a suspect or at the suspect's home or car or boat or anything connected to the suspect. Physical Properties of these trace substances can be used to describe and compare these items of interest.

The Locard Principle states that if one surface touches another, there will be an exchange of some physical material, which can be identified. Physical properties that describe substances can include: weight, volume, color, boiling or melting points, density and refractive indices. Glass and soil can be characterized by these characteristics.

The density (or types of specific gravity) of glass can be determined by obtaining the mass by weighing it on a balance and dividing by the volume determined by the water displacement method or by the Archimedes principle (p.13), "Criminalistics" Lab Manual by Meloan and Saperstein. The densities of different glass samples can be compared and used to determine if there is a match between glass connected to a suspect and glass found at the crime scene. Another physical property that can be used to compare glass samples is the refractive index. In general, different glasses and different liquids have specific refractive indices. If a glass sample is put into liquids of the same refractive index, it will not be visible as in the demonstration described below. If the refractive indices are different a dark edge or boundary can be seen with the naked eye and under a microscope. This line or edge is called the Becke line. If the line is not seen the refractive index of the glass and the liquid are the same giving the forensic scientist another physical characteristic for the glass sample to be identified.

Analysis of soils can include determination of pH, fluorescence, and sedimentation rates in different solvents by eye or in a spectrophotometer.