Forensic Chemistry

Laboratory Manual Revision 7.1

Dr. Bruce McCord Department of Chemistry

Florida International University

Spring 2005

This document is dedicated to the memory of Anthony J. Andrews (1965-2001) Professor of Forensic Chemistry, Ohio University.

Laboratory Schedule Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15 Footprints and Fingerprints 1 Footprints and Fingerprints 2 Seized Drugs: Spot tests Seized Drugs: microcrystal tests Seized Drugs: FTIR/UV/GC/ Documents and Trace: Inks by TLC Documents and Trace: Fibers by PLM Documents and Trace: Glass Explosives: Low Explosives 1 Explosives: Low Explosives 2 Toxicology:GC/MS 1 Toxicology: GC/MS 2 Arson 1 Arson 2 Presentation of Results *Rotation Order 1a 1b 2a 2b 2c 3a 3b 3c 4 4 5 5 6 6 7

Gr
1 2 3 4

1 1a 1a 1a 1a

2 1b 1b 1b 1b

3 2a 2a 2b 2b

4 2b 2b 2a 2a

5 2c 2c 2c 2c

6 3a 3a 3b 3b

7 3b 3b 3a 3a

8 3c 3c 3c 3c

9 4 5 6

10 4 5 6

11 5 6 4

12 5 6 4

13 6 4 5

14 6 4 5

15 mc mc mc mc

Table of Contents

Laboratory Procedures and General Information 1. Footprints and Fingerprints

2.

Drug analysis: microscopic tests, color tests, and instrumental

3. Trace Evidence: inks, paints and fibers 4. Glass Analysis 5. Explosives analysis: Spot tests, FTIR, Capillary electrophoresis 6. Drug extraction in urine: GC/MS 7. Arson analysis: GC-FID Appendix A, color test reagents .....46 Appendix B, Sample Lab Report.6 65

Notes 1. Each student will have one laboratory report for which the score will be thrown out 2. Removal from the labs or ingestion of controlled substances by any route, removal of equipment, glassware, and/or chemicals, cheating, dishonesty, plagiarism, or deception in fulfilling requirements will not be tolerated. Penalties include failing the course, and criminal prosecution.

LABORATORY NOTEBOOK PROCEDURES 1. The first page of the notebook should have your name and the course name. 2. The second page of the notebook should be a table of contents. Each time an experiment begins, a page number entry for the experiment should be entered in the table of contents.
3.

When you enter the laboratory to start an experiment, the following should already be included in your notebook.

a) Experiment name. b) Written statement of purpose. c) Summary of procedure to be performed condensed from the experiment handout.
d)

Space set aside and labelled for the experimental data to be taken.

4. Data collected in the laboratory should be entered in the allocated space in the notebook. 5. As you conduct the experiment, note observations in your notebook. For example, note any unexpected problems with the equipment or procedures. 6. All notebook entries should be in ink. 7. Each page should be labelled with the date.
8.

Important: At the end of each period, have the instructor or TA initial your notebook pages.

GENERAL INFORMATION Students will work in assigned groups. Each student is required to write a formal report on each experiment. The formal report is to conform to the guidelines of the ACS style guide and is due 7 days after the completion of that experiment. 3 points will be deducted for each day that a laboratory report is late. Each report MUST be typed. Hand written reports will have 15 points deducted. The report should follow the outline below and point values are a guideline. Introduction A brief (100 words max.) description of the purpose of the experiment. Experimental Description of equipment and chemicals used. Appropriate information on the sample analysed (sample number, physical description, etc.) Results A summary of the results that you obtained. Answer any questions given in each experiment. Conclusions What you learned from the experiment. An example would be "The tests performed (name the tests) indicated that sample XYZ contained cocaine". Draw the correct conclusions from the results that you obtain. Include sources of error. References Include this document and any other resources you used. TA Assessment Typos, proofreading, general flow of lab report, lab behaviour including teamwork, cleaning of glassware, etc. Answers to Questions 4

Grading: Labs will be graded on a 100 point scale. Grading will be based on the following criterion
1)

Explanation and understanding of the science (10 points) - Does the report show sufficient background information for the reader to follow and understand the experiments? Grammar, organization, and references. (10 points) - Is the document clearly written, well organized and properly referenced? Figures and photographs (20 points) - Are there sufficient photographs, tables and graphs necessary to support the results. Are they properly labelled and referenced in the text? Experimental procedures .. (10 points) - Is sufficient information available to allow the reader to repeat the experiment? Was the laboratory experiment properly recorded in the notebook? (References to specific laboratory procedures in this handout are OK. Results (20 points) - Do the results of the experiment match the known and are sources of variance properly noted and explained? Are all the points noted in the discussion section covered? Answers to questions and evaluation of paper...(30 points)

2)

3)

4)

5)

6)

Note: Certain questions will require access to the library, to your supplemental readings, or to your class notes. See your instructor if you are having trouble finding source materials. You are responsible for washing glassware you used according to the instructions and to the satisfaction of the instructor before you leave the laboratory. Always rinse used glassware thoroughly with tap water to remove the chemicals and other contaminants before placing them in a detergent solution or other cleaning solution. Use a distilled water final rinse. You are expected to keep the laboratories clean and orderly. Many of the compounds and experiments we will perform require attention to detail. Read experiments carefully prior to coming to lab and follow all safety precautions- gloves with controlled substances, goggles and lab coats at all times. Follow the laboratory safety rules and the instructions on each experiment on the disposal of waste materials and chemicals.

1. Footprints and fingerprints Scenario At the scene of the crime, a footprint was found in the mud. Also, several items were collected that may have the suspects fingerprints on them. Using the supplies and procedures below, determine which suspect matches the footprint and fingerprints found at the scene. For the fingerprint analysis, determine which suspect the print belongs to by highlighting the patterns of recognition provided in class notes and supplemental material. You may want to practice all of the techniques on your own fingerprints and determine which method works best, before analyzing the suspect prints. No specific footprint precautions or hazards. Footprint Introduction Calcium sulfate dehydrate or gypsum, dehydrates upon heating to form calcium sulfate hemihydrate or plaster of Paris. 2CaSO42H2O(s) + heat (CaSo4)2H2O(s) + 3H2O(g) gypsum plaster of Paris When enough water is added to plaster of Paris to make a paste, it quickly hardens as it reverts to gypsum. The mixture also expands as it hardens and forms a sharp impression of the object in contact with the mixture. The rate of hardening decreases as the amount of water used increases. The FBI now recommends the use of Dental Stone or Die Stone as they require less water and have greater hardness. This makes it unnecessary to reinforce the cast. Less material is required as well. Supplies and equipment Wooden box, earth and bag of die stone cast (about 1 kg). Procedure To hold the particles of earth in place to preserve the fine structure, allow a mist of lacquer to settle onto the print by spraying the lacquer horizontally above the shoeprint. Caution: Do not spray directly onto the shoeprint.
1.

2. Photograph the shoeprint in earth with oblique lighting with the film plane parallel to the shoeprint. Place a 6 to 12" rigid ruler next to the impression or at the bottom of the impression's surface. 3. Add approximately 280 g of water to the bag of Die Stone. Massage the mixture through the closed bag. The mixture should pour easily like a pancake batter but not too watery, it should be runny, but not too dry. Add more Die Stone or water if needed. 4. Note the time you start to add the mixture to the shoeprint. Carefully pour the mixture onto the shoeprint (from ground level). Fill the impression completely so that the mixture overflows out of the impression. 5. Inscribe identifying information (your initials and date) on the surface of the cast while it is drying. Include any other relevant information. Use straight lines instead of cursive writing. Use the drying time to note the characteristics of the shoes collected from the suspects. 6. Note the time when the upper crust of the cast is hard. This will be at least 30 minutes. Carefully lift the cast from the box of earth. Do not attempt to clean the cast, allow it to air dry for 48 hours. Measure the cast of the shoeprint and the shoe used, and compare 6

the measurements. Record the measurements. Compare them with the measurements made on the photograph. 7. Carefully examine the cast for any unique marks such as cuts, tears, wears, and/or imbedded materials. Record such marks. You can wash the cast with water, it will not dissolve or crumble. Disposal No specific disposal instructions. Fingerprint Precautions Ninhydrin is rubefacient and a poison, use the hood. Do not look at the UV light. Fingerprint Introduction Fingerprints on paper, cardboard, unpainted wood, and other absorbent surfaces are hard to obtain by dusting with fingerprint powder and are much better developed by chemical methods. Each of the following chemicals reacts with a different substance, which may be present in the latent print a. iodine reacts with the double bonds in unsaturated fatty acids. b. ninhydrin reacts with the free amino and carboxyl groups in proteins and peptides. c. silver nitrate reacts with chloride ions. Any one or all three of the chemicals may be used on most absorbent surfaces. When all three chemical methods are used, they must be used in the above sequence (a, b, and c). If the chemical methods are to be used, fingerprint powders should not be applied to the articles because powders cannot be removed from paper and may interfere with some types of document examination. Fingerprints on plastic baggies and rubber objects cannot be obtained by powder or the above chemicals but can be obtained by fuming with the vapour of cyanoacrylate in "superglues". The image may be seen with or without dusting with powder or staining with dyes. Supplies and equipment a General supplies: Tweezers, disposable gloves, scissors, electric steam iron b c d Iodine fuming chamber method: Iodine, TLC tank and lid, blank glass TLC plate, scotch tape, and glass tray for warming TLC tank with hot water. Nihydrin method: Ninhydrin solution: ninhydrin (5 g) in methanol (100 mL), nylon brush, steam iron and distilled water, and cardboard and blotter to protect table top from steam iron. Silver nitrate method: Silver nitrate (3 g) in distilled water (100 mL). Store in a brown bottle. Glass tray, nylon brush, and blotters, high intensity ultraviolet lamp and UV protective goggles. Cyanoacrylate method: Superglue, sodium hydroxide (10 % aqueous solution), TLC tank, 2 beakers (150 mL), string, scotch tape, paper clips, aluminium weighing cups, cotton gauze pad.

Procedure Preparation of fingerprint samples Prepare the following samples for your own experiments. Print your initials with a felt-tip marker on the upper right hand corner of each sample. Be careful to handle the items while wearing gloves. In dry weather, you can improve the fingerprints by wiping your fingers on sweaty parts of your body or putting moisturising lotion or cream on your hand. Each group may want to prepare a stock of the items listed below and use as required.

a. One plastic baggie for the superglue method. b. One aluminium can or rubber object. c. Three 3x5 index cards. d. Three 3x5 pieces of brown wrapping paper. e. Three 3x5 pieces of white letter paper. Recording a Photography. b Record in words: (1) experimental conditions (such as time, temperature, etc), (2) observations and results (the speed of development, colour and quality of the developed print, problems encountered and remedies proposed or attempted, etc), and (3) discussion (relative merits of the different methods, the effects of the different surfaces have on the results, etc). c From your results compare all the chemical methods of development of latent fingerprints in all aspects, including speed, reliability, clarity of prints, effects of the materials on which the prints were made and ease of application. Iodine method The image formed by the iodine method is not permanent and should be photographed as soon as the print is legible. a Prepare an iodine-fuming chamber by placing sublimated iodine crystals ( teaspoon)on the bottom of a TLC development tank with cover. Tape the samples on a piece of blank TLC glass and place the glass with the samples facing the bottom of the tank. Place the tank in a shallow pan of hot water to speed up the iodine vaporisation. Do not heat by any other method as this will break the TLC tank. Ninhydrin method Brush the solution on your sample using a nylon brush or spray the solution. Heat the brushed or sprayed sample with steam from a steam iron held about 1 inch above the paper. Record the duration of heating required to bring out the print. Silver nitrate method Place the silver nitrate solution in a glass tray in an illuminated room but not in direct sunlight. Immerse the specimen in the solution until the surfaces are completely moistened. Remove it, place it between blotters to remove the excess solution, and dry it. (sample can be slightly damp). Expose the treated specimen to sunlight, a 1 000 watt photoflood lamp, or a high intensity ultraviolet lamp. As soon as the ridge details of the prints are clearly visible, remove the paper from the light and photograph it promptly. Silver nitrate treated prints become illegible in a few hours when exposed to light but will keep for a long time in total darkness. Larger objects may be treated by brushing with the solutions. Cyanoacrylate method Attach a piece of string across the opening of the TLC tank to suspend other objects with paper clips. Use either of the following methods and determine the conditions of the development. a. Place a small aluminium dish of superglue next to or above a beaker of hot water in the TLC tank with cover. (It may be necessary to use a small heating mantle or hot plate to warm the superglue.) b. Soak cotton gauze pads with NaOH (10 %) and hang them up to dry in the hood. When ready to fume objects, place a pad in an aluminium dish and drop superglue on the pad and close the tank. View the developed latent prints in oblique light, photograph the resulting print, and record in words. Dust the print before photographing if necessary. To improve the print, spray or dip the print area in Ardrox stain (1% in isopropanol). After the print area is dry, wear UV protective glasses and view it under a 100 watt mercury vapour lamp.1
1

J.F. Fallano, Kodak Techbits 1992.

Discussion 1. For each unknown finger and foot print attach a figure and point our note 5 distinguishing features. Which lighting and developing techniques were able to best reproduce these features? 2. For each technique, explain using full chemical reactions, the mechanism for developing the latent prints. 3. Discus how you would convince a jury that your results uniquely identifies a particular shoe or fingerprint.

Disposal Ninhydrin and silver waste should be placed in the containers provided for this waste. Questions 1. What are the advantages and disadvantages of plaster of Paris as a casting material?
2. 3.

In your opinion, what minimum number of identifying features constitutes an identity? Why? How would you validate the ninhydrin test for finger prints, ie what tests would you perform to show that the tests always give the same results for a given sample and that the reagents always work? Go to the library. Find and copy one or more current articles on the use of laser induced fluorescence in the analysis of fingerprints or some other aspect of latent fingerprint analysis. Write a brief (1-page) review of the article which needs to be a summary and critique of the article in your own words. The article must be from a peer-reviewed journal. Internet and company articles are not acceptable. Please contact your instructor if you have trouble finding an article. Attach the article to your lab report.

4.

2. Drug analysis: microscopic tests, color tests, and instrumental analysis Scenario A raid was performed on a drug dealers home. Several solid drugs were found, however it is believed the dealer was also mixing the drugs with other materials to improve his profits (herbs mixed with marijuana, aspirin mixed with cocaine, etc.) You have been given a variety of samples to identify. Using morphology, spot tests, microcrystal tests and instrumental techniques, determine which drugs and/or diluents are present in your samples. Micro-chemical crystal tests, IR, MS and UV-VIS spectroscopy can be useful techniques in forensic chemistry. This experiment should give some indication of the usefulness and limitations of these different methods and illustrate their application in quantitative analysis

Precautions As detailed in Appendix A for the appropriate color tests. Chloral hydrate is harmful when ingested. Petroleum ether is extremely flammable. Introduction Cannabis sativa (Cannabinaceae), marihuana, is an annual plant of 3 to 16 ft in height. The "resin" which contains the psychoactive chemicals (principal OH component is 9-tetrahydrocannabinol, C23H30O4, MW = 358.5, CAS 23978-85-0) occurs mainly in the flowering H3C area, the leaves and the stem, particularly at the top of O CH3 the plants, with the highest amount found in the H3C flowering area. Only the roots and the lower parts of the stalks are usually free from the resin. Even tiny seedlings that have just developed their first true leaves contain some resin. The male and female plants produce resin nearly equally up to the time of flowering. The male plants die shortly after shedding their pollen. The major metabolites are 11hydroxy-9-tetahydrocannabinol and 11-nor-9-carboxy-9-tetahydrocannabinol. Marihuana is a schedule 1 drug. CH3 The leaves of marihuana are compound and consist of 5 to 11 separate leaflets. Each leaflet has characteristic hairs, veins, and serrated edges. The most characteristic feature is the hairs. There are two types of hairs 1. Cystolith hairs have deposits of calcium carbonate at their base. These hairs are mostly onecelled. 2. Gladular hairs contain and secrete the resin. They are short and may be unicelluar or multicellular. Larger glandular hairs have a multicellular stalk with heads containing 6 to 8 cells. Properties Form 9 -THC M.Pt. /C viscous oil H2O Insoluble Solubility ethanol chloroform 1 in 1 Readily soluble ether

Amphetamines CH2CHCH3 NH2 Amphetamine, (1-phenyl-2-aminopropane, C9H13N, MW = 135.2, CAS 300-62-9), is commonly available as the racemic mixture or the pure d10

isomer which are both used as a central nervous system (CNS) stimulant and in treating obesity, nacrolepsy, and hypertension. The d-isomer is 3 to 4 times more effective than the 1-isomer. The metabolites are norephedrine, phenylacetone, and benzoic acid. Amphetamines are schedule II drugs. Methamphetamine, (1-phenyl-2-methylaminopropane, C10H15N, MW = 149.2, CAS 537-46-2), is anorexic and is used in treating attention deficit disorder with hyperactivity. The hydrochloride of the d-isomer is used in the treatment of obesity. The 1-isomer, which is purported to have weaker CNS stimulant activity and greater peripheral sympathomimetic activity, is used as a decongestant in certain over-the-counter (OTC) inhalers. The principal metabolite is amphetamine. Properties Form Amphetamine Methamphetamine M.Pt. /C liquid Liquid H2O 1 in 50 Slightly soluble Solubility ethanol very soluble miscible chloroform very soluble miscible ether very soluble miscible

Ephedrine, l-isomer of 1-phenyl-l-hydroxy-2-methylaminopropane, occurs naturally in the Chinese drug mahuang (Ephedra vulgaris, E. Sinica). It is a sympathomimetic amine, which has large peripheral stimulant activity and mild CNS stimulant effect. Its chloride or sulphate is used as a bronchodilator. The metabolite is norephedrine. Pseudoephedrine (Sudafed), d-isomer of ephedrine, is used as a nasal decongestant and is found in OTC cold and allergy drugs in combination with antihistamines and analgesics. The metabolite is norpseudoephedrine.

Heroin Heroin (3,6-O-diacetylmorphine, C21H23NO5, M.W = 369.4, CAS 561-27-3) is a semi-synthetic opiate produced from morphine (a naturally occurring substance in the opium poppy Papaver somniferum). The major metabolites are 6-monacteylmorphine O then morphine (and glucuronides) and codeine. Morphine is a NCH3 potent narcotic analgesic, and its primary clinical use is in the management of moderately severe and severe pain. After heroin, morphine has the greatest dependence liability of the narcotic CH3COO analgesics in common use. Other semi-synthetic analogues of morphine are also available as painkillers in the US. Heroin is a schedule II drug. CH3COO Properties Form base hydrochloride M.Pt. /C 170 229-233 H2O 1 in 1700 1 in 1.6 Solubility ethanol chloroform 1 in 31 1 in 1.5 1 in 12 1 in 1.6 ether 1 in 100 Insoluble

CH3 COOCH3

OOCC6H5 H

Cocaine (methyl benzoylecgonine, C17H21NO4, MW = 303.4, CAS 5036-2) is a tropane alkaloid obtained from coca, the dried leaves of Erythroxylum coca and other species of Erythroxylum. The South American species contains more cocaine than the Asian species. It can be synthesised from ecognine or simpler starting materials. The natural product is optically active. The major metabolites are ecgonine, ecgonine methyl ester, and benzoylecgonine. Two major forms are 11

found in the US, the hydrochloride salt (coke) and the free base (crack). Cocaine is a schedule II drug. Properties Form base hydrochloride M.Pt. /C 96-98 197 (decomp) H2O 1 in 600 1 in 0.5 Solubility ethanol chloroform 1 in 7 1 in 0.5 1 in 4 1 in 15 ether 1 in 4 insoluble

H2C CH H

HO CH3O

H H

Street drugs are commonly mixed with other materials to increase bulk and dilute the active ingredients. Cocaine and heroin are the drugs to which diluents are most commonly added. However, diluents may be added to any drug that they possess similar physical appearance to. The most common diluents are sugars, inositol (an isomer of glucose), talcum powder, starch, caffeine and quinine (shown). It is important to be able to distinguish the diluents from the drug compound.

Supplies and equipment Several samples of plant material provided by the TA. Appropriate solutions prepared as in Appendix A. Procedure Microscopic tests Record your observations, draw diagrams, and photograph the characteristic hairs and adjacent structures so that you know where to find the hairs. a Place a small portion of plant material on a microscopic slide. Add one to two drops of distilled water and flatten the material with a cover glass. Inspect at 30x to 100x magnification. Add one to two drops HCl (1 M) to the materials under the cover glass and watch for carbon dioxide bubbles formed at the base of the cystolith hairs. Do not leave the slide with HCl on the microscope stage. b Prepare a similar sample and treat it with 3 drops of saturated aqueous chloral hydrate solution. Cover with a cover glass and warm gently over a hot plate in hood. Chloral hydrate removes coloured materials. Examine at 60x to 200x for more detailed information on the structure of the plant tissues Repeat a and b with other plant materials. Carry out color tests on the following compounds as described in the attached article by ONeal.
Duquenois-Levine Marquiss X X X X X X Mandelin X X X X X Cobalt isothiocyanate Nitric Acid Mecke X X X X X X X X X

Methamphetamine Marihuana Nutmeg Aspirin Codeine

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Cocaine Acetaminophen Epehdrine Tea Quinine Sugar Caffeine Unknown

X X X X X X X

X X X X

X X X X X X X X X X X X

X X

X X X

Supplies and equipment

Introduction For absorption in the IR region there must be change in dipole moment (polarity) of molecule. Diatomics must have permanent dipole. So N2/Cl2 do NOT absorb. Other molecules need not have permanent dipole, they can exhibit a dipole by vibration: IR spectroscopy is very useful in forensic chemistry because each molecule has a unique absorption spectrum, hence a 'fingerprint' pattern is obtained. IR is widely used for qualitative purposes, though quantitation is difficult. To perform quantitative IR one must remember that beers law holds here and that the thickness of your KBr pellet must be constant (not really possible). Instead an internal standard such as calcium carbonate, napthalene or sodium nitrite is usually added to the KBR mixture to perform quantitative analysis. Alternatively, if a mixture of two substances is present, the ratio of two peaks, one from component A and one from component B can be utilized to determine % concentration with a calibration curve. Note that you must draw a baseline across the bottom of the peak and use the peak height in your measurements. Precautions - Sulphuric acid is corrosive. Sodium hydroxide is corrosive. Microchemical tests introduction An older but still viable method for determination of unknown drugs is the microchemical crystal test. In the procedure a drop of test reagent is added to a solution containing a small amount of the unknown drug. The procedure is performed by placing a drop of the test solution and a drop of the reagent solution on a microscope slide. Magnification is set at 100- 400X The two drops are brought together using a small glass rod or spatula. Specific crystals are formed by the reaction and can be characterized using a microscope. Experimental Procedure Prepare certain reagents from the list below (ask TA for availability of reagents). Test your samples by adding the drug directly to the reagent or by dissolving a small amount of the compound in the test solution minus the reagent and placing a drop of each solution onto the microscope slide. Use a spatula to move the two drops together. Observe crystal formation at the interface between the two solutions. IR Procedure Prepare disks of the test compounds (2 mg) in KBr (100 mg). Prepare a KBr blank as well. (if available you may use the ATR system instead.) Obtain an IR spectrum for each compound between the wave numbers 400 and 4000. Collect at least 64 one second scans (4096 data points/scan). Compare the obtained spectra with the provided reference spectra. 13

1) Prepare and print spectra of the blank, drugs and diluent samples (blank may contain an internal standard.) 2) Examine the spectra of the unknown and using spectral subtraction, determine its identity and the identity of the diluent it is mixed with. 3) Prepare a calibration curve consisting of mixtures of drug and diluent. 4) Determine the % drug in your unknown, using the ratio of specific peaks of each component. 5) Determine the total quantity of drug in your unknown. Disposal - Dissolve solid waste in dilute acid and dispose of down the drain with copious amounts of water. UV-Vis Procedure Perform UV spectral analysis of a set of pure drugs and your unknown drug in methanol. Does the unknown known appear pure? Samples should be very dilute (approx. 1 mg/L.) Collect UV spectra over the wavelengths 220 nm to 340 nm. Be very careful to avoid contamination or peak overload. Use blanks and perform several dilutions to make sure your data is on scale and not overloaded. If your sample appears pure, perform a quantitation using Beers Law. Show your results to your instructor. Be aware that some diluents will not absorb in the UV. How will this affect your results? Discussion 1. Make a table listing each drug you analyzed and the specific results from each test you employed. 2. List significant interferences for each test and explain why they occur 3. In your table, compare the utility of each technique and give advantages and disadvantages of each. 4. Detail how you arrived at your conclusion regarding the identity of your unknown and how you arrived at that conclusion. 5. Using your results with known compounds what was the effect of the diluent on your analysis by the different techniques?

Disposal Chlorinated solvent and other organic solvent waste must be placed in the appropriate container. Other waste may be disposed of down the drain with copious amounts of water.

Questions Assuming a linear calibration curve, calculate the % purity of each unknown. How accurate will this result be in the presence of the diluent? Figure a way to compensate for the presence of the diluent and get an accurate result.

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Vist the SWGDRUG web site, www.swgdrug.org and look up the section on methods of analysis. Explain why there are three distinct catagories of analytical methods and describe the difference between them. 2. If the drug you were given did not match up with any of the spectra or color tests you used, what could you do to identify the drug? 3. What determines the wavelength of absorption maxima for UV spectra?
1.

4. Suppose you were using IR to analyze these drug mixtures. Describe how you would quantitate the concentration of cocaine in a mixture with sugar using this technique? Be specific. 5. Codeine, heroin, and morphine have very similar structures. Draw these structures and explain how you could tell them apart by IR spectroscopy. 6. Visit the SWGdrug site and review the comments on validation. Describe what you consider to be the most important tests you would perform to validate a spot test for cocaine. 7. Find an article describing a thin layer chromatography (TLC) or other chromatographic test for the determination of marihuana. Write a brief (1-page) review of the article which needs to be a summary and critique of the article in your own words. The article must be from a peer-reviewed journal, (1pp.)

15

3. Trace analysis: inks, fibers, and glass Scenario A ransom letter was found in a plastic bag inside the home of a kidnapped child. A suspect was arrested, and several pens found in their home were collected and labelled. Using thin layer chromatography, determine if the ink from any of the pens gathered match that used in the ransom note. Furthermore, several small fibers and glass particles were discovered in the plastic bag. Using chemical tests, polarizing light microscopy and FTIR, identify the composition of the fibers and compare them to materials collected from the suspects home. Analyze glass particles using refractive index measurements and atomic spectroscopy. Precautions Avoid looking directly at UV light 3a. TLC Introduction The analysis of inks and dyes is an important technique in the arsenal of the document examiner. Ink analysis can be used to detect forgery of documents, to check for backdating of documents or falsification of signatures. In its more sophisticated format, ink analysis has even been used to provide estimate of the date at which a particular document was signed. Some reasons for forensic ink analysis include tests for the adulteration of numbers to increase the amount of money being charged, tests to determine if the ink used in two parts of a document came from the same pen, or tests to compare the writing used in a threatening letter with pens taken from a suspects home. Liquid inks used in ballpoint pens contain a mixture of substances, which include dyes, stabilizers, solvents, and resinous binders. Certain manufacturers also add fluorescent markers or heavy metals to aid in product identification. Thin layer chromatography (TLC) is the preferred technique for ink analysis. In TLC, a uniform layer of silica, alumna or some other adsorbant is placed on a glass plate. The ink samples are punched out of the paper, extracted in pyridine, and spotted onto the TLC plate using a glass capillary as a transfer pipette. The plate is then placed in a glass tank containing a layer of solvent at the bottom. This solvent mixture is the chromatographic eluent used to separate the mixture of dyes present in the sample. The distance a particular dye moves relative to the solvent front is known as the Rf value. Supplies and Equipment: A TLC tanks, silica TLC plates, beakers, scissors or syringe punch (a wide bore syringe needle cut flat for punch holes in paper, Plastic or wood sheet for use with punch. Glass capillary tubes for spotting plates, 1.8ml tubes with v bottoms. Glass transfer pipettes and bulbs, UV light boxes. B. Reagents: pyridine, ethyl acetate, butanol, ethanol, and methanol. Procedure: Experimental Setup: 1) Prepare 2 solvent mixtures for the TLC experiments: Mixture 1 contains 14 ml of ethyl acetate, 7 ml of ethanol and 6 ml of water. Mixture 2 contains 10 ml of butanol, 2ml of ethanol and 3 ml of water. Place each mixture in a separate TLC tank in the hood and cover with a glass lid. 2) Cut or punch out 10 small paper dots from the inked line in the ransom letter. These dots must be extremely small and contain only the inked part of the paper. Use a syringe needle that has been squared off as a punch. On another separate piece of paper make test writings with the pens obtained from the suspects apartment. Cut or punch dots from each letter. Place each set of dots in to a small conical vial. In the hood, add 1- 2 drops of pyridine to each tube. (Warning pyridine has a stench! 16

Keep it in the hood! Dispose all solvent in the hood as well) Be sure to use a minimal amount of pyridine or the sample will be too dilute. Let sit for 10-15 minutes. If necessary gently warm or vibrate each sample to aid the extraction. Add additional pyridine if necessary. 3) Prepare two TLC plates by lightly drawing a straight line with a pencil across each plate 1 inch from the bottom. 4) Spot the plates using the capillary tubes by pacing them into the extracted samples and drawing out a small amount of solution. Apply a small spot, let dry and repeat until a very small, clearly visible spot appears on the plate. Be careful to not let the spot become too big or your experiment will be ruined. 5) Examine the level of solvent in the TLC tank. The solvent can not be above sample spots when the plate is placed in the tank, it should be approximately inch up in the tank. 6) Place the plates in each tank; be sure each sample is properly labelled in pencil at the top of the plate. Watch as the solvent front moves slowly up the plate. When the front is or so up the plate, remove the plate, mark the solvent from with a pencil, and place in the hood to dry. 7) Take the dry plate and examine using both visible and UV light. Calculate Rf for each band. Rf = distance from origin of sample band/distance from origin of sample front. Which inks have common origin? Do any show fluorescent bands? 3b. Fiber Analysis 1. Using the chemical tests listed below, attempt to determine the composition of the fibers in your baggie. ( If the amount of fiber is limited, perform the non-distructive tests listed below first. ) 2. Examine your unknown fiber and the known fibers provided by your instructor under the polarized light microscope. (take pictures if possible) For each fiber examine using the following conditions: a) no polars note morphology b) one polar rotate stage and note any effects on color due to orientation c) crossed polars rotate stage, note positions of extinction, note color effects. Check for birefringence. 3. Take your fibers to the infrared microscope and obtain spectra of each fiber. Identify major peaks and correlate with functional groups present in the polymeric matrix of the fiber.

17

Saferstein, R. Criminalistics Lab Manual, Prentice Hall, 1998, p. 162

3c. Glass Analysis This experiment involves a series of demonstrations on the analysis of glass. Please review your handouts on glass analysis and report to Dr. Almirals lab for further instructions.

Disposal Place organic waste in proper containers. Acids and bases may be disposed of down the drain with copious amounts of water. Questions: 1. Can TLC be used to determine conclusively whether any two pens are the same? 2. What advantages does TLC have over other chromatographic techniques for chemical analysis? 3. What was the difference between the two solvent systems and why did they produce different results? 4. Describe the procedure you would use to determine the difference between a bank note and a counterfeit note produced by a color photocopier. 18

TLC can be used to date inks. Explain how this is done. See the article by Cantu et al. in your handouts or perform a literature search 6. Draw the polymeric structure of each fiber you identified. 7. Compare the information obtained by IR to that from polarized light microscopy. 8. Fiber analysis has been a key factor in the solution of a number of important criminal cases. Explain how experts can answer the question - what is the possibility these sets of fibers appeared in the suspect's house purely by chance? (1page.) You may use a scientific article to back up your claims. Attach the article to your report.
5.

19

4. Explosives analysis: Spot Tests, FTIR, Capillary electrophoresis Scenario A bomb was placed in the department chairs mailbox and all the grade forms were destroyed. A search was performed of the likely culprit and some commercial fireworks were recovered. Your job will be to analyze a sample of the firework recovered from the scene and determine its components. You will then burn the material to determine if it is an explosive and if the residue pattern matches that recovered from an extract of the grade forms. Introduction Spot tests, IR spectroscopy, and capillary electrophoresis are valuable techniques in the analysis of inorganic explosives. Typically an inorganic explosive consists of inorganic oxidizers such as potassium nitrate, potassium perchlorate, potassium chlorate, and fuels such as Al, Fe, S and C. A variety of spot tests and microcrystalline tests can be used to detect the oxidizers and fuels, including the greiss reagent and diphenyl amine for nitrates, Silver nitrate and methylene blue for chlorates and the formation of Prussian blue for the detection of iron. For FTIR there are specific bands indicative of nitrate (1383 cm-1) and chlorate (ca 900cm-1). Sulphates, perchlorates and other components will also produce results. Lastly the inorganic oxidizers will produce specific ions which can be determined by capillary electrophoresis or ion chromatography. The goal for this experiment is to use your laboratory skills to determine if the material is explosive, and if so the nature of the oxidizer and fuels. Precautions - Sulphuric acid is corrosive. Sodium hydroxide is corrosive. Mix in open containers A. Flame test. Place a small amount of the material on the tip of a spatula. Place under a flame and observe reaction. B. Microscopy (for both burned and unburned material) 1) Wearing gloves, examine the evidence by carefully cutting it open with a knife or scalpel. (Rinse blade with DI water prior to use) 2) Recover the explosive material and examine it under the microscope. Note morphology and look for crystalline material. Examine under crossed polars and note any specific features. 3) Prepare or obtain the following reagents a) Greiss reagent for nitrites: Prepare 0.25 g napthol in 80 mL of 30% acetic acid and 0.5 g sulfanilic acid in 50 mL of 30% acetic acid. Add one drop of each to test plate. Look fro Orange color. b) Diphenlyamine test for nitrates Prepare 5mg diphenyl amine in 50ml of concentrated HCL, look for blue color as indicator of nitrate. c) Perchlorate and chlorate Add 1 drop conc. H2SO4 (look for brown gas indicative of chlorate), add a small amount of Zn to unknown to reduce the CLO4-. Add Ag NO3 and look for white precipitate (Cl). Also place 2 drops of solution of unk. in spot plate. Add 2 drops of saturated zinc sulphate, add 2 drops of 10g potassium nitrate in 40mL water (beware contamination) and add 1 drop 0.03g methylene blue in 100mL of water d) Aluminium 20

Place known or unknown in 3 wells of a spot plate. Test samples with conc. HCL, 1M NaOH, and conc. HNO3. Use microscope to test for effervescence. Al will not effervesce with HNO3. e) iron Place concentrated K4Fe(CN)6 in a glass slide add drop of unknown dissolved in HCl . Move two drops together under microscope and look for blue color. f) Sulfur Place material on a capped slide. Heat gently with burner and look for sublimation of sulphur/odours. IR Procedure Obtain an IR spectrum for each of the following compounds potassium perchlorate, potassium chlorate, potassium sulfate potassium nitrite and potassium nitrate between the wave numbers 400 and 4000 cm-1. Collect at least 64 one second scans (4096 data points/scan). Compare the spectra of your burned and unburned unknown with the above results. Look for specific spectral bands. Hint: separation of components by recrystallizing out of methanol may improve results. Be sure MeOH is completely gone prior to analysis. CZE Procedure Dissolve a very small amount of your known and unknown samples in DI water. Filter and dilute 1/50. Place in each in a glass vial and take upstairs to room 371 for analysis by capillary electrophoresis. Determine individual ions via comparison with standards Method for Anions: 50 cm fused silica capillary 75um ID 1.8 mM Potassium dichromate visualizing agent (for indirect detection) Diethylene triamine osmotic flow modifier (to reverse osmotic flow) Borate buffer 2mM borate 40mM boric acid (pH to 7.8 with diethylene triamine) Wavelength 205,280 nm for detection of ions by indirect (280) and direct (205) methods. Analyze at 20kV with a 2 second pressure injection. Discussion 1. Which set of techniques provided the most definitive information in your experiments? 2. Give the specific bands for the individual components in the FTIR analysis. 3. What differences did you see between pre and post blast analysis and why? 4. Was the fuse material different from that of the bulk explosive? How could you tell? What other differences did you see? 5. How definitive were the spot tests compared to the instrumental techniques?

Questions: 1) Sulfur is a material added to many low explosives. What role might it play in the composition of the explosive? 2) It is also important to look for different cations in low explosives. Ammonium nitrate is a common explosive in mining. Potassium salts are used in black powder. Describe two different methods you could use to tell the difference between ammonium and potassium. 3) Contamination from nitrates used in foods and other commercial products is a real problem in explosives residue detection. If you used these experiments to determine whether someone had 21

handled explosives, what controls would you use in your experiments to avoid contamination from these and other sources? 4. For an explosive composed of potassium perchlorate, sulphur and sugar, what ions would you expect to find in the post blast residue? 5. Another type of explosive commonly used in the US is smokeless powder. Find a paper describing a method of analysis for smokeless powders and give the paper a critical evaluations. (1 page)

Disposal Chlorinated solvent and other organic solvent waste must be placed in the appropriate container. Other waste may be disposed of down the drain with copious amounts of water.

22

5. Drug extraction in urine: GC-MS Scenario - A urine sample will be provided for you from the pilot of the plane you will take to Florida next week. A blank urine sample will also be provided. Perform a drug screen by performing a liquid/liquid extraction of basic drugs from urine followed by separation and identification using GC/MS. If drugs are present, report the concentration found in the urine. Precautions BSTFA is flammable and harmful by inhalation. Introduction Gas Chromatography (GC) is a widely used technique in forensic chemistry. Compounds that are not highly volatile or likely to decompose at the higher temperature used in GC are usually H3C Si CH3 derivatised prior to analysis. The most common type of derivative is a methyl silane. This functional group increases the volatility of CH3 O the compound. There are a number of different silyating reagents available. The choice of reagent depends upon the functional CF3 C N Si CH3 groups to be derivatised. The reagent used here (N-Obis(trimethylsilyl) trifluororacetamide, BSTFA) is a multi-purpose CH3 reagent which is capable of derivatizing a wide range of functional groups. The mass spectral detector is probably the most important detector for GC in forensic chemistry. This is partly due to the low limits of detection that the detector has, but mainly because of the structural information provided by the fragmentation ions produced. CH3 Procedure Remove 5 ml of urine and adjust to a pH of 10-11 using NH4 OH (approximately 0.8ml) Place in a small separatory funnel. Add 5ml 1-chlorobutane. Shake, remove 4 ml of the organic layer into a test tube, and evaporate to dryness by blowing a steady stream of nitrogen over the tube. Make sure your sample is completely dry. Add 25ul BSFTA to the dry tube. The reaction will not work if any moisture is present. Cap and let stand 10 minutes. Warm if necessary. Inject 0.5 L of this onto the GC/MS. Repeat 2 more times. Determine the precision of your analysis by calculating the standard deviation of the peak areas. Determine the identity of your drug using the MS library and through the injection of the samples provided by the TA Prepare an external standard calibration graph. Using the stock solutions provided by the TA dilute this solution so that your calibration graph covers the concentration range 1 g/mL to 10 g/mL. Start your calibration standard preparation at the derivatization stage. Your calibration graph should have at least 5 points in it. Discussion Was your derivatization successful? Why or why not? Provide a table of the specific peaks and give fragmentation mechanisms for three of these peaks in your mass spectrum which convinced you that you had correctly identified your drug of interest 3. For this particular drug what might a typical concentration be in blood or urine? 4. Explain why it was necessary to adjust the pH of the urine to 10-11? 5. Give the slope, the correlation coefficient and standard deviation of the y estimate for your results and use this data to provide an estimate of the reliability of your result.
1. 2.

23

Disposal Chlorinated solvent and other organic solvent waste must be placed in the appropriate container. Other waste may be disposed of down the drain with copious amounts of water.

Questions 1. Using the paper in your supplementary notes describe 3 different methods for the derivatization of drugs for GC analysis. Which procedures are recommended for cocaine, cannabis, morphine and amphetamine and why?
2. 3.

What factors influence the recovery of a liquid/liquid extraction? Derivatization is especially important for the analysis of drug metabolites. Explain the reason metabolites are more polar than their parent drug and why this makes them easier to derivatize.

4. Give the structure of BSTFA. Show the reaction of BSTFA with benzoylecgonine.
5.

Derivatization alters the molecular structure. Sometimes this is an advantage in MS analysis, why? List this and other advantages of the use of derivatization reagents. Two mass spectrometric techniques sometimes used in toxicological laboratories to aid in drug analysis are selective ion monitoring and MS/MS. Explain what these are and how they can be useful in toxicological analysis. GC/MS is used as a confirmatory test in drug analysis. Immunoassay is commonly used as an initial, screening tool. Describe how immunoassay functions and explain how some drug users can adulterate their urine to fool this test.

6.

7.

8. Find and critique an article on the use of liquid-chromatography/mass spectrometry for forensic drug analysis.

24

6. ARSON ANALYSIS: GC-FID Scenario A restaurant was burned to the ground and the fire inspector suspects arson. Samples of the restaurant carpet were believed to be treated with an accelerant. You have been provided with a sample from the restaurant, as well as blank and spiked burnt carpet samples to determine if accelerant was used, what type, and the amount. Introduction There are a number of mechanisms that can be postulated to result in a fire of unknown origin. One of these is arson. It is the role of the fire investigator to seek the source of the fire. If arson is suspected the investigator will collect debris from suspect locations and place it in a metal paint can or specially formulated sealed plastic bag. Upon arrival at the laboratory, a sample of the headspace of the paint will be taken and a charcoal strip will be suspended above the debris. The can will then be gently heated or simply left sit overnight. The charcoal strip is then extracted using a small amount of CS2 and a small amount of the extract is injected into the gas chromatograph. In order to interpret the results of an arson analysis it is important to understand the how crude oil is refined and manufactured. The following table illustrates the nomenclature used in the petroleum distillation process: Class # 1 Light Petroleum Distillates (LPD) 2 Gasoline 3 Medium PD 4Kerosene 5 Heavy PD 0 Unclassified peak spread based on n-alkane carbon #s C4-C8 C4-C12 C8-C12 C9-C16 C10-C23 Variable Examples Petroleum ethers, pocket lighter fuels, rubber cement solvents, lacquer thinners Automotive gasoline, some lantern fuels Charcoal starters, paint thinners, mineral spirits, dry cleaning fluids, torch fuels # 1 fuel oil, jet-A (aviation) fuel, insect sprays, charcoal starters # 2 fuel oil, diesel fuel Single compounds such as alcohols, acetone, or toluene, xylenes, isoparaffinic mixtures some lamp oils, camping fuels, lacquer thinners, duplicating

Supplies and equipment Procedure Carpet (2" X 2") samples were all cut from one large piece. Samples consist of a Pyrolyzed carpet, and carpets spiked with the following accelerants: Gasoline (250 l), Lighter Fluid (250 l), Turpentine (250 l) and Diesel Fuel (250 l). The carpet sample from the house was taken from a 10X20 ft. room. All carpet samples were bagged in nylon with activated charcoal strips (Albrayco Laboratories, 500 Corporate Row, Cromwell, CT 06416; Ph: 860-635-3369). The Pyrolyzed sample was heated by propane torch until the carpet started to burn. Accelerant samples had listed amounts applied to carpet and were ignited by match. Samples were burned to consume half of the accelerant. Burn time was estimated by burning 250 l of the accelerant only, in a 3" diameter open dish. All pyrolyzed and accelerant samples were bagged directly after burning. All samples were heated @ 100 C for 90 min. Analysis Conditions 25

column temp ECD gas

DB-5MS 15 m x 0.25 mm x 0.25 m 40 C (2min) 15 C/min to 265 C

N2 carrier detector temp Injector temp

35 cm/sec

split flow make up gas

100 ml/min

Check to make sure these conditions are what your system is using to avoid delays. Before starting check to make sure the GC column is properly functional and clean. Bake column and run blanks if necessary. Prepare a sample of gasoline, 60% evaporated gasoline, pristane and phytane, and diesel fuel NOTE: For this laboratory you will be injecting standards and samples that are diluted (.50 l of sample in 10 ml methylene Chloride). The C5-C20 n-alkane standard is diluted in pentane. Experimental: 1. Analyze 1.0 l of the C5-C20 standard with the above temperature program. 2. Take the charcoal strips from nylon bags and place in 1.8 ml vials with appropriate label. 3. In the hood using syringes provided add 500 l methylene chloride to a vial containing a charcoal strip. 4. Analyze all carpet samples by GC. Due to lack of time do not run blank GC analyses between samples unless the column has been overloaded. Inject 1 l of sample into the GC. 5. Analyze 1.0 l sample of a gasoline sample which has been evaporated to 60% of its original volume and then diluted. Compare this result to a fresh sample. 6. Run a diesel fuel standard as well. Discussion 1. What were the complete conditions for sample preparation and instrument operation? 2. What is the range of peaks that are present, light medium or heavy? Is there a broad or narrow distribution of peaks? 3. Are characteristic class features for all of the compounds present? 4. Do you see peaks in the unknown that correspond with peaks in the C5-C20 standard? 5. Use these features to limit your possibilities and then run standards to test and confirm your hypothesis on the identity of your unknown. 6. What was the effect of the weathered (evaporated) gasoline? Would you have difficulty in distinguishing evaporated gasoline from diesel fuel? What could help you in recognition of differences? 7. What was the effect of the burned carpet on the gasoline analysis? Did it complicate the results?

Questions 1. There are several methods for the detection of arson. These include passive headspace (this lab) dynamic headspace, solid phase microextraction and distillation. Describe each method and give advantages/disadvantages of each. 2. In this experiment you used a burned carpet as a control. From your results, is it necessary is it to obtain a control, or would a neat sample of the accelerant do? 3. Suppose your suspect claimed that she had spilled an lighter fluid last week on the carpet. How would you verify her claim? 4. Pristane & Phytane are indicators of a heavy petroleum distillate. In which accelerant should you easily find them? What particular n-alkanes do they elute near? Pristane (2,6,10,14-tetramethylpentadecane) Mol. Wt. 268.53 CAS. NO. 1921-70-6 Phytane (2,6,10,14-tetramethylhexadecane) Mol. Wt. 282.55 CAS. NO.638-36-8
5.

Find an article that discusses the use of GC/MS in arson analysis and the advantages of single ion monitoring. Write a brief (1-page) review of the article which needs to be a summary and 26

critique of the article in your own words. The article must be from a peer-reviewed journal, internet and company articles are not acceptable. Please see a TA if you have trouble finding an article. Attach the article to your lab report.

27

APPENDIX A: COLOUR TEST REAGENTS Cobalt isothiocyanate Test Precautions No specific precautions. Reagent Prepare a 2% w/v solution of cobalt isothiocyanate in water. Method 1. Add reagent to the test substance in a test tube. Indications Development of a blue-green colour indicates that cocaine (or surrogates) may be present. Duquenois-Levine Test Precautions Acetaldehyde boils at 21 C, is highly flammable and harmful. Avoid inhaling it and handle it in the hood. Chloroform is harmful, use it in the hood. Dispose of chloroform waste in marked containers provided. Reagent Dissolve vanillin (1 g) in ethanol (95%, 50 mL) in a glass-stoppered bottle. Add fresh acetaldehyde (0.15 mL). Store the reagent in a cool, dark place. Discard the reagent when it acquires a deep yellow colour. Acetaldehyde is shipped in a sealed glass bottle. Before you break the seal, cool the bottle in ice water to reduce the vapour pressure inside the container. Score the glass tubing on top of the bottle with a triangular file and break the tubing carefully. Pour the contents into a brown glass bottle and store in a cool, dark place. Method 1. Place a small amount of plant material in a small test tube. Add 12 drops of the reagent and stir. Decant the liquid into another test tube and record the liquid colour. 2. Add an equal volume of concentrated HCl to the above liquid and mix. Record the mixture colour. 3. Add a few drops of chloroform to the above mixture and agitate. Identify the layers and record the colour of each layer. Indications A colour change from grey to green through blue to violet blue suggests the presence of cannabis. Distinction from roasted coffee and patchouli oil is required. Only with cannabis is the violet colour extracted into the chloroform layer. Compound Cannabis Coffee (roasted) Initial Colour Violet-blue Violet-brown 28 Colour extracted by CHCl3 layer Violet None

Patchouli Oil Tea (leaves)

Violet Green-Blue

None None

Other natural products such as basil, bay leaf, marjoram, nutmeg, rosemary, sage, thyme or tobacco give no colour. Mandelin Test Precautions Sulphuric acid is corrosive. Make all dilutions in an open beaker!!! Reagent Dissolve ammonium vanadate (0.5 g) in water (1.5 mL) and dilute to 100 mL with sulphuric acid. Method Add a drop of reagent to the sample on a white tile. Indications Various colours across the entire visible spectrum are given by a large number of compounds. The colour given by Liebermann's test should also be taken into account when interpreting the result. Hydrochlorides give a red colour. Red Brown-red Orange Red-orange Green-orange Brown-orange Yellow Orange-yellow Green-yellow Green
Ajmaline, azacyclonol, chlorprothixene, diperodon (green), dofamium (brown), flupenthixoi, gelsemine (green), ndapamide, mequitazine, methotrexate, nialamide. pericyazine, prajmalium, prolintane, sodium cromoglycate, thiothixene, xylometazoline Nadolol Dropropizine (slow), ethylnoradrenaline, hydrastinine (green), lachesine (green), levamisole (greygreen), methanthelinium, methixene, methyldopa, methyldopate, methylpiperidyl benzilate (brown green), noradrenaline, orphenadrine, pipenzolate (green), poldine methylsulphate (greenviolet), propantheline, proquamezine (violet), solanidine (violetblue), solanine (violetblue), sulindac, thenalidine (brown) Cotamine (brown) 5-Methyltryptamine Mexiletine Azaperone, benztropine, broxaldine, chelidonine (green), conessine, deptropine, desipramine (blue), dihydralazine, diphenhydramine, diphenidol, diphenylpyraline, dropropizine (orange), halquinol, homidium, lidoflazine, methacycline (orange-violet), paraphenylenediamine, penicillamine, protokylol (brown), tofenacin, tylosin (yellow-brown), veratrine (orangeviolet-brown), viprynium Hexoprenaline Methoxamine Acepromazine (red), adiphenine (blue), benorylate, bephenium hydroxynaphthoate, bibenzonium buclosamide (blue rim), bunamidine, chlorpromazine (violet), clefamide (brown), codeine, coichicine, cyclazocine, cyclomethycaine (brown), debrisoquine, diaveridine, dibenzepin, diethazine (violet with excess reagent), diethylthiambutene (greenblue), dimethindene, dimethoxanate (brown), dimoxyline, dipipanone (blue), dothiepin, doxorubicin, doxycycline (yellow), ethopropazine (violet), fenpiprane, guanoxan, harman, hydroxyephedrine, isoxsuprine, metanephrine, methadone (blue), methdilazine (violet), methocarbamol, methoxyamphetamine, methylenedioxyamphetamine (blue), -methyltryptamine (orange), metopimazine, monocrotaline, niclosamide, nitmxotine, norharman (yellow), norrnetanephrine, obidoxime (blue), oleandomycin, oxymetazoline, pecazine (violet), pentazocine, perazine (violet), phenazone, phenazopyridine, phenformin, phenindamine, phenoxybenzamine (violet), phenyltoloxamine, pindolol, piperacetazine (redviolet), pipoxolan (brown), prenylamine, proflavine, promazine (violet), pmmethazine (violet), propranolol, reserpine, ritodrine, thenium, thenyldiamine, thiocarlide (yellow), tranylcypromine (violet), trifluomeprazine (red-violet) Normethadone, opipramol Benzoctamine, berberine (brown), edrophonium, hydroxystilbamidine, ketobemidone, methoxyphenamine, phentolamine, profadol (green), viloxazine Benzydamine, chlorphenesin Alverine, azapropazone, benzhexol, diamphenethide, diethyltryptamine (yellow), dihydrocodeine, guaiphenesin, hordenine, levomethadyl acetate, norinorphine, oxyphencyclamine, papaverine, terbutaline Bamethan (green), clomipramine, deserpidine (green), desferrioxamine (violet), doxapram, droperidol (green), harmine (green), imipramine (add water), maprotiline, mebhydrolin, metaraminol, phenaglycodol, phenyramidol, pyridoxine (grey-green), salbutamol (blue rim brown rim), thioridazine (violet), trimipramine (add water), triphenyltetrazolium (slow), xipamide, xylazine, yohimbine (green)

Yellow-green Blue-green Brown-green Grey-green Blue

29

Green-blue Violet

Chlophedianol, labetalol Amidephrine, benperidol, bezitmmide (orange), bisacodyl, captodiame, cephaloridine, chloropyrilene (orange), clomiphene (orange-brown), clomocycline (brown), denatonium, dipyridamole, guanoclor (orangebrown-yellow), guanoxan, hexobendine, hydromorphone (orange), mepacrine (yellow), mepyramine, methisazone (yellow), mianserin, morantel, naloxone (brown), oxyclozanide (orange), oxyphenisatin, oxytetracycline (redorange), penthienate, perphenazine, phenylbutazone, pizotifen (green), prilocaine, primaquine (orange), propiomazine, pyrantel, pyffobutamine, rolitetracycline (redorange), strychnine, tetracycline (redorange), thiethylperazine, thiopropazate, triacetyloleandomycin (slow), tridihexethyl, trimeprazine, trimetazidine Antazoline, carphenazine, dimethothiazine, histapyffodine, thonzylamine Alcuronium, hexocyclium, methotrimeprazine Alprenolol, bitoscanate, butaperazine, naphazoline Methoserpidine, oxprenolot, tricyclamol Methapyrilene Amitriptyline (green), azapetine, bamipine, carbetapentane (slow), clidinium (green), cyclopentolate, diphemanil, dipyrone, doxepin, embutramide, fluanisone, fluphenazine, isoetharine, isometheptene, isoprenaline, methindizate, methyl benzoquate, methysergide, metoclopramide, norpipanone (blue), nortriptyline (green), phenclzine, phenylephrine, pimozide, piperidolate, prochlorperazine (violet), propoxycaine, rescinnamine, salinazid, stanozolol, tetrabenazine, thioproperazine (green violet), tolnaftate, tolpropamine, tramazoline, tubocurarine Benzthiazide, clioxanide, cycrimine, decoquinate, diclofenac, ethomoxane, fluopromazine, hydrastine (red), trifluoperazine Metoprolol Rifampicin, spiramycin, thebaine Clemastine, clofazimine, physostigmine, rifamycin SV, trimethoprim, tripelennamine Etenzamide, harmaline, lysergic acid, mesoridazine, narceine, syrosingopine Chlortetracycline (yellow), cyproheptadine, demeclocycline, dihydroergotamine, ergotamine, lymecycline (yellow), methylergometrine, nicergoline (brown), octaphonium, oxethazaine, protriptyline, trimethobenzamide Dextropropoxyphene, mephenesin carbamate Dihydromorphine, diprenorphine, etilefrine (greenbrown), ibogaine (violet), indomethacin, lobeline, lysergide, oxypertine, propranolol, trazodone (violet) Alphaprodine, diamorphine, morphine Procyclidine

Red-violet Blue-violet Brown-violet Grey-violet Black-violet Brown

Red-brown Pink-brown Orange-brown Yellow-brown Green-brown Violet-brown Grey-brown Grey Blue-grey Black Marquiss Test Precautions

Sulphuric acid is corrosive. Make all dilutions in an open beaker!! Reagent Mix 1 volume formaldehyde solution (formalin) with 9 volumes of sulphuric acid. Method Add a drop of reagent to the sample on a white tile. Indications Various colours across the entire visible spectrum are given by a large number of compounds. Red
Alprenolol, benzylmorphine (violet), buphenine, dimethothiazine, etenzamide, etilefrine, fenclofenac (slow), fenpiprane, fluphenazine, flurbiprofen, hexoprenaline, labetalol (brown-red), maprotiline, mephenesin carbamate, mequitazine (slow), mesoridazine (violet), methoxyphenamine, metopimazine, mexiletine, nadolol, pentazocine (green), pericyazine, phenazopyridine, phenoperidine, phenylephrine, piperacetazine, prenylamine, thebaine (orange), thiethylperazine (green), thioproperazine, thiothixene, tolpropamine, tranylcypromine (brown), vinblastine Alverine, bethanidine, diphemanil, flupenthixol Thioridazine (blue-green) Alphaprodine Fenoprofen, metoprolol Adrenaline (violet), aletamine, amphetamine (brown), anileridine (slow), benactyzine (green blue), benzethonium, benzilonium (green blue), benzphetamine, bunamidine (red), carbetapentane (slow), carphenazine (red-violet), clidinium (blue), cyclandelate (slow), cycrimine (red), dehydroemetine, dimethyltryptamine, dipyridamole, ethacridine (red), ethoheptazine, ethylnoradrenaline (brown), famprofazone, fenbufen (brown), fencamfamin, fenethylline, fentanyl, harmine, indapamide (violet), indomethacin, isothipendyl, ketobemidone, lachesine (green

Orange-Red Violet-Red Brown-Red Pink Orange

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Red-Orange Pink-Orange Yellow-Orange Brown-Orange Yellow

blue), lymecycline, mepenzolate (transient), mephentermine (brown), mescaline, metanephrine (violet-brown), methacycline, methanthelinium, methindizate (green), methylamphetamine, methylpiperidyl benzilate (green blue), 5-methyltryptamine (brown), -methyltryptamine (brown), N-methyltryptamine, nefopam (brown), nomifensine (slow), normetanephrine (violet-brown), oxeladin, oxytetracycline, pentapiperide, pethidine, phenethylamine, phenformin (brown), phentermine, piminodine, pipenzolate (green blue), piperidolate, pizotifen (red), poldine methylsulphate (greenblue), primaquine, profadol (red-brown), prolintane (brown), propantheline, prothipendyl, psilocybin, rolitetracycline, spiramycin, tetracycline, trimethoprim, trimethoxyamphetamine, tryptamine, veratrine, xylometazoline Chlorprothixene Diuron Orphenadrine, pipradrol Amitriptyline Acriflavine (red), amiloride, azacyclonol, benzquinamide, benztropine, bromodiphenhydramine, broxaldine, broxyquinoline, caramiphen, chlordiazepoxide, chlorphenoxamine (green), chlortetracycline (green), chlorthalidone, cinchophen, clefamide, clemastine (green rim), colchicine, conessine (orange), cyclizine, demeclocycline (green), deptropine, diethyltryptamine (brown), diphenhydramine, diphenidol, diphenylpyraline, DOM, doxycycline, ethoxzolamide, ethylmorphine (violet black), furaltadone, halquinol, hydrocodone (brownviolet), hydromorphone (red violet), hydroxyephedrine, isoetharine (orange), lidoflazine, lorazepam, mepacrine, methyldopa (violet), methyidopate (violet), norcodeine (violet), orciprenaline, oxycodone (brown violet), oxyphenbutazone, phanquone, phenbutrazate (slow), phentolamine, phenyramidol, pindolol (brown), pramoxine (green), proflavine (orange), salbutamol, salinazid, sodium cromoglycate, solanine (violet), terbutaline, tetrabenazine, thebacon (violet), tofenacin, triamterene, trimetazidine (fades), vancomycin, viprynium embonate, zomepirac (100, orange) Stanozolol Berberine, carbaryl, chelidonine, harman, norharman, oleandomycin, propranolol, protriptyline, pseudomorphine, sulindac (slow) Acepromazine (red), verapamil (grey) Tolnaftate Harmaline Cyproheptadine, deserpidine, naphazoline, oxypertine, phenindamine, protokylol, rescinnamine, reserpine (brown) Clofibrate, embutramide, nicergoline (grey) Mebhydrolin, 1-naphthylacetic acid Apomorphine (black), azatadine, benorylate, bisacodyl, buprenorphine, butriptyline, captodiame, chloropyrilene, chlorpromazine, clofazimine, codeine, diamorphine, diethylthiambutene, dihydrocodeine, dimethindene (blue), dimethoxanate, doxorubicin, doxylamine, ethopropazine, ethoxazene, guaiphenesin, guanoxan, hexocyclium methylsulphate, mepyramine, 6-monoacetylmorphine, morphine, nalorphine, normorphine, oxprenolol, oxyphenisatin, pecazine, penthienate, perazine, perphenazine, phenoxybenzamine, phenyltoloxamine, pholcodine, pimozide, pipoxolan (grey), prochlorperazine, procyclidine, promazine, promethazine, proquamezine, solanidine, thenium, thiopropazate, tricyclamol, trimeprazine, viloxazine Acetophenazine, benzoctamine, bephenium hydroxynaphthoate, cephaloridine, chlophedianol (brown), dihydromorphine, ethomoxane, fluopromazine, isoxsuprine, lobeline, methdilazine, propiomazine, tramazoline, trifluomeprazine, trifluoperazine, trimeperidine Methocarbamol, methotrimeprazine, morantel, neopine, noscapine (fades), pyrantel Butaperazine, dopamine, tridihexethyl Benzhexol, diprenorphine, oxymorphone, pyrrobutamine, thenalidine Dextropropoxyphene (green), methapyrilene, thenyldiamine Bibenzonium, carbidopa, cyclazocine (green), diclofenac (slow), dimoxyline, dothiepin, doxepin, ergometrine, ergotamine, erythromycin, hordenine (green), ibuprofen (100, orange), isoprenaline (violet), lysergamide, lysergic acid, naloxone (violet), naproxen, narceine (green), noradrenaline, phenazocine, rimiterol (black), serotonin (slow), syrosingopine, tyramine (green) Biperiden, debrisoquine, methyl benzoquate, oxethazaine, phenprobamate, trimetozine, tripelennamine Benethamine (brown), clomocycline, nortriptyline Ritodrine, thymoxamine, triacetyloleandomycin, tylosin Alcuronium, bufotenine, psilocin Clomiphene, diethazine, levomethadyl acetate (grey-green), methoxamine (green) Dihydroergotamine, methylergometrine, octaphonium Butorphanol, diaveridinc (violet-brown), ibogaine (orange), lysergide, methoserpidine, methyscrgide, pholedrine (green) Acetorphine (yellow-brown), etorphine (yellow-brown) Methylenedioxyamphetamine

Orange-Yellow Green Yellow-Green Blue-Green Brown-Green Grey-Green Blue Grey-Blue Violet

Red-Violet Blue-Violet Brown-Violet Grey-Violet Black-Violet Brown Red-Brown Orange-Brown Yellow-Brown Green-Brown Violet-Brown Grey-Brown Grey Blue-Grey Black Blue-Black

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Nitric Acid Test Precautions Nitric acid is highly corrosive. Wear goggles and gloves when performing this test Reagent Concetrated nitric acid. Method Add a drop of acid to the sample on a white tile. The Mecke reagent is prepared from 1g of selenious acid in 100 mL of concentrated sulfuric acid. Mecke Test Precautions Sulphuric acid is corrosive. Make all dilutions in an open beaker!! Reagent Mix 1 gm of selenious acid in 100 mL of concentrated sulphuric acid. Method Add a drop of reagent to the sample on a white tile.

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APPENDIX B: SAMPLE LAB REPORT

Experiment 1 Jane Doe High Performance Liquid Chromatography Forensic Chemistry January 8, 1994

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INTRODUCTION In this experiment, high performance liquid chromatography (HPLC) was used to separate a mixture of three similar alkaloids: caffeine, theobromine, and theophylline. The retention times of the three compounds were measured, and a calibration model for caffeine was constructed based on the chromatographic peak heights produced by a series of six caffeine standard solutions. The derived calibration model was then employed in the prediction of caffeine content in a commercial soft drink.

PROCEDURE Instrumentation and Equipment. A modular HPLC setup was used in this experiment, consisting of (1) a MiltonRoy Model 1100 pump, (2) a 20 L sample injector, (3) an Altex C8 reverse phase HPLC column, (4) an ultraviolet detector operating at 254 nm, and (5) an output chart recorder. In solution preparation, a Mettler AE160 analytical balance was used to weigh solid reagents. Class A volumetric glassware was used throughout the experiment. Solution Preparation. For the study of the retention times of the three alkaloids, separate 1 L solutions of caffeine, theophylline, and theobromine were prepared in deionized water. The weights of solute used were 0.1011 g, 0.1022 g, and 0.0998 for caffeine, theophylline, and theobromine, respectively. In constructing the calibration model for caffeine, six standard solutions were prepared by serial dilution from a caffeine stock solution. The stock solution was prepared by dissolving 0.5000 g of caffeine in enough deionized water to make 1 L of solution. Employing 5, 10, 15, and 20 mL volumetric pipets, aliquots of 5, 10, 15, 20, 30, and 40 mL were removed from the stock solution and diluted to 100 mL with deionized water. The unknown for the caffeine determination was a sample of "Pepsi", a commercial soft drink. Data Collection and Analysis. A mixture of acetonitrile (20% v/v) in deionized water was used as the mobile phase. This solution was degassed prior to being used. A flow rate of 1.0 mL/min was employed during the equilibration and during subsequent injections of the samples. For the study of the retention times of the three alkaloids, three injections of each solution were made. In constructing the caffeine calibration model, three injections were made of each of the six standard solutions. Three injections of the "Pepsi" unknown were then made. No dilution of the unknown was made before injection. The recorded chromatograms were analyzed by measuring peak heights with a ruler.

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EXPERIMENTAL DATA Retention Time Study Retention Time (sec, + 0.1 sec) Compound Caffeine Theobromine Theophylline Trial 1 180.1 154.6 244.7 Trial 2 181.3 155.3 245.8 Trial 3 180.5 154.9 245.2

Caffeine Calibration Study Aliquot Volume (mL) 5.0 10.0 15.0 20.0 30.0 40.0 Unknown Peak Height (cm, + 0.05 cm) Trial 1 2.10 3.82 5.02 8.40 11.90 14.55 6.23 Trial 2 1.25 3.20 5.09 8.35 10.25 15.34 6.58 Trial 3 1.50 3.80 5.60 7.70 11.05 13.80 6.61

CALCULATIONS AND RESULTS Retention Time Study The retention times of caffeine, theobromine, and theophylline were analyzed to compute the mean retention time, standard deviation of retention time, and % relative standard deviation. Means and standard deviations were computed by use of functions resident on a HewlettPackard Model 42S electronic calculator. Compound Caffeine Theobromine Theophylline Mean Retention Time (s) 180.6 154.9 245.2 35 Standard Deviation (s) 0.6 0.4 0.6 % RSD 0.39 0.23 0.22

Caffeine Calibration Study Calculation of Exact Concentrations of Standards Cstock = (0.5000 g caffeine/1.0 L) x (1000 mg/g) = 500.0 mg/L = 500.0 ppm For a given standard, Cstd = (A)(Cstock)/100 mL where A is an aliquot volume in mL. Example: (Standard #1) Cstd = (5 mL)(500.0 ppm)/(100 mL) = 25.0 ppm

Table I Exact Concentrations of Standards Standard # 1 2 3 4 5 6 Concentration (ppm) 25.0 50.0 75.0 100.0 150.0 200.0

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Calculation of Mean Peak Heights and Standard Deviations (HP 42S calculator): Table II Mean Peak Heights and Standard Deviations Concentration (ppm) 25.0 50.0 75.0 100.0 150.0 200.0 Mean Peak Height (cm) 1.62 3.61 5.24 8.15 11.07 14.56 S. Deviation (cm) 0.44 0.35 0.32 0.39 0.83 0.77

Calculation of Calibration Model A leastsquares calculation was used to compute the slope and intercept of the best calibration model for the above data. The resident leastsquares functions on the HP 42S calculator were used for this calculation. Peak Height = 0.0486 + (0.074220)(Concentration) To evaluate the quality of the calibration model, the correlation coefficient was computed: The built in function of the HP 42S calculator was used. r = 0.996 On the next page, the calibration curve is plotted. In the plot, the mean peak heights are indicated by circles, and the leastsquares line is indicated by the solid line. Computation of Unknown Caffeine Concentration The mean peak height of the unknown was calculated to be 6.47 cm. This value was used with the computed slope and intercept to determine the concentration of caffeine in the sample of "Pepsi". Cunk = (6.47 + 0.0486)/0.07422 = 87.9 ppm

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Calibration Curve for Caffeine Determination

18 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 Caffeine Concentration (ppm) Mean Peak Height (cm)

Figure I. Calibration curve for Caffeine Determination. Error bars are based on a 95% Confidence Interval. DISCUSSION The calibration model produced an excellent fit to the experimental data, as evidenced by the high value of the correlation coefficient. While no formal test of accuracy was performed, the successful calibration model lends confidence to the determined caffeine value. The retention time study indicated clearly that the three compounds could be separated using the C8column. This study also revealed that the retention times were quite reproducible (rsd < 1 %). No anomalies were encountered during the experimental procedure. QUESTIONS 1. Two explanations for constituents not showing up in the chromatograms are (1) their possible co elution with other peaks in the chromatogram or (2) their being permanently retained on the column. It is also possible that constituents may not absorb light at 254 nm, the wavelength used by the detector employed here. 2. Enhanced qualitative structural information would best be obtained by use of an alternate detector. Mass spectrometric detection would perhaps

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