forensic chemistry - bienvenidosmatematicas.udea.edu.co/~carlopez/forense/lab3-3.pdf · chemistry...

Chemistry 487

Forensic Chemistry

Laboratory Manual Revision 3.3

Anthony Andrews and Bruce McCord

Department of Chemistry and Biochemistry

Ohio University

Spring 2001

2

Laboratory Schedule

27 March Casts of Footprints 29 March Development of Latent fingerprints 3 April Visualization of Punched Markings on Metal 5 April Firearms Residue on Victims 10 April Marihuana, Amphetamine and Morphine 12 April Cocaine, Barbiturate, Diluent, and Unknown 17 April Thin layer Chromatography *Rotation Order

Gr 19 Apr

24 Apr

26 Apr

1 May

3 May

8 May

10 May

15 May

17 May

22 May

24 May

29 May

1 8 14 13 13 18 15 11 11 10 17 9 9 2 16 16 8 14 13 13 18 10 11 11 15 17 3 10 18 16 16 8 14 13 13 17 15 11 11 4 11 11 10 12 16 16 17 15 18 13 12 8 5 18 8 11 11 10 17 16 16 15 14 13 13 6 13 13 18 8 11 11 10 17 16 16 8 15 31st May Laboratory checkout

3

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 labeled with the date.

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

4

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 guide1 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 5 points deducted. The report should follow the outline below (number of points in parenthesis) Introduction (1) A brief (100 words max.) description of the purpose of the experiment. Experimental (3) Description of equipment and chemicals used. Appropriate information

on the sample analysed (sample number, physical description and any other relevant details).

Results (6) A summary of the results that you obtained. Answer any questions given in each experiment.

Conclusions (2) What you learnt 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.

References (1) As appropriate. Answers to Questions (7)

Note:

Certain questions will require access to the library, to your supplemental readings or to Ohiolink to find and discuss articles on topics in forensic chemistry. Use the following procedure to find an article by Ohiolink: (Note: you must have adobe acrobat reader in order to read the PDF file. Download acrobat reader at http://www.adobe.com if you don't)

Next goto http://www.ohiou.edu, go to library, go to ohiolink, go to full text services, go to electronic journal center, go to search, search forensic AND toolmarks or some other combination of words. (Note: the AND is capitalized), go to the bottom of the page and print the full text (PDF file) of the article/s of your choice. Other locations for articles include the main library for J. Forensic Science and the Chemistry Library for J. Anal. Toxicology and Forensic Science Reviews.

1) The ACS Style Guide: A Manual for Authors and Editors, Second Edition, Janet S. Dodd, Editor, American

Chemical Society, Washington DC 1997.

5

Attach the yellow copies from your lab book to the written report. Labs will be graded out of 20 points. 3 points will be deducted for yellow sheets without the initials of a TA or the course instructor. Follow the laboratory safety rules and the instructions on each experiment on the disposal of waste materials and chemicals. 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. You are expected to keep the laboratories clean and orderly. Academic misconduct: 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, referral to University Judiciaries (see Ohio University Bulletin), and criminal prosecution. Tampering with safety equipment at any time violates Student Conduct Code A.13 (Ohio University Student Handbook).

6

1. CASTS OF FOOTPRINTS

Precautions

No specific hazards.

Introduction

Calcium sulfate dehydrate or gypsum, dehydrates upon heating to form calcium sulfate hemihydrate or plaster of Paris.

2CaSO4•2H2O(s) + heat → (CaSo4)2•H2O(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 1. Fill a shallow (7.5 x 15.5 x 34 cm) wooden box with 5 cm of soft earth. Use this

opportunity to observe the difference between shoeprints made in walking, running, or standing, etc. Smooth the earth and have another group make a shoeprint in the earth (out of your sight). One of your group members should note the footwear characteristics of the members of the group that made the shoeprint.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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 the measurements. Record the measurements. Compare them with the measurements made on the photograph.

7

8. 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.

Questions

1. Can you identify the person who made the footprint?

2. What are the advantages and disadvantages of plaster of Paris as a casting material?

3. Why it is not easy to use this method on impressions in snow?

4. Discus how you would convince a jury that your casting uniquely identifies a particular shoe.

5. Go to the library or to Ohio link. Find and copy a current article on the analysis of toolmarks in forensic science. Write a brief review of the article. Attach the article to your lab report.

Disposal

No specific disposal instructions.

8

2. DEVELOPMENT OF LATENT FINGERPRINTS

Precautions

Ninhydrin is rubefacient and a poison, use the hood. Do not look at the UV light.

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

b 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.

c 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.

d 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.

e 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 should prepare one plastic baggie and one aluminium can. Place several fingerprints on the baggie so that each member of the team can keep one developed print. Each team should 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.

9

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).

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 iodine crystals (½ 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.

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 with an electric hair dryer/heat gun. 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 a beaker of hot water in the TLC tank with cover. 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

Questions 1. Identify the general type of fingerprint and note 5 distinguishing features. Which technique

was able to best reproduce these features? 2. 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.

3. For each method of development describe the chemical/physical reaction used to develop the

print. Why do certain prints fade with time? 1) J.F. Fallano, Kodak Techbits 1992.

10

4. Go to the library or to Ohio link. 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 review of the article. Attach the article to your lab report.

Disposal Ninhydrin and silver waste should be placed in the containers provided for this waste.

11

3. VISUALISATION OF PUNCHED MARKINGS ON METAL AND EXAMINATION OF TOOLMARKS

Precautions

Hydrochloric acid is corrosive. Notes: Throughout the class time different individuals should break out of their group for part f which involves the use of the comparison microscope.

Introduction On metallic objects, the punched markings such as serial numbers, letters, or other designs, which have been filed off, may occasionally be visualised chemically. The punching process creates different degrees of compaction on the metal surface, with the indented numbers, letters, and other designs being the most compacted areas. A chemical that reacts with the metal will react more rapidly with the more compacted areas than the less compacted areas. If the reaction product is insoluble and has a different colour from the colour of the metal, the numbers, letters, and other designs can be differentiated from the background. Iron and alloys of iron reacts with a concentrated aqueous solution of copper(II) chloride in hydrochloric acid as follows

Fe(s) + CuCl2(aq) → FeCl2(aq) + CuCl(s)

Copper and alloys of copper reacts with a concentrated aqueous solution of Fe(III) chloride in hydrochloric acid as follows

Cu(s) + FeCl3(aq) → CuCl(s) + FeCl2(aq) In both reactions, the slightly soluble product is CuCl(s). It is important that the surface in question be smoothed by polishing with very fine grit sandpaper before the reaction. On a rough surface, the ridges with the largest surface to volume ratio will react the fastest creating an image of the ridges, which may obscure the punched images.

Supplies and equipment

Cotton Q-tips, silicon carbide sandpapers of 1500 grit and 600 grit. For new samples with rougher surface, coarser sandpapers are necessary for the initial smoothing down. Paper towels, light machine oil, aqueous sodium hydrogen carbonate solution in a beaker, and metal samples provided by the TA.

Unknown samples Metals samples of brass, soft steel, stainless steel and aluminium have been prepared. A single identification letter or number has been stamped on one surface. One to three letters and/or numbers have been punched on the opposite surface and ground off on a grinding wheel in the shop. The ground surface has been smoothed off and coated with light machine oil to prevent corrosion.

Reagents Dispense the following in small dropping bottles. a Iron(III) chloride reagent: Dissolve iron(III) chloride (20 g) in concentrated HCl (20 mL), add

distilled water (80 mL). b Copper(II) chloride reagent: Dissolve copper(II) chloride (40 g) in concentrated HC1 (54 mL),

add distilled water (45 mL). c Wash acetone

12

Procedure You will do three unknown metallic samples, one brass and two different iron alloys. Do one sample at a time until you finish.

a Record the identification letter or number on the sample and the name of the alloy.

b Examine the smoothest surface of the sample. If the surface is smooth and not corroded, give it a finishing polish with the 1500-grit silicon carbide paper. Place a cardboard or paper towel on the tabletop and place a narrow strip of the silicon carbide sandpaper, grit side up, on the cardboard or paper towel. Polish the sample by moving the sample back and forth on the sandpaper. Turn the sample a little from time to time. Rinse off the surface with acetone. Do not use the same piece of sandpaper for different alloys.

c During this step, record how much reagent you use, how you apply the reagent, any changes you observe, approximately how long it takes for the image to appear, your observations, and the colour of the image that appears, and the colour of the background. Dab (don't rub) the polished surface with the appropriate reagent on a Q-tip. Watch carefully for changes on the surface. The letter or number will emerge as a faint image. When you are certain you have a usable image, rinse the sample quickly with some distilled water to stop the reaction. Record the image by drawing a diagram of the smooth surface of your sample on your lab record. Show the sample and your diagram to the TA and ask the TA to initial the diagram on your lab record to confirm that you have obtained the image. If you use a different method of application to achieve the desired results, describe your method in full.

d Dip the sample briefly in a beaker containing a dilute aqueous solution of sodium hydrogen carbonate to neutralise the acid. Rinse the sample thoroughly in tap water. Dry the sample with paper towel. Polish off the image with a fresh piece of the 1500 grit silicon carbide paper. Coat the sample with some light machine oil and returned to a designated container.

e Repeat steps a, b, c, and d with the other samples.

f Another example of a toolmark is to examine stryations on bullets. Utilize the comparison microscope to determine if the two exemplar bullets were fired from the same weapon. List and describe (draw) the number of similarities.

Questions 1. Rank the alloys in the ease of visualisation. 2. Write the 1/2 reactions for each chemical reaction and balance the equations. 3. Why would the method work or fail if the indented serial number or identification marks have

been moulded rather than punched? 4. What is the purpose of washing the sample with acetone? 5. Why should the same piece of sandpaper not be used with the different alloys? 6. Describe the procedure you would use if you had to recover a serial number stamped in wood?.

Disposal

Waste may be disposed of down the drain with copious amounts of water.

13

4. FIREARM RESIDUES ON VICTIMS CLOTHING

Precautions

Wear rubber gloves throughout the nitrite test. Do all spraying in the hood for the lead test.

Introduction When a handgun has been fired within two meters of a victim, the pattern of residues on the victim's clothing around a bullet hole can be used to estimate the distance at which the handgun has been discharged. The closer the distance, the denser the residue. The residues consist primarily of inorganic nitrites and lead. The nitrites are the ignition products of cellulose nitrate in the smokeless powder used in cartridge type ammunition. The lead comes from several sources, most from the lead compounds in the cartridge primer mixture. A significant amount can also come from the friction between the bullet and the gun barrel and erosion of the bases of bullets. Visible and invisible lead residues may be left when a bullet "wipes" a surface or the immediate perimeter of a bullet hole.

The chemical reactions for the nitrite test are 1. Nitrite reacts with acetic acid to form nitrous acid. 2. Nitrous acid reacts with sulfanilic acid to form a diazonium salt of sulfanilic acid. 3. The diazonium salt couples with the 1-naphthol to form an yellow water-soluble azo dye. The dye formed on the victim's clothing is transferred to the gelatin coating of a dye-transfer paper in contact with the clothing. The chemical reaction for the lead test is the formation of blue-violet coloured lead(II) rhodizonate (pink/red-brown with barium) with sodium rhodizonate.

NITRITE TEST

Supplies and equipment 1. 11 x 14 inches plastic photographic tray. 2. String and plastic cloth pins for hanging up papers to dry. 3. A household electric iron. 4. Four or more pieces of nitrite- free cheesecloth (or thin cotton fabric) slightly larger than 11 x 14

inches. 5. Corrugated cardboard larger than 11 x 14 inches. 6. Four or more sheets of 11 x 14 inches, double weight, smooth surfaced (F- or glossy surface)

dye transfer paper. If dye transfer paper is not available, use double weight, smooth surfaced photographic paper. Treat the paper in advance in a concentrated sodium thiosulfate solution or photographic "fixer" solution without hardener to remove silver halides. Wash the paper thoroughly in water to remove sodium thiosulfate and dry the paper as if drying a photographic print.

7. Nitrite test cotton swabs. Soak the cotton end in the sodium nitrite solution and set the swabs aside to dry. Stored in a sealed container.

8. Beakers: 1 L and 100 mL.

Samples These will be provided by the TA.

Solutions Prepare sulfanilic acid (or 4-aminobenensulfonic acid, 0.5 g in 100 mL distilled water), 15% acetic acid (150 mL glacial acetic acid diluted to 1 L with distilled water), sodium nitrite (0.69 g in 100 mL distilled water) and either a or b. a 1-Naphthol (or 1-naphthalenol, 0.4 g in 100 mL methanol).

14

b N-(I-naphthyl)ethylenediamine dihydrochloride (or N-1-naphthalenyl-1,2-ethandiamine, 0.5 g in 100 mL methanol).

Procedure A 1. Mix equal volumes of sulfanilic acid solution and 1-napthol (or naphtyl-ethylenediamine)

solution in a plastic photographic tray. 2. Soak one at a time the dye transfer paper in the mixture until the paper is thoroughly wet. Pick

up the paper by two corners and let the excess solution drain in the tray. 3. Hang the paper on the string with cloth pins to dry. 4. Store the dried paper in a dark and dry place. 5. Just before using, test a sheet of treated paper for nitrite sensitivity by dabbing the four corners

with a nitrite testing swab saturated with 15% acetic acid.

B 1. Place a treated dye transfer paper, gelatin side up, on corrugated cardboard. 2. Place the sample cloth, residue covered side down on the dye transfer paper. 3. Soak a cheesecloth in 15% acetic acid in a 1 L beaker. Pick up a cheesecloth, squeeze out the

excess liquid, and spread it out evenly on the back of the sample fabric. 4. Press the above sandwich from the back of the cheesecloth with a hot iron. 5. Peel off and discard the cheesecloth. 6. Mark the bullet hole. Mark the edges of the fabric if the fabric is smaller than the dye transfer

paper. In actual cases, mark with a pencil important features of the sample fabric: suspected bullet holes, tears, cuts, rips, button holes, buttons, seams, pockets, etc., for possible future reference in court.

7. Peel off the sample fabric and save it for the lead test. 8. When the dye transfer paper is dry, mark it on one edge in ink with your initials and case and/or

file number.

LEAD TEST

Supplies and equipment Spray bottles, test strips containing soluble calcium, strontium, barium, and lead salts.

Solutions Saturated sodium rhodizonate (or 5,6-dihydroxy-5-cyclohexene-1,2,3,4-tetrone disodium salt) in buffer (50 mL), the solution should be prepared fresh each day. HC1 (5 mL concentrated HCl and 95 mL distilled water) and pH 2.8 buffer (sodium hydrogen tartrate monohydrate (1.90 g) and tartaric acid (1.50 g) in distilled water (100 mL).

Procedure A. Direct application to white sample fabrics 1. Spray the appropriate area with the saturated sodium rhodizonate solution. Pink colour

indicates the presence of lead(II) and other metal ions. 2. Spray the same area with HCl solution. If the pink colour turns to a blue colour, the area has

lead(II). 3. Repeat steps 1, 2, and 3 on test strips containing known metallic elements. Record and

compare the results.

B. Indirect application to coloured sample fabrics 1. Place a piece of filter paper on the area of interest, include any suspected bullet holes. Record

the location of the fabric covered by the filter paper. 2. Spray 15% acetic acid on the filter paper until it is uniformly damp. 3. Cover the dampened filter paper with several layers of dry filter paper and press a hot iron on

the filter paper until the paper is dry.

15

4. Remove the filter paper that is in direct contact with the evidence fabric and treat as in Part A. Remember that any coloured images developed is a mirror image of the materials on the fabric.

5. Mark the filter paper with your initial and case and/file number.

Other methods of examining firearm residues on victims 1. Infrared photography of victim's clothing or body. metallic elements.

Questions 1. Write the equations for the nitrite reactions. 2. Write the equations for the lead reactions. 3. Explain the experiment you would use to determine the distance and angle that the subject was

from the barrel of the firearm. 4. What materials might interfere with each of these tests? 5. Organic nitrites may also be present in gunshot residue. Find an article/s that describes the

detection of organic gunshot residue and write a brief review of the procedure.

Disposal All lead solutions should be placed in the appropriate waste container. Other waste may be disposed of down the drain with copious amounts of water.

16

5. MARIHUANA, AMPHETAMINE AND MORPHINE ANALYSIS

Precautions

As detailed in Appendix A for the appropriate colour 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 component is ∆9-tetrahydrocannabinol, C23H30O4, MW = 358.5, CAS 23978-85-0) occurs mainly in the flowering area, the leaves and the stem, particularly at the top of the plants, with the highest amount found in the 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 the ir 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 11-hydroxy-∆9-tetahydrocannabinol and 11-nor-9-carboxy-∆9-tetahydrocannabinol. Marihuana is a schedule 1 drug. 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 one-

celled. 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 Solubility

Form M.Pt. /°° C H2O ethanol chloroform ether ∆9-THC viscous oil Insoluble 1 in 1 Readily soluble


Several samples of plant material provided by the TA. Appropriate solutions prepared as in Appendix A.

Procedure

Microscopic tests Record your observations and draw diagrams of 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

CH3

O

OH

H3C

H3C CH3

17

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.

Colour tests Carry out the following colour tests as detailed in Appendix A on the plant material that you have received. 1. Duquenois-Levine 2. p-Dimethylaminobenzaldehyde 3. Corinth test

Toxi•Lab Analyse the organic layer from the Duquenois-Levine test by Toxi•Lab as detailed in Appendix C.

Questions

1. How specific are each of the colour tests are for marihuana? 2. Would you be satisfied with a visual test and a spot test for the identification of marihuana -

why or why not? 3. The toxilab is a TLC test for the determination of THC. Go to the literature, find and describe

(1-2 pp) a TLC or other chromatographic test for the determination of THC.

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.

18

Amphetamines

Introduction

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


Form M.Pt. /°° C H2O ethanol chloroform ether Amphetamine liquid 1 in 50 very soluble very soluble very soluble Methamphetamine Liquid Slightly soluble miscible miscible 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.


Samples provided by the TA. Appropriate solutions prepared as in Appendix A. TOXI•LAB A, blank TOXI-GRAM A and TOXI-DISC A, special procedure standard TOXI-DISC no. 129 SA, dry acetone, potassium sulphate, fresh concentrated ammonia.

Procedure Colour tests Carry out the following colour tests as detailed in Appendix A on the material that you have received. 1. Liebermans test 2. Marquis test 3. Ninhydrin test

Toxi•Lab Perform a Toxi•Lab test as detailed in Appendix C upon the material provided. The special procedure standard disc no. 129 SA contains five amines. Locate them using the following approximate Rf values.

CH2CHCH3

NH2

19

Drug Rf Methamphetamine 0.20 Phentermine 0.40 Amphetamine 0.70 Phenylpropanolamine 0.85 Ephedrine/Pseudoephedrine 0.90

Hints Amphetamines are heat labile, sample discs should be removed from the evaporation plate immediately after drying. Heating of TOXI-GRAM A beyond dryness after development may result in diffusion and/or reduced size of amphetamine spots. Stacking or overlaying of developed chromatograms can result in diffusion of amphetamine and methamphetamine from one chromatogram to another. Notes Normeperidine, metabolite of meperidine, which cannot be distinguished from ephedrine and pseudoephedrine in the routine TOXI•LAB A procedure, has a Rf value of 0.18 in this procedure. Phenylethylamine has a Rf value of 0.70 in this procedure, overlapping amphetamine.(1)

Questions 1. How useful do you think the colour tests are for amphetamines? 2. What is detected by these color tests? 3. What are some legal compounds that might produce a false positive for illegal amphetamines?


1) B.A. O'Brien, J. M. Bonicamp, and D. W. Jones, Differentiation of Amphetamines and its Major Hallucinogenic

Derivatives Using Thin Layer Chromatography, J. Anal. Toxicol., 1982, 6, 143.

20

Morphine

Introduction

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 then morphine (and glucuronides) and codeine. Morphine is a 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 analgesics in common use. Other semi-synthetic analogues of

morphine are also available as painkillers in the US. Heroin is a schedule II drug.


Form M.Pt. /°° C H2O ethanol chloroform ether base 170 1 in 1700 1 in 31 1 in 1.5 1 in 100 hydrochloride 229-233 1 in 1.6 1 in 12 1 in 1.6 Insoluble


Samples provided by the TA. Appropriate solutions prepared as in Appendix A.

Procedure

Colour tests 1. Liebermans test 2. Mandelins test 3. Marquis test

Toxi•Lab Perform a Toxi•Lab test as detailed in Appendix C upon the material provided. Be sure to list Rf values for each unknown.

Questions

1. How useful do you think the colour tests are for morphine? 2. List the two general categories of opiates and the members of these categories. 3. What metabolites of heroin are found in urine? What species is most commonly present in

urine? 4. What are some legal compounds that might produce a false positive for morphine?


O

CH3COO

CH3COO

NCH3

21

6. COCAINE AND BARBITURATE ANALYSIS

Precautions

As detailed in Appendix A for the appropriate colour tests.

Introduction Cocaine (methyl benzoylecgonine, C17H21NO4, MW = 303.4, CAS 50-36-2) 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

found in the US, the hydrochloride salt (coke) and the free base (crack). Cocaine is a schedule II drug.


Form M.Pt. /°° C H2O ethanol chloroform ether base 96-98 1 in 600 1 in 7 1 in 0.5 1 in 4 hydrochloride 197 (decomp) 1 in 0.5 1 in 4 1 in 15 insoluble



Procedure

Colour tests 1. p-Dimethylaminobenzaldehyde 2. Mandolin Test 3. Cobalt isothiocyanate test

Toxi•Lab Perform a Toxi•Lab test as detailed in Appendix C upon the material provided. Be sure to include Rf values for each unknown.

Questions

1. Why is the chemical difference between cocaine and crack? How would you distinguish them? 2. How useful do you think the colour tests are for cocaine? 3. What precautions should be taken when performing analysis of cocaine in samples of urine

which are several days old? 4. 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.

NCH3 COOCH3

OOCC6H5

H

22

Barbiturates

Introduction One of the most common barbiturates is phenobarbital (5-ethyl-5-phenylbarbituric acid, C12H12N2O3, MW = 232.2, CAS 50-06-0). It exists as colourless crystals or a white powder. Major metabolites are N-glucopyranosylphenobarbitone and 4-hydroxyphenobarbitone (and glucuronide). Other common barbiturates are secobarbital, pentobarbital and amobarbital. Barbital, the first drug of this class to be synthesised, was introduced into medicine in Germany in 1903. Barbiturates rapidly gained a

wide usage as tranquillisers, sedatives and hypnotics (sleep inducers) which continues to this day. In the past half-century, over 2 000 different barbiturates have been synthesised, although less than a dozen make up the bulk of current use. Barbiturates are schedule IV drugs.


Form M.Pt. /°° C H2O ethanol chloroform ether Base 174-178 1 in 1 000 1 in 10 1 in 40 1 in 40 Sodium 175 1 in 3 1 in 25 Insoluble Insoluble



Procedure

Colour tests 1. Liebermans test 2. Koppanyi-Zwikker Test

Toxi•Lab Perform a Toxi•Lab test as detailed in Appendix C upon the material provided. Be sure to include Rf values for each unknown.

Questions 1. How useful do you think the colour tests are for barbiturates? 2. Barbiturates are acidic drugs. Explain why this is so? 3. UV analysis of barbiturates is performed under basic conditions, why?


NH

N OO

O

C6H5

C2H5

H

23

6. DILUENT AND UNKNOWN ANALYSIS

Precautions

As detailed in Appendix A for the appropriate colour tests.

Introduction 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.

Frequently laboratories are required to analyse body fluid samples from suspects or accident victims. Blood is the most appropriate fluid, especially for determining the degree of

intoxication. Urine can also be used. If the sample gives an initial positive result for a controlled substance, this result is then confirmed by another analysis method.



Procedure Perform the following colour tests on each of the solid samples that you have been provided with

Colour tests 1. Liebermans test 2. Koppanyi-Zwikker Test 3. Mandelins test 4. Marquis test 5. p-Dimethylaminobenzaldehyde

Perform a Toxi•Lab screen for opiates, barbiturates, cocaine, amphetamines and marihuana upon the urine sample that you have been given. Include standards of these drugs for comparison purposes. Be accurate in this screening, as the result that you obtain today will determine the experiments you do in the future on this urine sample.

Questions 1. Make a flow chart describing the use of the five tests for the determination of opiates,

barbiturates, cocaine, amphetamines and marihuana. You may do each compound separately or put together a combined chart.

2. List which of the above compounds are acidic, basic or neutral. How does this affect their analysis in urine?

3. Would any of the above diluents give a positive colour test indicating a controlled substance? 4. What was the identity (if any) of the drug in the urine sample that you were given? 5. Another method for screening unknown drugs is enzyme immunoassay. Write a brief synopsis

on the use of immunoassay as a screen for the 5 classes of compounds.


N

CH3O

H

N

H

HO

HCHH2C

24

7. THIN LAYER CHROMATOGRAPHY OF INKS

Precautions

Use standard laboratory procedures and equipment. Avoid looking directly at UV light

Introduction The analysis of inks and dyes are 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 suspect’s 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. In this experiment you will extract and analyze the ink present in a threatening letter. In this scenario an umpire has been received a threatening letter from an over exuberant fan. The police have developed several suspects by observing last weeks TV feed and have recovered several pens from each suspect’s apartment. It is your job to determine which pens match the ink used in the letter.

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) Obtain the threatening letter. Cut or punch out 10 small paper dots from the inked line in the letter. 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. 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 drawing a straight line with a pencil across each plate 1 inch from the bottom.

25

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 small, clearly visible spot appears on the plate. Be careful to not let the spot become too big.

5) Examine the level of solvent in the TLC tank. If it is higher than the level of the sample spots, reduce the amount until it is about ½ inch above the top of the tank.

6) Place the plates in each tank; be sure each sample is properly labeled in pencil at the top of the plate. Watch as the solvent front moves slowly up the plate. When the front is ¾ 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 us ing both visible and UV light. Calculate Rf for each band. Rf = distance from origin of sample band/distance from origin of sample front.

Questions:

1. Compare the known ink samples with those from the note. 2. Which inks have a common origin? Do any inks show fluorescent bands? 3. Can TLC be used to determine conclusively whether any two pens are the same? What

advantages does TLC have over other techniques for chemical analysis? 4. What was the difference between the two solvent systems and why did they produce different

results? 5. Describe the procedure you would use to determine the difference between a bank note and a counterfeit note produced by a color photocopier. 6. TLC can be used to date inks. Explain how this is done.

26

8. ALCOHOL ANALYSIS

Precautions

Ethanol and propanol are toxic.

Introduction Alcohol is commonly determined in breath or blood samples. However, because of the problems with obtaining accurate gaseous alcohol samples and the risk of infection when using blood samples, alcohol content in urine will be determined. A conversion factor ([UrineEtOH]/[BloodEtOH] = 1.3/1) relates the alcohol content in urine to blood alcohol content. This will be used in the final calculation.

Supplies and equipment A urine sample from a DUI suspect. Blank urine, ethanol and n-propanol. Carry out the alcohol analysis on the HP5890 GC in Clip 080 using the FID.

Procedure

Construct two calibration graphs using the external standard method (plot area of standard vs concentration) and the internal standard method (plot area of sample divided by area of standard.vs conc.) Do this by mixing blank urine (1 mL), n-propanol (200 µL, IS) and an appropriate amount of ethanol in a headspace vial. Your low ethanol concentration should be 0.05 g/100 mL and the high concentration should be 0.3 g/100 mL. The area of your IS should be within the range of those produced in your calibration curve. There must be at least 4 points on your calibration graph. Run each concentration in duplicate. Prepare a real sample in exactly the same manner, again in duplicate. Mix vials thoroughly and allow to equilibrate for 30 minutes. Run all samples with 3 blank samples (IS only, no ethanol) in a random order. Make injections by taking 1 mL of headspace gas from each vial with a gastight syringe and injecting into the GC.

Analysis Conditions

column DB-624, 0.53 mm x 30 m x 3 µm Temperature 35 °C N2 or He carrier 50 cm/s

H2 ~40-50 mL/min detector temp 250 °C make up gas 30 mL/min

air ~450 mL/min injector temp 250 °C split ratio 50:1

Questions 1. What problem can occur in the interpretation of % alcohol in urine? 2. Why might packed columns be a better choice for the analysis of blood alcohol? 3. What are the major interferences in blood alcohol analysis? 4. Was the suspect in this case legally intoxicated? 5. Why do you run blank and control samples? 6. It has been reported that women become intoxicated more easily than men. Is this true? What determines the concentration of alcohol in blood? 7. Blood alcohol can be determined by portable instrumentation which measure breath alcohol.. Find a paper on breath alcohol determination. Compare and contrast that approach with the GC technique. Include a copy of your paper.

Disposal Waste may be disposed of down the drain with copious amounts of water.

27

9. HPLC ANALYSIS

Precautions

Methanol and acetonitrile are toxic. Sulphuric and phosphoric acids are corrosive.

Introduction High performance liquid chromatography (HPLC) is commonly used to separate thermally labile compounds that are subject to degradation and decomposition at the high temperatures frequently found in GC. It is widely used in the pharmaceutical industry and chemical industry. HPLC has not found such widespread use in forensic chemistry, although it can be used to separate all the drugs commonly encountered by forensic labs.


Your urine sample provided in experiment 10. A blank urine sample, solutions as detailed in the appropriate drug section in Appendix B. Mobile phase flow rates should be 1 mL/min and the detector wavelength should be 240 nm. Injection volume should be 20 µL.

Procedure Carry out the HPLC analysis for the tentative drug identity that you obtained in Experiment 10.

Questions 1. Did the HPLC analysis support the data from the urine screen? 2. How does HPLC analysis compare to GC analysis for the determination of drugs of abuse? 3. What are the advantages and disadvantages of the diode-array detector? 4. If you had a choice of chromatographic parameters explain why you chose the conditions that

you did. 5. Why is HPLC not as commonly used as GC in forensic chemistry labs for drug determination? 6. An alternative technique for a drug screen is capillary electrophoresis. Describe this technique

and explain its advantages/disadvantages

Disposal

HPLC waste must be disposed of into the containers provided. Other waste may be disposed of down the drain with copious amounts of water.

28

10. GC-ECD ANALYSIS

Precautions

Pesticides are toxic.

Introduction Pesticides are commonly used in agriculture. Their widespread availability has led to a number of cases of suicide and attempted poisoning using pesticides. The determination of pesticides can be accomplished by a number of methods. In the case of chlorinated pesticides a suitable technique is gas chromatography with electron capture detection (GC-ECD). In this experiment the pesticide must be extracted from the sample matrix prior to analysis. The most common form of extraction is liquid/liquid extraction. However,

solid phase extraction can be used.

Supplies and equipment A sample of tea suspected to be poisoned with pesticides. Blank sample. Maxi-Clean C18 SPE cartridges. Standard solution of chlorinated pesticides.

Procedure Pre-activate the Maxi-Clean C18 SPE cartridges by passing methanol (HPLC grade, 2 x 2 mL) through them followed by water (HPLC grade, 3 x 2 mL). Do not allow the SPE cartridge to dry. Pass the sample (10 mL) through the cartridge. Air dry the cartridge by passing air though it. Elute the analytes with ethyl acetate (HPLC grade, 2 x 1 mL). Repeat using a new cartridge with the blank sample. Inject 1 µL of your extract from both extractions into the GC for analysis. Identify the pesticide peak (if any).

Analysis Conditions

Column DB-5, 0.32 mm x 30 m x 0.1 µm N2 carrier 50 cm/s split ratio 50:1

Temp 150-275 °C @ 8 °C/min Detector temp 300 °C make up gas 60 mL/min

ECD gas 10 % of make-up gas flow Injector temp 250 °C

Questions

1. What pesticide (if any) was found in the tea? 2. Ask your TA for the concentration of the pesticide in the unknown and in the standard.

Determine the % recovery for the extraction. 3. What are potential sources of error in quantitating this compound by GC? 4. What are the main toxicological effects of chlorinated pesticides? 5. List the advantages of SPE over liquid/liquid extraction and explain each. 6. Assume you have a very dirty sample. How would you change the selectivity of the extraction

procedure to eliminate unwanted species in the final extract?

Disposal All waste containing chlorinated pesticides must be placed into the appropriate container. Vials must be rinsed with methanol into this container prior to disposal.

O

Cl Cl

ClCl

ClCl

29

11. GC-MS ANALYSIS

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 derivatised prior to analysis. The most common type of derivative is a methyl silane. This functional group increases the volatility of the compound. There are a number of different silyating reagents available. The choice of reagent depends upon the functional groups to be derivatised. The reagent used here (N-O-bis(trimethylsilyl) trifluororacetamide, BSTFA) is a multi-purpose 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. Your goal in this experiment will be to perform a drug screen by performing a liquid/liquid extraction of basic drugs from urine followed by separation and identification using GC/MS.

Supplies and equipment 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. You will be using GC/MS for your analysis.

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. Add 25ul BSFTA to the dry tube. Reaction will not work if any moisture is present. Cap and let stand 10 minutes. Warm if necessary. Inject 0.5 ul 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 µg/mL to 10 µg/mL. Start your calibration standard preparation at the derivatization stage. Your calibration graph should have at least 5 points in it. Calculate the slope, correlation coefficient, and the standard deviation of the calibration data by using the deviation of each point from the line as the error. Determine the concentration of the drug in mg/L in your urine sample. Time permitting, examine the spectra of your drug and perform an analysis using selected ion monitoring and/or MS/MS.

Questions 1. Discuss the errors that may cause deviations from linearity in your calibration plot. Consider

the GC injector, the split ratio and the mass spectrometer. 2. How realistic is the concentration of the drug in the urine? Determine what a typical

concentration would be for a user of this material. 3. Explain the reason derivatization is especially useful for drug metabolites.

CF3 C N Si

CH3

CH3

CH3

O

Si

CH3

CH3H3C

30

4. Write the reaction mechanism for BSTFA with your drug and using your spectra as a guide, determine which (if any) sites were modified. 5. Using the paper in your supplementary notes describe 3 different methods for the derivitiztion of drugs for GC analysis. Which procedure is recommended for cocaine, cannabis, morphine and amphetamine and why? 6. Derivitization alters the molecular structure. Sometimes this is an advantage in MS analysis, Why? List some disadvantages to the use of derivatization reagents. 7. Describe how selective ion monitoring and MS/MS can be useful in toxicological analysis. 8. Describe the precautions that must be taken in collecting a urine sample for forensic casework. Include problems with stability and sample contamination.

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.

31

12. IR ANALYSIS OF DRUGS

Precautions


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, quantitation is difficult though.


You will be given samples of 5 drugs and 3 diluents (or have already been given these samples for experiment 16). Reference spectra for all these drugs will be provided. The IR instrument is a Perkin-Elmer in Clip 232. Potassium bromide.

Procedure Prepare disks of the test compounds (2 mg) in KBr (100 mg). Prepare a KBr blank as well. Obtain an IR spectrum for each compound between the wavenumbers 400 and 4 000. Collect 64 one second scans (4096 data points/scan). An instruction sheet for the instrument will be provided with the reference spectra. Compare the obtained spectra with the provided reference spectra. Identify the 5 drugs and the 3 diluent samples

Questions

1. How important is IR spectroscopy in the identification of unknown drug samples? 2. How does it compare to other spectroscopic techniques such as UV-Vis and fluorescence for

drug identification? 3. If the drug you were given did not match up with any of the spectra you were given what could

you do to identify the drug? 4. Describe 4 different sampling techniques for IR analysis of Drugs 5. Draw a picture of the effect of 20% glucose would have on a spectra of cocaine.

Disposal Dissolve solid waste in dilute acid and dispose of down the drain with copious amounts of water.

32

13. AA DETERMINATION OF METALS IN GUNPOWDER

Precautions

Lead is toxic.

Introduction ICP has the advantage of being able to conduct multi-element determination simultaneously. The increased temperature of the plasma gives decreased limits of detection (LOD) compared to a conventional flame atomic absorption system. Some typical LODs are indicated below. However, our ICP is very temperamental and so we will use AA for the metal ion determination.

Element Determination Limit (µg/L)

Pb 2.5 Zn 0.13 Cd 0.25 Ni 0.5 Mn 0.06 Fe 0.25 V 0.38 Cu 0.5

Supplies and equipment Cotton swabs, four sample swabs containing suspected gunpowder residue. Polystyrene/polypropylene 15 x 75 mm tubes with polyethylene snap caps.

Procedure Prepare a series of mixed standards using 1000 ppm stock solutions of the following metals Pb, Ba, Fe, and Sb in 10% HNO3. Use the following concentration range 25-250 ppm per metal.

Extraction Cut off the cotton swab, leaving a short piece of shaft, into the sample vial. Add HNO3 (10% v/v, 2 mL), close cap, vortex. Loosen cap and heat at 80 °C in oven for 90 minutes. Close cap, vortex, cool and centrifuge for 5 minutes. Take 1 mL of supernatant and dilute to 5 mL with water prior to analysis. Construct a calibration graph for each metal over the desired concentration range. Determine the the quantity of each metal on the swabs in micrograms from this graph.

AA operating conditions

Philips SP9 Atomic Absorption Spectrometer Operation

This is a brief check- list for single element, fixed-wavelength quantitative determinations. Consult the Operations Manual for more details.

The SP9 is a single-beam D2-background corrected AAS. It has an Ebert-style diffraction grating (20 x 20 mm area, 1200 lines/mm, 250 nm blaze) for the range 190-853 nm. The focal length is 174 mm, with a linear dispersion of 4.7 nm/mm (f 7.7). The detector is a photomultiplier tube. The wavelength accuracy is + 1.0 nm.

NOTE: Samples with an organic component MUST be ashed/mineralized.

1. Turn the power on. In the compartment on the left front, set the D2 Lamp to high and set the

source current to the specific value for the element to be determined (see the Philips Data Book,

33

e.g., 4 mA for Cu).

2. Align the HCDT (source lamp) as described in the manual.

3. Set the damping to 0.5 s and the bandpass to 0.5 nm (see the Philips Data Book for

4. Element specific settings). Set to ABS (absorbance mode). Ignore the wavelength scan and scale expansion buttons

5. Open the air and acetylene tanks – the source pressure should be ~30 psi for air and ~9 psi for acetylene.

6. Press the air/fuel check button: it should read approximately 20/40/40. The airflow is about 2 L/min and the acetylene is about 3 L/min.

7. Once the pressure sensor goes off, the start button lights -- press it.

8. Check to be sure that the waste line is in its proper position. While aspirating the aqueous solvent, press the zero button. While aspirating a standard solution, adjust the lamp height to maximise the signal.

9. After shutdown, bleed off all compressed gases using the switch at the left rear.

Questions 1. Is there a difference in trace metal content between the two gunpowders evaluated? 2. Which metallic elements are most commonly used to show that a person has discharged a

firearm? 3. Why are these particular elements chosen? 4. Describe the 4 main methods for detection of GSR. Which method is the most specific? 5. Name some potential interferences in GSR analysis of metals. 6. Explain how GSR particles are formed and how SEM is used in GSR analysis. 7. Disposal Lead and antimony waste must be placed in the containers provided. Other waste may be disposed of down the drain with copious amounts of water.

34

14. ARSON ANALYSIS

Precautions

Introduction To gain experience in arson analysis technique by sampling of fire debris samples and making observations about the chromatographic information (specifically pattern recognition). 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 can 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 # “peak spread” based on

n-alkane carbon #s Examples

1 Light Petroleum Distillates (LPD)

C4-C8 Petroleum ethers, pocket lighter fuels, rubber cement solvents, lacquer thinners

2 Gasoline C4-C12 Automotive gasoline, some lantern fuels 3 Medium PD C8-C12 Charcoal starters, paint thinners, mineral

spirits, dry cleaning fluids, torch fuels 4Kerosene C9-C16 # 1 fuel oil, jet-A (aviation) fuel, insect

sprays, charcoal starters 5 Heavy PD C10-C23 # 2 fuel oil, diesel fuel 0 Unclassified Variable Single compounds such as alcohols, acetone,

or toluene, xylenes, isoparaffinic mixtures some lamp oils, camping fuels, lacquer thinners, duplicating


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 µl), Lighter Fluid (250 µl), and Diesel Fuel (250 µl). 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 µl 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

column DB-5MS 15 m x 0.25 mm x 0.25 µm N2 carrier 35 cm/sec split flow 100 ml/min

temp 40 °C (2min) 15 °C/min to 265 °C detector temp make up gas

35

ECD gas Injector temp

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 µl of sample in 10 ml methylene Chloride). The C5-C20 n-alkane standard is diluted in pentane.

Experimental:

1. Analyze 1.0 µl 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 250 µl methylene chloride to a vial containing

charcoal strip. 4. Analyze carpet samples and the 3 solution standards of accelerants by GC. Due to lack of time

do not run blank GC analyses between samples unless the column has been overloaded. Inject 1 µl of sample into the GC.

If time permits: 5. Analyze 1.0 µl 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 present?

a) For light petroleum distillates: rapid elution and narrow boiling point range b) For medium petroleum distillates: 2-3 n- alkanes normally present c) For gasoline: ethyl toluenes and 1,2,4 trimethyl benzene present

4. napthalene and methyl napthalene present 5. For heavy petroleum distillate: Pristane and phytane present 6. Use these features to limit your possibilities and then run standards to test and confirm your

hypothesis on the identity of your unknown.

Questions

1. Discuss your results. Which sample produced the best match? 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. 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?

6. What was the effect of the burned carpet on the gasoline analysis? Did it complicate the results?

36

8. Discuss the use of GC/MS in arson analysis. Explain the advantages of single ion monitoring

37

15. SPECTROSCOPIC/FLUORESCENCE ANALYSIS

Precautions

Sulphuric acid is corrosive. Sodium hydroxide is corrosive.

Introduction IR, UV-VIS and fluorescence spectroscopy can be useful techniques in forensic chemistry. This experiment should give some indication of the usefulness and limitations of these two methods and illustrate their application in quantitative analysis


You will be given samples of salicylic acid, cocaine, and amphetamine and two diluents - sugar, and caffeine. You will also be given a sample of 2 unknown drugs each mixed with a diluent. Reference spectra for all these drugs, concentrated NaOH solution and H2SO4 (0.2 M). will also be provided. The UV instrument, a HP 8451A, and the fluorescence instrument are both in Clip 080. Your job will be to determine the % drug in each mixture.

Procedure Prepare a spectra for the pure drug and the mixture of drug-diluent drug in H2SO4 (0.2 M). Samples should be very dilute (approx. 1 ppm.) Collect UV spectra for the acidic solutions over the wavelengths 220 nm to 340 nm. Obtain fluorescence spectra on the acidic solutions immediately after obtaining the UV spectra. Plot the fluorescence spectra of each compound at the UV absorbtion maxima and at one other wavelength. Take note of the maximum absorbance and fluorescence signal. Once both sets of spectra have been obtained make the solution basic by the addition of several drops of concentrated NaOH solution. Obtain both UV and fluorescence spectra for the basic solutions. Discussion 1. From your comparison of the known and diluted compounds spectra, what is the effect of the diluent on the spectra? 2. 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.

Questions 1. What determines the wavelength of absorption maxima for UV absorption? 2. Did the fluorescence spectra change with the absorption wavelength? 3. What is the advantage of fluorescence detection over UV analysis? 4. UV/vis and fluorescence spectra cannot be used in court to identify a compound, why? 5. Petroleum jelly has a specific fluorescence spectral signature that has been used in forensic

investigations of rape. What compounds are present that would produce this signature? 6. Describe a procedure you would use to connect a bottle of Vaseline captured from a suspect's

house with that found on a bed sheet at a crime scene? Name some potential interferences and explain how would you eliminate them?

7. 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.

Disposal

38

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.

39

16. QUANTITATION OF CAFFEINE IN URINE BY HPLC-UV

Precautions


Introduction One of the most common toxicological and clinical tests performed is the analysis of drugs in urine. Several screening tests and methods have been developed for the determination of drugs and urine is one of the easier samples to work with because it is generally free of protein and lipids. Therefore, it can be extracted directly with organic solvent or solid phase extraction techniques without prefiltering or extensive cleanup steps. There are several considerations to be made when measuring drugs in urine. For example, pH can affect the excretion of some compounds because urine has a wide pH range of 5.5 to 7. Additionally, some drugs are not directly measured in practice because the ir metabolites or conjugates are in much higher concentrations than the parent drug. Most drugs have a limited time window over which they can be most easily quantified before they fade to below limits of detection. Another complication arises due to the variability of urine volume over time. However, most tests are designed for the use of specific sample volumes and can only give results in terms of concentration. More meaningful values can be obtained if the total urine volume is multiplied by the concentration, which gives the amount of substance excreted over a period of time. In the following experiment the concentration of a parent drug, caffeine, will be determined in urine or cola using a simple solid phase extraction method and HPLC with UV detection. Persons ingesting coffee, tea or chocolate will excrete caffeine and its analogs caffeine, theophyllene, and theobromine in their urine. A sample of caffeine-free urine will also be extracted and analyzed as a control.

Supplies and equipment Unknown and control urine samples are provided. Calibration standards of caffeine, theophyllene, and theobromine in methanol are also included.

Procedure Extraction 1. Attach the small end of a Sep-Pak C18 solid phase extraction cartridge (~240 mg) to the tip of a

disposable syringe and remove the plunger. 2. Add 5 ml of methanol into the syringe using a disposable glass Pasteur pipet and reinsert the

plunger. 3. Condition the column packing by depressing the syringe and eluting the methanol through the

cartridge and into an organic waste container. 4. Remove the cartridge from the syringe tip and then remove the syringe plunger. 5. Repeat the conditioning steps above with 5 ml deionized water (into aqueous waste container).

Be careful not to let the column dry out. 6. Remove the C18 cartridge from the syringe and then remove the syringe plunger. 7. Reattach the cartridge to the syringe body. 8. Using a graduated glass pipet, add 5.0 ml of the urine sample into the syringe barrel. 9. Install the plunger and elute the sample at a rate of a drop every 2-3 seconds through the

packing and into an aqueous waste container. 10. Wash the packing by passing 5.0 ml of 5% methanol in deionized water through column (aq.

waste). 11. Dry the column by forcing air through the column with the syringe. 12. Elute the caffeine from the packing with 1.0 ml of methanol and collect in a centrifuge tube. 13. Transfer 0.1 ml of sample to a vial and add 0.9 ml of deionized water. Label the vial

40

appropriately.

Analysis According to Johnson and Stevenson (Basic Liquid Chromatography, 1978): the efficiency of the LC column is greatest for compounds with capacity factor (k) values between 2 and 6, but on a practical basis, k values within the range of 1 to 15 are used. An isocratic binary system of water and 20-50% methanol should yield a capacity factor (k) >3 for caffeine to provide adequate resolution from early components in urine.

1. Select a suitable UV detector wavelength based on the caffeine spectra of your standard. 2. Experiment with solvent ratios to achieve baseline resolution with minimal analysis time. 3. Analyze the control and suspect samples along with necessary standards to allow quantitation.

Use a 5 point calibration curve for caffeine and a one point calibration for the other standards. 4. Provide your answer in Fg/ml. Be sure to relate your answer to the concentration in the original

sample prior to extraction.

Questions

1. What wavelength did you choose for your analysis and why?

2. HPLC-UV spectral libraries can be used to help identify eluted compounds. This data is not acceptable for use in court, why?

3. Why is solid phase extraction of drugs in urine more selective than liquid- liquid extraction?

4. Even more specificity can be obtained using a mixed mode extraction. Describe this technique

5. What can you conclude about the caffeine source based on the composition of your unknown?

5. Find a paper on the use of solid phase extraction in drug analysis and write a brief review of the technique.

41

17. PRESUMPTIVE DRUG SCREENS PART I Ion Mobility Spectrometry Introduction Ion mobility spectrometry (IMS) is used as a screening method for explosives and drugs of abuse. IMS has the advantages of high sensitivity, detection in the parts per billion range, and short analysis times. Determining reduced mobilities, K0, identifies peaks in IMS. The equation used to determine the K0 of the target peak is: K0 target = (K0 calibrant * t calibrant) / t target

K0 calibrant is the reduced mobility of the internal calibrant (nicotinamide for narcotics mode), which is a standard. t calibrant is the experimental drift time of the calibrant, and t target is the experimental drift time of the target peak. Experimental Procedure Outline for conducting an experiment on the Barringer Ionscan 350 in narcotic (positive ion) mode: 1) Record Ionscan instrumental parameters:

a) View IMS status display window by pressing 1 on the control panel. b) Record tube temperature, inlet temperature, and desorber temperature.

c) Press enter to return to Operator’s status display window. 2) To view the Channel status display window (provides a complete summary of the status of the

calibrant and any response to target channels after an analysis) press enter. 3) Place a Teflon filter into the wand. 4) Swipe the wand with the filter in place over the surface under investigation. 5) Remove the filter from the wand and place it into a filter holder. 6) Place the filter holder into the cartridge slide assembly.

7) Make sure that the Ionscan is Ready (displayed in top right corne r) and slide the assembly into place above the desorber heater and below the inlet system. If the reading is flashing Ready, you must wait until it stops flashing.

8) The desorber anvil will raise the desorber heater and form a closed system as the sample

is heated. The yellow (analyzing) light will be on. 9) Within seconds, one of two things will happen: a) An alarm will sound and the red (fail) light will be on. b) The green (pass) light will be on.

42

10) Press enter to view the Channel status display window if the alarm sounded to view the narcotics detected.

11) Use the information obtained on the laptop computer to determine the reduced mobility, K0, of

the target peaks for positive identification. PARTII Microchemical crystal 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.

43

Deforest et al., Forensic Science, McGraw-Hill, 1983, p.138. Questions: 1. Describe the operation of an ion mobility spectrometer. Explain how ions are formed, separated

and detected. 2. For detection of explosives in airports, the direction of the electric field is reversed. Why? 3. Why is ion mobility a presumptive test? 4. Compare the results of the microchemical crystal test and the IMS. Do you think a positive

result obtained using both tests would constitute an identity? 4. Many explosives have extremely low vapor pressures. Why do you think IMS and dogs can

detect trace quantities of these materials?

44

18. ANALYSIS OF FIBERS BY IR MICROSCOPY

Precautions


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, quantitation is difficult though. IR microscopy permits the analysis of very small samples such as paint chips and fibers recovered from crime scenes. Examples of situations in which fiber evidence is recovered include hit and run, abductions, rape, and robbery. A variety of techniques are used in the identification of fibers

Supplies and equipment You will be given examples of several different fibers obtained from a hypothetical crime scene, and from a potential suspect's clothing. Your job is to match and identify known and questioned fibers.

45

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

Procedure:

Using a microscope sort and match the sample fibers by color and appearance. If sufficient fibers are present a distructive "burn test" and certain chemical tests may be performed. See chart below. Accompany TA upstairs to the IR microscope for further testing. TA will demonstrate the mounting of the fiber and operation of the IR microscope. Obtain spectra for at least three sets of known and questioned fibers. Perform library search and identify the chemical composit ion. If possible compare your results with a fiber of similar composition from the reference collection.

Questions 1. How useful was IR spectroscopy in the identification of unknown fibers? 2. Draw the polymeric structure of each fiber you identified. 3. Compare the information obtained by IR to other techniques such as microspectrophotometry and polarized light microscopy. 4. If the fiber you were given did not match up with any of the spectra you were given, what techniques could you use to further characterize the fiber? Give 3 and describe each. 5. Fiber Analysis has been a key factor in the solution of a number of important criminal cases. Explain how how experts can answer the question - what is the possibility these sets of fibers appeared in the suspect's car purely by chance?

Disposal

Dissolve solid waste in dilute acid and dispose of down the drain with copious amounts of water.

Appendix A

47

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.

Corinth Test

Precautions

Petroleum ether is flammable.

Reagent 1% Fast Blue B salt in anhydrous sodium sulphate. Petroleum ether (40 °C-60 °C fraction).

Method 1. Place small amount of drug sample on a piece of filter paper. Cover with another piece of

paper and add a few drops of petroleum ether. 2. After extraction (5 mins), remove upper paper, dry, add about 1 mg of Fast Blue B salt (fast

blue is 1.0% in anhydrous sodium sulfate) and a few drops of water.

Indications Development of a red-violet colour indicates that cannabinoids may be present. No pink colour should be present in the control.

p-Dimethylaminobenzaldehyde

Precautions

Sulphuric acid is corrosive

Reagent Dissolve p-dimethylaminobenzaldehyde (0.5 g) in a mixture of ethanol and sulphuric acid (50 mL, 60:40). Reagent should be freshly prepared.

Method Add reagent to sample. Warm if necessary. Observe colour produced and carefully dilute with water.

Indications

Red (violet on dilution) Cannabinols, phenazone (100°, 5 min), pindolol, tryptamine

Red ( no violet on dilution) Benserazide, cocaine (100°, 3 min), feprazone, harmine, phencylidine (100°, 3 min),

Orange (violet on dilution) Dobutamine, dopamine, orciprenaline, phenol, terbutaline, tyramine

Violet Dihydroergotamine, ergometrine, ergotamine, ergotoxine, lysergide, methysergide

Appendix A

48

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 Initial Colour Colour extracted by CHCl3 layer

Cannabis Violet-blue Violet Coffee (roasted) Violet-brown None Patchouli Oil Violet None Tea (leaves) Green-Blue None

Other natural products such as basil, bay leaf, marjoram, nutmeg, rosemary, sage, thyme or tobacco give no colour.

Appendix A

49

Koppanyi-Zwikker Test

Precautions

Ethanol is toxic

Reagent Solution of cobalt nitrate (1%) in ethanol.

Method Dissolve the sample in 1 ml of ethanol, add 1 drop of the reagent followed by 10 µL of pyrrolidine, and agitate the mixture.

Indications A violet colour is given by substances containing the following structures. Imides, in which >C=O and >NH are adjacent in a ring (e.g. barbiturates, glutethimide, oxyphenisatin, saccharin). Sulphonamides and other compounds with free -SO2 - NH2 on a ring (e.g. clopamide, frusemide, sulphanilamide, thiazides), or with-SO2 - NH2 in a side-chain (e.g. chlorpropamide), or with -SO2. NH2 linking a benzene ring with another ring which is other than a pyrazine, pyridazine, pyridine, or pyrimidine ring (e.g. sulphafurazole, sulphamethoxazole). These latter structures give pink or red-violet colours (e.g. sulphadiazine, sulphadimethoxine). No response is obtained with compounds where there are other substituents on the nitrogen atom. Anomalous responses are obtained with paramethadione and theophylline (violet), and with cycloserine, idoxuridine, methoin, niridazole, riboflavine (no response). Note. Hydrochlorides give a blue colour before the addition of pyrrolidine.

Appendix A

50

Liebermann's Test

Precautions


Reagent Add sodium nitrite (5 g) to sulphuric acid (50 ml) with cooling and swirling to absorb the brown fumes.

Method Add 2 or 3 drops of the reagent to the sample on a white tile. Occasionally it is necessary to carry out the test in a tube and heat in a water-bath at 100 °C Many substances give colours with sulphuric acid alone, and the test should be repeated using sulphuric acid instead of the reagent in order to ascertain that the colour seen is due to the reagent.

Indications 1. Orange colours are given by substances containing a mono-substituted benzene ring not joined to >C=O, >N-C(=O)-, or to a ring containing a >C=N-O- grouping. 2. Orange or brown colours are given by some substances containing two mono-substituted benzene rings (or some di-substituted compounds where fluorine is the second substituent) which are joined either to one carbon atom or to adjacent carbon atoms 3. A wide range of colours is given by compounds containing OH, O-alkyl, or -O-CH2. Groups attached to a benzene ring or to a ring in a polycyclic structure containing a benzene ring. The benzene ring must not bear -NO2, nor be halogenated, nor contain an -O- substituent ortho to the oxy groups. Compounds containing ring sulphur give a similar range of colours. A yellow colour is given by a variety of other compounds.

Red Ajmaline, alprenolol, arninacrine (100°), antazoline, brucine, chlorprothixene, clopenthixol, flupenthixol, mestranol, oxytetracycline, prajmalium, thiazinamium, thiothixene, tolmetin (100°), trifluopemzine, xylazine

Violet-red Indapamide

Brown-red Methylchlorophenoxyacetic acid

Pink Trichlorophenoxyacetic acid (→brown)

Brown-pink Prazosin (100°→red-orange)

Orange Aletamine, alverine, ampicillin, atropine methobromide, atropine methonitrate, baclofen, benactyzine (→brown), bethanidine (→brown), broxyquinoline, butanilicaine, chloroquine (100°), clidinium (→brown), cyclandelate, cyclizine, dazomce, decoquinate (slow), diethylthiambutene (100°), dimefline, diuron, doxapram, dyclonine (100°), fenclofenac (100°→brown), fenitrothion, fenpipramide, glibenclamide (100°, 15 s), hyoscine butylbromide, hyoscine methonitrate, linuron, loxapine (50°-60°), methindizate (→brown), methylphenidate, metolazone (→green-brown), monolinuron, nomifensine, phenazone (100°), phenelzine, propham, salinazid, sulphinpyrazone, tolazoline, trimetaphan, tripelennamine (→brown), triprolidine, xipamide, zomepirac (100°)

Red-orange Acetanilide, amphetamines, aniline, atropine, bamipine, beclamide, benethamine, caramiphen, carbetapentane, chlorcyclizine, cinchophen, cycrimine, diphenylpyraline, doxylamine, dropropizine, ephedrines, famprofazone, fencamfamin, glutethimide, hyoscine, hyoscyamine, isoaminile, isocarboxazid, levamisole, meclozine, mephentermine, methixene, methoin, methyl benzoquate, methylphenobarbitone, metomidate, morazone, nialamide, pentapiperide, pethidine, phenacemide, phenbutrazate, phendimetrazine, phenglutarimide, pheniramine, phenmetrazine, phenobarbitone, phensuximide, phenylmethylbarbituric acid, phenytoin, prolintane, tofenacin, tranylcypromine, triamterene, triphcnyltetrazolium, warfarin

Brown-orange Ambutonium, bumetanide, diphenhydramine, fenuron, feprazone (100°→brown), ibuprofen, labetalol, mepivacaine, methadone, nefopam (→brown), tetrahydrozoline

Yellow Amicarbalide (100°), clonidine (100°→orange), dequalinium (100°→orange), diethylpropion, diloxanide, ethoxzolamide, fenfluramine (100°), flavoxate, gliclazide, metoclopramide, nifenazone (100°), piroxicam, propachlor, tropicamide

Brown-yellow Amiodarone

Green Bialamicol, chlorotrianisene, colchicine, dextromoramide (100°), diamthazole, hydrastine, mequitazine, naphthols, phenol, phenothiazine, thiocarlide

Blue-green Hydrochlorothiazide, hydroflumethiazide, pindolol

Brown-green Cyclopenthiazide

Grey-green Azapropazone

Black-green Naproxen

Blue Amethocaine (100°), amidopyrine (100°), bendrofluazide, benzonatate (100°), chromonar (100°, 3 min), clomipramine, dipyrone (100°), imipramine, mefenamic acid, mefruside, oxypertine, padimate (100°), procarbazine (100°, 15 s),

Appendix A

51

dipyrone (100°), imipramine, mefenamic acid, mefruside, oxypertine, padimate (100°), procarbazine (100°, 15 s), propyphenazone (100°) (red with water), yohimbine

Green-blue Amiphenazole (100°)

Violet Methocarbamol, mianserin, paracetamol, penthienate methobromide, phenacetin, propiomazine, resorcinol, timolol (100°), trazodone (100°) (transient)

Red-violet Chloroxuron

Black-violet Methoxychlor

Brown Acepromazine, acetophenazine, adiphenine, azacyclonol, barban, benzilonium, benzyl nicotinate, biperiden, clemastine, clomiphene, cyclothiazide, dextropropoxyphene, dichlorprop, dicophane, diperodon, diphemanil, diphenidol, emepronium, etenzamide, fenpiprane, flurbiprofen, haloperidol, mepenzolate, methylpiperidyl benzilate, mexiletine, nadolol, penfluridol, phenaglycodol, phenylbutazone (100°), phosalone, pimozide, pipazethate (100°→red), pipoxolan, pyrrobutamine, rotenone, sotatol (100°), sulindac, veratrine, zimeldine

Red-brown Benzthiazide, bisacodyl, carphenazine, chlorpromazine, diclofenac, dothiepin, ethopropazine, etisazole, fenbufen, fenoprofen, methapyrilene, perphenazine, polythiazide

Pink-brown Metoprolol

Orange-brown Benazolin, diphenadione, maprotiline, methiocarb, piperidolate

Green-brown Methdilazine, norbormide, promazine, thiopropazate

Violet-brown Bamethan, clofibrate, dichlorophen

Black-brown Mecoprop

Grey Isopropamide

Black Acetomenaphthone, aloin, aminophenols, amodiaquine, apomorphine, atenolol, benorylate, benzquinamide, buprenorphine, butorphanol, carbaryl (→green), carbidopa, cephaëlne, chloroxylenol, chlorphenesin, clomocycline, clorgyline, codeine, cotarnine, cresol, cyclazocine, dextromethorphan, diamorphine, dibromopropamidine, diprenorphine, doxepin, emetine, ethamivan, ethinyloestradiol, etilefrine, frusemide, glycopyrronium, guaiphenesin, hexobendine, hydroxyephedrine, hydroxystilbamidine, ibogaine, indomethacin, levallorphan, mebeverine, mescaline, methylchlorophenoxyacetic acid, methylenedioxyamphetamine, morantel, morphine, naloxone, 1-naphthylacetic acid, narceine, nicergoline, normetanephrine, noscapine, noxiptyline, octaphonium, oestradiol, oestriol, oestrone, oxprenolol, oxyphenisatin, papaverine, pholcodine, pizotifen, practolol, profadol, propanidid, protokylol, pyrantel, rimiterol, ritodrine, rotenone, salbutamol, terbutaline, tetrabenazine, tetracycline, thymol, trimethobenzamide, trimetozine, tubocurarine, verapamil, viloxazine.

Appendix A

52

Mandelin Test

Precautions


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 Ajmaline, azacyclonol, chlorprothixene, diperodon (→green), dofamium (→brown), flupenthixoi, gelsemine (→green), ιndapamide, mequitazine, methotrexate, nialamide. pericyazine, prajmalium, prolintane, sodium cromoglycate, thiothixene, xylometazoline

Brown-red Nadolol

Orange Dropropizine (slow), ethylnoradrenaline, hydrastin ine (→green), lachesine (→green), levamisole (→grey→green), methanthelinium, methixene, methyldopa, methyldopate, methylpiperidyl benzilate (brown →green), noradrenaline, orphenadrine, pipenzolate (→green), poldine methylsulphate (→green→violet), propantheline, proquamezine (→violet), solanidine (→violet→blue), solanine (→violet →blue), sulindac, thenalidine (→brown)

Red-orange Cotamine (→brown)

Green-orange 5-Methyltryptamine

Brown-orange Mexiletine

Yellow Azaperone, benztropine, broxaldine, chelidonine (→green), conessine, deptropine, desipramine (→blue), dihydralazine, diphenhydramine, diphenidol, diphenylpyraline, dropropizine (→orange), halquinol, hom→idium, lidoflazine, methacycline (→orange-violet), paraphenylenediamine, penicillamine, protokylol (→brown), tofenacin, tylosin (→yellow-brown), veratrine (→orange→violet -brown), viprynium

Orange-yellow Hexoprenaline

Green-yellow Methoxamine

Green 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 (→green-blue), 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), α-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 (→red→violet), pipoxolan (→brown), prenylamine, proflavine, promazine (→violet), pmmethazine (→violet), propranolol, reserpine, ritodrine, thenium, thenyldiamine, thiocarlide (→yellow), tranylcypromine (→violet), trifluomeprazine (→red-violet)

Yellow-green Normethadone, opipramol

Blue-green Benzoctamine, berberine (→brown), edrophonium, hydroxystilbamidine, ketobemidone, methoxyphenamine, phentolamine, profadol (→green), viloxazine

Brown-green Benzydamine, chlorphenesin

Grey-green Alverine, azapropazone, benzhexol, diamphenethide, diethyltryptamine (→yellow), dihydrocodeine, guaiphenesin, hordenine, levomethadyl acetate, norinorphine, oxyphencyclamine, papaverine, terbutaline

Blue 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)

Green-blue Chlophedianol, labetalol

Violet Amidephrine, benperidol, bezitmmide (→orange), bisacodyl, captodiame, cephaloridine, chloropyrilene (→orange), clomiphene (→orange-brown), clomocycline (→brown), denatonium, dipyridamole, guanoclor (→orange→brown-yellow), guanoxan, hexobendine, hydromorphone (→orange), mepacrine (→yellow), mepyramine, methisazone (→yellow), mianserin, morantel, naloxone (→brown), oxyclozanide (→orange), oxyphenisatin, oxytetracycline (→red→orange), penthienate, perphenazine, phenylbutazone, pizotifen (→green), prilocaine, primaquine (→orange), propiomazine, pyrantel, pyffobutamine, rolitetracycline (→red→orange), strychnine, tetracycline (→red→orange), thiethylperazine, thiopropazate, triacetylolean→domycin (slow), tridihexethyl, trimeprazine, trimetazidine

Appendix A

53

Red-violet Antazoline, carphenazine, dimethothiazine, histapyffodine, thonzylamine

Blue-violet Alcuronium, hexocyclium, methotrimeprazine

Brown-violet Alprenolol, bitoscanate, butaperazine, naphazoline

Grey-violet Methoserpidine, oxprenolot, tricyclamol

Black-violet Methapyrilene

Brown Amitriptyline (→green), azapetine, bamipine, carbetapentane (slow), clidinium (→green), cyclopentolate, diphemanil, dipyrone, doxepin, embutramide, fluanisone, fluphenazine, isoetharine, isometheptene, isopren→aline, methindizate, methyl benzoquate, methysergide, metoclo pramide, norpipanone (→blue), nortriptyline (→green), phenclzine, phenylephrine, pimozide, piperidolate, prochlorperazine (→violet), propoxycaine, rescinnamine, salinazid, stanozolol, tetrabenazine, thioproperazine (→green →violet), tolnaftate, tolprop→amine, tramazoline, tubocurarine

Red-brown Benzthiazide, clioxanide, cycrimine, decoquinate, diclofenac, ethomoxane, fluopromazine, hydrastine (→red), trifluoperazine

Pink-brown Metoprolol

Orange-brown Rifampicin, spiramycin, thebaine

Yellow-brown Clemast ine, clofazimine, physostigmine, rifamycin SV, trimethoprim, tripelennamine

Green-brown Etenzamide, harmaline, lysergic acid, mesoridazine, narceine, syrosingopine

Violet-brown Chlortetracycline (→yellow), cyproheptadine, demeclocycline, dihydroergotamine, ergotamine, lymecycline (→yellow), methylergometrine, nicergoline (→brown), octaphonium, oxethazaine, protriptyline, trimetho→benzamide

Grey-brown Dextropropoxyphene, mephenesin carbamate

Grey Dihydromorphine, diprenorphine, etilefrine (→green→brown), ibogaine (→violet), indomethacin, lobeline, lysergide, oxypertine, propranolol, trazodone (→violet)

Blue-grey Alphaprodine, diamorphine, morphine

Black Procyclidine

Appendix A

54

Marquiss Test

Precautions


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

Orange-Red Alverine, bethanidine, diphemanil, flupenthixol

Violet-Red Thioridazine (→blue-green)

Brown-Red Alphaprodine

Pink Fenoprofen, metoprolol

Orange 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 →blue), lymecycline, mepenzolate (transient), mephentermine (→brown), mescaline, metanephrine (→violet -brown), methacycline, methanthelinium, methindizate (→green), methylamphetamine, methylpiperidyl benzilate (→green →blue), 5-methyltryptamine (→brown), α-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 (→green→blue), primaquine, profadol (→red-brown), prolintane (→brown), propantheline, prothipendyl, psilocybin, rolitetracycline, spiramycin, tetracycline, trimethoprim, trimethoxyamphetamine, tryptamine, veratrine, xylometazoline

Red-Orange Chlorprothixene

Pink-Orange Diuron

Yellow-Orange Orphenadrine, pipradrol

Brown-Orange Amitriptyline

Yellow 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 (→brown→violet), 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)

Orange-Yellow Stanozolol

Green Berberine, carbaryl, chelidonine, harman, norharman, oleandomycin, propranolol, protriptyline, pseudomorphine, sulindac (slow)

Yellow-Green Acepromazine (→red), verapamil (→grey)

Blue-Green Tolnaftate

Brown-Green Harmaline

Grey-Green Cyproheptadine, deserpidine, naphazoline, oxypertine, phenindamine, protokylol, rescinnamine, reserpine (→brown)

Blue Clofibrate, embutramide, nicergoline (→grey)

Grey-Blue Mebhydrolin, 1-naphthylacetic acid

Appendix A

55

Violet 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

Red-Violet Acetophenazine, benzoctamine, bephenium hydroxynaphthoate, cephaloridine, chlophedianol (→brown), dihydromorphine, ethomoxane, fluopromazine, isoxsuprine, lobeline, methdilazine, propiomazine, tramazoline, trifluomeprazine, trifluoperazine, trimeperidine

Blue-Violet Methocarbamol, methotrimeprazine, morantel, neopine, noscapine (fades), pyrantel

Brown-Violet Butaperazine, dopamine, tridihexethyl

Grey-Violet Benzhexol, diprenorphine, oxymorphone, pyrrobutamine, thenalidine

Black-Violet Dextropropoxyphene (→green), methapyrilene, thenyldiamine

Brown 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)

Red-Brown Biperiden, debrisoquine, methyl benzoquate, oxethazaine, phenprobamate, trimetozine, tripelennamine

Orange-Brown Benethamine (→brown), clomocycline, nortriptyline

Yellow-Brown Ritodrine, thymoxamine, triacetyloleandomycin, tylosin

Green-Brown Alcuronium, bufotenine, psilocin

Violet-Brown Clomiphene, diethazine, levomethadyl acetate (→grey-green), methoxamine (→green)

Grey-Brown Dihydroergotamine, methylergometrine, octaphonium

Grey Butorphanol, diaveridinc (→violet-brown), ibogaine (→orange), lysergide, methoserpidine, methyscrgide, pholedrine (→green)

Blue-Grey Acetorphine (→yellow-brown), etorphine (→yellow-brown)

Black

Blue-Black Methylenedioxyamphetamine

Appendix A

56

Ninhydrin Test

Precautions

Ninhydrin is rubefacient and a poison, use the hood.

Reagent Dissolve ninhydrin (0.5 g) in acetone (40 mL).

Method Dissolve sample in methanol. Place one drop on filter paper. Add one drop of reagent. Dry in a current of hot air.

Indications A violet colour, appearing rapidly, indicates the presence of an aliphatic primary amine or amino acid group. The presence of an aromatic ring inhibits the response. Inhibition increases the nearer the amino group is to the ring, e.g. amphetamine (pink-orange), procainamide and proxymetacaine (both yellow). If the amino group is associated with a saturated ring a positive but weak pink-violet colour is obtained (amantadine, rimantadine). Gentamicin gives a violet colour after heating for 4 minutes.

Appendix B

57

CHROMATOGRAPHIC CONDITIONS

GC Use the following treatment for your urine sample depending upon the result of your preliminary urine analysis.

Amphetamine Sample extraction Prepare a pH 10.2 borate buffer by mixing sodium borate (Na2B4O7.10H2O, 250 mL, 0.025 M) and sodium hydroxide (18.0 mL, 0.1 M). Adjust pH to 10.2 by the addition of sodium hydroxide. Pre-treat the SPE extraction cartridges with MeOH (HPLC grade, 3 mL), water (HPLC grade, 2 x 3 mL) and borate buffer (2 mL). Add borate buffer (5 mL) to urine (2 mL) and vortex for 1 minute. Add the liquid to the pre-conditioned cartridge. Wash the cartridge with water (HPLC grade, 3 mL) and allow to dry. Elute the compounds of interest with MeOH (HPLC grade, 2 x 1 mL). Evaporate solvent and derivatise sample.

Derivatisation Without Solvent 1. Combine sample and 0.1-0.5 mL of BSTFA in a clean dry 3 mL small reaction vial. 2. Cap, mix well and let stand for 5-10 minutes or until reaction is complete. 3. Inject an appropriate size sample for column and detector. In many cases, derivatizations are effectively completed at room temperature and without solvent. When there is no information available for a particular compound, it is recommended that these conditions be tried first. If derivatization is not complete under these conditions, either higher temperatures or solvent can be employed.

With Heat 1. Combine sample and 0.1-0.5 mL of BSTFA in a clean, dry 3 mL small reaction vial. 2. Cap, mix well and heat at 70 °C for 15 minutes. 3. Cool to room temperature and inject an appropriate sample.

With Solvent 1. Dissolve sample in 1.0 ml of a suitable solvent (see solvent suggestions, on instruction sheet)

in a clean, dry 3 mL reaction vial. 2. Add 0.1-0.5 ml of BSTFA. 3. Cap, mix well and let stand for 5-10 minutes. 4. Inject an appropriate sample.

With Heat and Solvent 1. Dissolve sample in 1.0 mL of a suitable solvent in a clean, dry 3 mL small reaction vial. 2. Add 0.1-0.5 mL of BSTFA. 3. Cap tightly, mix well and heat at 70 °C for 15 minutes. 4. Cool to room temperature and inject an appropriate sample.

Analysis Conditions

Column DB-5, 30 m x 0.32 mm x 0.25 µM Detector Temp 280 °C Flow rate ~1 mL/min

Temp. 50 to 275 °C @ 15 °C/min Split ratio 50:1 Injector temp 250 °C

Barbiturate Sample extraction Extract sample using the procedure as detailed under amphetamines, except adjust buffer pH to 9.2.

Derivatisation

Appendix B

58

Derivatise with BSTFA according to the instructions under amphetamines.

Analysis Conditions



Cocaine Sample extraction Extract sample using the procedure as detailed under amphetamines, except adjust buffer pH to 9.2.

Derivatisation Derivatise with BSTFA according to the instructions under amphetamines.

Analysis Conditions



Marihuana Sample extraction Extract sample using the procedure as detailed under amphetamines.


Analysis Conditions

Column DB-5, 30 m x 0.32 mm x 0.25 µM Detector Temp Flow rate ~1 mL/min

Temp. 100 t0 300 °C @ 25 °C/min Split ratio Injector temp 250 °C

Opiate Sample extraction Extract sample using the procedure as detailed under amphetamines, except adjust buffer pH to 9.2.


Analysis Conditions


Temp. 100 t0 300 °C @ 25 °C/min Split ratio 50:1 Injector temp 250 °C

Appendix B

59

HPLC Use the following treatment for your urine sample depending upon the result of your preliminary urine analysis. Amphetamine Sample extraction Extract sample according to the instructions given under amphetamines in GC analysis section. Reconstitute sample in mobile phase (1 mL).

Analysis Conditions

System HA1 Column: Silica (Spherisorb S5W, 5 µm, 25 cm x 4.6 mm id). Eluent: A solution containing ammonium perchlorate (1.175 g, 0.01 M) in methanol (1 000 ml); adjust to pH 6.7 by the addition of sodium hydroxide in methanol (1 ml of 0.1 M).

System HB2 Column: ODS-silica (ODS-Hypersil, 5 µm, 25 cm x 4.6 mm id). Eluent: A solution containing 19.60 g (0.2 M) of phosphoric acid and 7.314 g (0. 1 M) of diethylamine in 1 000 ml of a 10% v/v solution of methanol; adjust the pH to 3.15 by the addition of sodium hydroxide solution.

System HC3 Column: Silica (Spherisorb, 5 µm, 25 cm x 4.6 mm id). Eluent: Methanol: ammonium nitrate buffer solution (90:10). To prepare the buffer solution add strong ammonia solution (94 ml) and nitric acid (21.5 ml) to water (884 ml) and adjust to pH 10 by the addition of strong ammonia solution.

System K' values HA HB HC Adrenaline — — 0.63 Amphetamine 0.9 8.48 0.99 Benzphetamine 1.2 — 0.15 Caffeine 0.2 — 0.26 Cathine 1.0 4.39 0.83 Chlorphentermine 0.9 — 0.82 Diethylpropion 1.7 — 0.16 Dimethylamphetamine — 11.08 1.89 DOM — — 1.13 Ephedrine 1.0 5.68 1.79 Fencamfamin 1.3 — 0.72 Fenethylline — — 0.27 Fenfluramine 1.3 — 0.88 Hordenine — 2.00 — Hydroxyamphetamine — 2.24 1.11 Hydroxyephedrine — 0.73 — Mazindol 1.8 — 0.20 Mephentermine 1.5 — 2.48 Mescaline 1.3 16.82 2.17

1) I. Jane et al., J. Chromatogr., 1985, 323, 191-225. 2) R. Gill et al., J. Chromatogr., 1981, 218, 639-646. 3) B. Law et al., J. Chromatogr., 1984, 301, 165-172.

Appendix B

60

Methoxyamphetamine — 14.95 — Methoxyphenamine 1.7 32.17 — Methylamphetamine 2.0 10.52 2.07 Methylenedioxyamphetamine — — 0.98 Methylephedrine 2.3 — 1.83 Methylphenidate 1.7 — 0.36 Noradrenaline — 0.10 — Normetanephrine — 1.09 Oxedrine — 0.27 — Pemoline 0.2 — 0.14 Phendimetrazine 0.9 — 0.32 Pheneizine 1.0 5.91 0.37 Phenethylamine 1.2 3.64 1.31 Phentermine 0.6 19.46 0.86 Phenylephrine 1.3 — 1.64 Phenylpropanolamine 0.9 3.87 0.70 Pipradrol 1.2 — 0.69 Prolintane 2.0 — 1.26 Pseudoephedrine 1.2 5.90 1.77 Tranylcypromine 1.0 — 0.26 Trimethoxyamphetamine — — 1.48 Tyramine 1.2 0.81 1.47

Appendix B

61

Barbiturates Sample extraction Extract sample according to the instructions given under amphetamines in GC analysis section. Reconstitute sample in mobile phase (1 mL).

Analysis Conditions

System HG4 Column: ODS-silica (ODS-Hypersil, 5 µM, 25 cm x 4.6 mm internal diameter). Eluent: Methanol: sodium dihydrogen phosphate (0.1 M, 11.998 g/litre) (40:60); adjust to pH 3.5 by the addition of phosphoric acid.

System HH5 Column: As for System HG, above.

Eluent: As for System HG except that the mixture is adjusted to pH 8.5 by the addition of sodium hydroxide solution.

System k' values

HG HH Allobarbitone 2.46 1.33 Amylobarbitone 10.91 7.05 Aprobarbitone 3.42 2.22 Barbitone 1.11 0.63 Brallobarbitone 3.09 1.72 Butalbital 6.17 3.48 Butobarbitone 5.43 3.42 Cyclobarbitone 5.25 2.61 Cyclopentobarbitone 6.00 3.84 Enallylpropymal 8.65 6.96 Heptabarbitone 9.90 4.93 Hexethal 34.28 20.39 Hexobarbitone 7.37 5.67 Ibomal 4.01 2.58 Idobutal 8.12 4.77 Metharbitone 2.69 1.99 Methohexitone 27.61 20.48 Methylphenobarbitone 7.27 3.84 Nealbarbitone 10.22 6.19 Pentobarbitone 10.96 8.07 Phenobarbitone 3.09 1.23 Phenylmethylbarbituric acid 1.48 0.94 Quinalbarbitone 16.28 11.47 Secbutobarbitone 4.89 3.32 Talbutal 7.25 4.67 Vinbarbitone 4.83 2.32

4) R. Gill et al., J. Chromatogr., 1981, 204, 275-284. 5) R. Gill et al., J. Chromatogr., 1981, 226; Biomed. Appl. 15, 117-123.

Appendix B

62

Cocaine Sample extraction Extract sample according to the instructions given under amphetamines in GC analysis section. Reconstitute sample in mobile phase (1 mL).

Analysis Conditions

System HQ6 Column: ODS-silica (ODS-Hypersil, 5 µm 25 cm x 4.6 mm internal diameter). Eluent: Methanol: water: 1% v/v solution of phosphoric acid: hexylamine (30:70:100:1.4).

System HR7 Column: As for System HQ, above. Eluent: Methanol: 1% v/v solution of phosphoric acid: hexylamine (100:100:1.4).

System k' values HA HQ HR Amethocaine 2.0 16.25 1.33 Benzocaine 0.1 20.06 1.61 Benzoylecgonine 0.9t 5.68 — Bupivacaine 0.9 7.19 0.86 Butacaine 1.2 8.97 — Butanilicaine — 4.42 — Chloroprocaine — 0.24 — Cinchocaine 1.9 — 5.51 Cocaine 2.8 2.68 — Cyclomethycaine — — 10.31 Dimethisoquin 2.2 — 11.24 Diperodon — — 2.48 Dyclonine — — 2.78 Lignocaine 0.6 0.79 — Mepivacaine 0.9 1.09 — Oxethazaine — — 4.14 Oxybuprocaine — 16.25 0.86 Piperocaine — 4.59 — Pramoxine 0.6 — 2.4 Prilocaine 1.0 1.38 — Procaine 1.9 0.00 — Propoxycaine — 1.09 — Proxymetacaine 2.1 1.38 —

t =tailing peak

6) R. Gill et al., J. Chromatogr., 1984,301,155-163. 7) R. Gill et al., ibid.

Appendix B

63

Marihuana Sample extraction Extract sample according to the instructions given under amphetamines in GC analysis section. Reconstitute sample in mobile phase (1 mL).

Analysis Conditions

System HL8 Column: ODS-silica (Spherisorb-ODS, 5 µm, 25 cm x 4.6 mm id). Eluent: Sulphuric acid(0.01 M):methanol:acetonitrile (4:11:9).

System k' values

HL Cannabichromene 19.09 Cannabicyclol 14.78 Cannabidiol 7.47 Cannabidiolic acid 8.76 Cannabigerol 8.18 Cannabinol 11.77 Cannabivarin 7.47 ∆8-Tetrahydrocannabinol 14.07 ∆9-Tetrahydrocannabinol 13.35 Tetrahydrocannabinolic acid 25.83 Tetrahydrocannabivaric acid 14.64 Tetrahydrocannabivarin 8.18

8) P.B. Baker et al., J. Anal. Toxicol., 1980, 4, 145-152.

Appendix B

64

Opiate Sample extraction Extract sample according to the instructions given under amphetamines in GC analysis section. Reconstitute sample in mobile phase (1 mL).

Analysis Conditions Systems HA or HC, previously described, may be used or System HS, below.

System HS 9 Column: Amino-propyl bonded silica (Spherisorb S5NH2, 5 µm, 25 cm x 4.6 mm id). Eluent: Acetonitrile: tetrabutylammonium phosphate (0.005M, pH 7.5) (85:15).

System k' values HA HC HS Acetylcodeine 0.78 0.78 0.50 Benzylmorphine 4.4t 1.03 — Buprenorphine 0.4 0.05 — Caffeine 0.2 — 0.21 Codeine 4.8t 1.21 1.90 Dextromethorphan 5.6t — — Dextromoramide 0.7 0.09 — Dextropropoxyphene 1.9 0.19 — Diamorphine 3.0t 0.66 0.35 Dihydrocodeine 7.2t 2.50 — Dihydromorphine 5.7t 2.75 — Diphenoxylate 0.2 — — Dipipanone 2.2 1.61 — Ethoheptazine 3.3 1.55 — Ethylmorphine 3.7t 1.06 1.45 Etorphine 0.6 1.11 — Fentanyl 0.8 1.11 — Hydrocodone 7.lt 2.17 — Hydromorphone 7.9t — — Ketobemidone 2.8t — — Levallorphan 1.9t 1.46 — Levorphanol 4.4t 3.20 — Methadone 2.2 1.03 — 6-Monoacetylmorphine 3.6t 0.80 1.00 Morphine 3.8t 1.30 5.16 Nalorphine 1.0 0.29 — Naloxone 1.4 0.17 — Norcodeine 3.lt 3.51 — Normethadone — 0.53 — Normorphine 2.9t 3.92 — Norpipanone — 0.35 — Noscapine 0.3 0.15 0.01 Oxycodone 6.9t 0.85 — Oxymorphone 6.7t — —

9) P.B. Baker and T.A. Gough, J. Chromatogr. Sci., 1981,19, 483-489.

Appendix B

65

Papaverine 0.3 0.16 0.04 Pentazocine 1.8 0.67 — Pethidine 2.8t 0.55 — Phenazocine 1.3 0.30 — Phenoperidine 0.8 0.10 — Pholcodine 6.0t 1.63 — Piritrarnide 0.6 0.14 — Quinine 2.4 — 2.02 Strychnine 1.0t — 2.43 Thebacon 3.7t 0.85 — Thebaine 4.6t 0.94 0.79

Appendix C

66

Toxi-Lab Information

Toxi-Lab is a standardized screening method for drugs based on the principles of thin layer chromatography

(TLC). The chromatogram or Toxi-Gram is run and developed in designated Toxi-Lab chambers containing

various solvents. The Toxi-Lab procedures found in the appendix are complete protocols for Toxi-lab

analysis. In this lab you may only need to use part of this procedure (i.e. extraction may not be required).

Use judgment and think carefully about what you are doing before you proceed.

Adapted from Liu, R. H.: Gadzala, D. E. Handbook of Drug Analysis, American Chemical Society,

Washington, D. C., 1997.

The chromatographic behavior of various drugs under different solvent systems has been well

characterized in several comprehensive studies. Although these references are valuable for system selection

and for comparison of results, maximal standardization of operation steps, including extraction-

concentration, developing and detection, is made possible through the use of commercial Toxi-Lab kits,

which include extraction tubes, evaporation discs, silica gel embedded fiberglass plates, and color-

developing solutions. The Toxi-lab system has been described in a recent book chapter, and training

programs and regular newsletters are available from the manufacturer. A typical operation of a Toxi-Lab kit

involves the addition of 5 mL of urine or 2 mL to an appropriate Toxi-Tube, followed by mixing for 1 min.

After centrifugation, the extract is evaporated onto a small disc of chromatographic media, and the dried disc

(now impregnated with the extracted drugs) is inserted into an open hole of a Toxi-Gram, which includes

various drug standards. The Toxi-Gram is then developed in a small jar containing the recommended

solvent system. Following development, the chromatogram is sequentially dipped into a series of reagents

the produce color changes for numerous drugs. The availability of Photo-Grams (photographs of drug and

metabolite detection characteristics) and standard drugs on discs for parallel development with the analyte

minimizes subjective data interpretation.

Two general analytical schemes are used in the Toxi-Lab system: A and B. System A is designed

for basic and neutral drugs, whereas System B is designed for acidic and neutral drugs. While these general

procedures are effective for many drugs, special procedures are needed for correctly detecting drugs and

metabolites that are:

1. Too polar for effective extraction:

2. Present in low concentration

3. Present mainly as conjugates and require prior hydrolysis: and

4. Present with other drugs having similar features and retention characteristics.

It should be noted that this standardized procedure cannot improve the inherent low sensitivity

associated with TLC technology. With some exceptions, it is suitable for detection toxic

concentrations of most commonly used and abused drugs. Many drugs in their therapeutic

concentrations can also be detected. Just as in most standardized procedure, modifications are

Appendix C

67

commonly needed for specific applications. Since coextracted lipids may cause interference, revised

extraction procedures are recommended when Toxi-Lab is adopted for the analysis of liver

specimens.

Points to Note:

§ When preparing toxi-disc of drug standards : ie pure cocaine powder, no extraction is

required… dissolve the a few grains powder in a small amount of liquid and apply to the

toxi disc directly.

§ Always run samples next to standard discs on the same Toxi-Gram so that direct

comparison can be made

§ Marijuana (THC) requires a separate protocol (TOXI-LAB THC) which is attached.

1 2 3 4 5 6

Schematic of Toxi-Gram: Holes at bottom are for Toxi discs

Appendix C

68

Abbreviated Instructions -TOXI•• LAB A

1. Briefly shake each TOXI-TUBES A extraction tube. add urine to the 5-mL arrow, cap, and mix by inversion for 2 min Centrifuge for 2-5 min.

2. Insert the appropriate number of disposable concentration cups into the wells of the OMEGA-12 extraction solvent concentrator. With a disc-handling pin, place one TOXI-DISCS Blank A in each cup

3. Transfer all the upper organic layer from each extraction tube to the appropriate cup. Then place the OMEGA-12 on the electric warmer and cover with the OMEGA-12 screen. Direct a gentle current of warm air from the heat gun across the top of the cups and evaporate the discs to dryness.

4. After solvent evaporation insert the dried discs into the centre holes of the TOXI-GRAMS A. Place the TOXI-GRAMS on the warmer with the disc ends slightly off the edge.

5. Transfer 3 mL of A developing solution into each chromatography jar, add the required amount of ammonium hydroxide, and swirl vigorously to mix. Place one of the TOXI-GRAMS in each jar and cover.

6. When the dye spots reach 9.5 cm (12-17 min), remove the TOXI-GRAMS and dry face down on the warmer for 30-60 sec.

7. Transfer the TOXI-GRAMS to TOXI-DIP A-1 for a minimum of 5 min, then remove and place the lower two-thirds on the warmer for no more than 5 sec.

8. Proceed with the following detection steps for each of the TOXI-GRAMS: Stage I -Slowly dip in and out of TOXI-DIP A-2 and hold over the jar for at least 15-60 sec. Record observations. Stage II -Dip in and out of the jar of water. Re-dip as required to bring out the full spectrum of colours. Record observations. Stage III -Lightly blot on a clean paper towel and view in the dark under long-wave ultraviolet light. Record observations. Stage IV-Place in TOXI-DI P A-3 for at least 1 0 sec. Remove and record observations.

Nonbiological Materials (pills, powders, capsules, liquids)

Crush pills to a fine powder using a clean mortar and pestle.

Add 2 to 3 mg of powder material or 2 to 3 µL of liquid material to a TOXI•TUBE A. Add deionized (or distilled) water to the 5.0-mL arrow. Cap, and mix by inversion for 2 min.

Centrifuge the tube for 2-5 min at a minimum of 2500 rpm.

Place a TOXI•DISC Blank A into each of two concentration cups in the Omega-12.

Transfer 2 to 3 drops of the A extract to one cup and approximately 20 drops to the other cup. Save the remainder of the extract in the tube.

Proceed as in step 3. Refer to the TOXI•LAB AB Instruction Manual for detailed instructions.

Appendix C

69

Abbreviated Instructions -TOXI•• LAB B

1. Using TOXI-TUBES B extraction tubes, add urine to the 4.5-mL arrow, cap and mix by inversion for 2 min, Centrifuge for 2-5 min.

2. Insert the appropriate number of disposable concentration cups into the wells of theOMEGA-12 extraction solvent concentrator. With a disc-handling pin place one TOXI•DISCS Blank B in each cup.

3. Transfer all the upper organic layer from each extraction tube to the appropriate cup. Then place the OMEGA-12 on the electric warmer and cover with the OMEGA-12 screen. Direct a gentle current of warm air from the heat gun across the top of the cups and evaporate to dryness.

4. After solvent evaporation insert the dried discs into the centre holes of the TOXI-GRAMS B. Place the TOXI-GRAMS on the warmer with the disc ends slightly off the edge.

5. Transfer 3 mL of B developing solution into each chromatography jar, add the required amount of ammonium hydroxide, and swirl vigorously to mix. Place one of the TOXI-GRAMS in each jar and cover.

6. When the dye spots reach 9.5 cm (12-17 min), remove the TOXI-GRAMS and dry face down on the warmer for 30-60 sec.

7. Proceed with the following detection steps for each of the TOXI-GRAMS:

8. Dip in and out of TOXI-DIP B-1 and place in the drying rack until all the dichloromethane has evaporated. Stage I -Dip in and out of TOXI-DIP B-2 and immediately transfer into TOXI-DIP B-3, agitating until the background clears. Record observations. Stage 11 -View in the dark under long-wave ultraviolet light. Record observations.

Nonbiological Materials (pills, powders, capsules, liquids)

Crush pills to a fine powder using a clean mortar and pestle.

Add 2 to 3 mg of powder material or 2 to 3 µL of liquid material to a TOXI•TUBE B. Add deionized (or distilled) water to the 4.5-mL arrow. Cap, and mix by inversion for 2 min.

Centrifuge the tube for 2-5 min at a minimum of 2500 rpm.

Place a TOXI•DISC Blank B into each of two concentration cups in the Omega-12.

Transfer 2 to 3 drops of the B extract to one cup and approximately 20 drops to the other cup. Save the remainder of the extract in the tube.

Proceed as in step 3. Refer to the TOXI•LAB AB Instruction Manual for detailed instructions.

Appendix C

70

APPENDIX VII

SAMPLE LAB REPORT

Experiment 1

High Performance Liquid Chromatography

Chemistry 325

January 8, 1994

Appendix C

71

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 Milton–Roy Model 1100 pump, (2) a 20 µL sample injector, (3) an Altex C–8 reverse phase HPLC column, (4) an ultraviolet detector operating at 254 nm, and (5) an output chart recorder. In solution preparation, a Mettler AE–160 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 centimeter ruler.

Appendix C

72

EXPERIMENTAL DATA

Retention Time Study

Retention Time (sec, + 0.1 sec)

Compound Trial 1 Trial 2 Trial 3

Caffeine 180.1 181.3 180.5

Theobromine 154.6 155.3 154.9

Theophylline 244.7 245.8 245.2

Caffeine Calibration Study

Aliquot Volume

Peak Height (cm, + 0.05 cm)

(mL) Trial 1 Trial 2 Trial 3

5.0 2.10 1.25 1.50

10.0 3.82 3.20 3.80

15.0 5.02 5.09 5.60

20.0 8.40 8.35 7.70

30.0 11.90 10.25 11.05

40.0 14.55 15.34 13.80

Unknown 6.23 6.58 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 Hewlett–Packard Model 42S electronic calculator.

Compound Mean Retention Time (s) Standard Deviation (s) % RSD

Caffeine 180.6 0.6 0.39

Appendix C

73

Theobromine 154.9 0.4 0.23

Theophylline 245.2 0.6 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 # Concentration (ppm)

1 25.0

2 50.0

3 75.0

4 100.0

5 150.0

6 200.0

Appendix C

74

Calculation of Mean Peak Heights and Standard Deviations (HP 42S calculator):

Table II

Mean Peak Heights and Standard Deviations

Concentration (ppm) Mean Peak Height (cm) S. Deviation (cm)

25.0 1.62 0.44

50.0 3.61 0.35

75.0 5.24 0.32

100.0 8.15 0.39

150.0 11.07 0.83

200.0 14.56 0.77

Calculation of Calibration Model

A least–squares calculation was used to compute the slope and intercept of the best calibration model for the above data. The resident least–squares 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 least–squares 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

Appendix C

75

Calibration Curve for Caffeine Determination

02468

10121416

10 30 50 70 90 110 130 150 170 190 210Caffeine Concentration (ppm)

Mea

n P

eak

Hei

ght

(cm

)

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 give the most structural information.

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 C8–column. This study also revealed that the retention times were quite reproducible (rsd < 1 %). No anomalies were encountered during the experimental procedure.

forensic chemistry - bienvenidosmatematicas.udea.edu.co/~carlopez/forense/lab3-3.pdf · chemistry...

Documents