Forensic Chemistry - Laboratory Manual ANDREWS 2001 PDF

Chemistry 487
Forensic Chemistry
Laboratory Manual
Revision 3.3
Anthony Andrews and Bruce McCord

Department of Chemistry and Biochemistry
Ohio University
Spring 2001
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
19 24 26 1 3 8 10 15 17 22 24 29
Gr Apr Apr Apr May May May May May May May May 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
2
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.
3
GENERAL INFORMATION
Students will work in assigned groups. Each student is required to write a formal report on each
experiment. The formal report is to conform to the guidelines of the ACS style guide 1 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.
4
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 witho ut 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 instructio ns 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).
5
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.
2CaSO42H2O(s) + heat (CaSo4 )2 H2 O(s) + 3H2 O(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.
6
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.
7
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.
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.
8
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 ( 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.

9
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.
10
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.
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
11
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.
12
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 wit h 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
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).
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b N-(I-naphthyl)ethylenediamine dihyd rochloride (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
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.
14
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 guns hot 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.
15
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
CH3 annual plant of 3 to 16 ft in height. The "resin" which
contains the psychoactive chemicals (principal
OH
component is 9 -tetrahydrocannabinol, C23 H30O4 , MW
= 358.5, CAS 23978-85-0) occurs mainly in the
H3C flowering area, the leaves and the stem, particularly at
H3C O CH3 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 H2 O ethanol chloroform ether
-THC
9
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
16
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
ToxiLab
Analyse the organic layer from the Duquenois-Levine test by ToxiLab 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.
17
Amphetamines
Introduction
Amphetamine, (1-phenyl-2-aminopropane, C9 H13 N, MW = 135.2, CAS
CH2CHCH3 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
NH2 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, C10 H15N, MW = 149.2, CAS 537-46-2), is
anorexic and is used in treating attention deficit disorder with hyperactivity. The hydrochloride of
the d-isomer is used in the treatment of obesity. The 1- isomer, which is purported to have weaker
CNS stimulant activity and greater peripheral sympathomimetic activity, is used as a decongestant
in certain over-the-counter (OTC) inhalers. The principal metabolite is amphetamine.
Properties
Solubility
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. TOXILAB 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
ToxiLab
Perform a ToxiLab 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.
18
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 TOXILAB 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?
Disposal
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.
19
Morphine
Introduction
Heroin (3,6-O-diacetylmorphine, C21 H23NO5 , M.W = 369.4, CAS
CH3COO 561-27-3) is a semi- synthetic opiate produced from morphine (a
naturally occurring substance in the opium poppy Papaver
somniferum). The major metabolites are 6-monacteylmorphine
O then morphine (and glucuronides) and codeine. Morphine is a
NCH3 potent narcotic analgesic, and its primary clinical use is in the
management of moderately severe and severe pain. After heroin,
CH3COO 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.
Properties
Solubility
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
ToxiLab
Perform a ToxiLab 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?
Disposal
20
6. COCAINE AND BARBITURATE ANALYSIS
Precautions
Introduction
Cocaine (methyl benzoylecgonine, C17 H21 NO4 , MW = 303.4, CAS 50-
CH3 COOCH3 36-2) is a tropane alkaloid obtained from coca, the dried leaves of
N
Erythroxylum coca and other species of Erythroxylum. The South
American species contains more cocaine than the Asian species. It can
OOCC6H5 be synthesised from ecognine or simpler starting materials. The natural
product is optically active. The major metabolites are ecgonine,
H
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.
Properties
Solubility
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
2. Mandolin Test
3. Cobalt isothiocyanate test
ToxiLab
Perform a ToxiLab 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
21
Barbiturates
Introduction
One of the most common barbiturates is phenobarbital (5-ethyl-5-
H phenylbarbituric acid, C12 H12 N2O3 , MW = 232.2, CAS 50-06-0). It exists as
O N O
colourless crystals or a white powder. Major metabolites are N-
C6H5 glucopyranosylphenobarbitone and 4- hydroxyphenobarbitone (and
NH
C2H5 glucuronide). Other common barbiturates are secobarbital, pentobarbital and
amobarbital. Barbital, the first drug of this class to be synthesised, was
O
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.
Properties
Solubility
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
ToxiLab
Perform a ToxiLab 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?
Disposal
22
6. DILUENT AND UNKNOWN ANALYSIS
Precautions
Introduction
Street drugs are commonly mixed with other materials to
H2C CH H 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
H N diluents are sugars, inositol (an isomer of glucose), talcum
HO
powder, starch, caffeine and quinine (shown). It is important to
H be able to distinguish the diluents from the drug compound.
CH3O
Frequently laboratories are required to analyse body fluid
samples from suspects or accident victims. Blood is the most
N 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
Perform a ToxiLab 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.
Disposal
23
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 suspects home.
Liquid inks used in ballpoint pens contain a mixture of substances, which include Dyes, stabilizers,
solvents, and resinous binders. Certain manufacturers also add fluorescent markers or heavy metals
to aid in product identification.
Thin layer chromatography (TLC) is the preferred technique for ink analysis. In TLC, a uniform
layer of silica, alumna or some other adsorbant is placed on a glass plate. The ink samples are
punched out of the paper, extracted in pyridine, and spotted onto the TLC plate using a glass
capillary as a transfer pipette. The plate is then placed in a glass tank containing a layer of solvent
at the bottom. This solvent mixture is the chromatographic eluent used to separate the mixture of
dyes present in the sample. The distance a particular dye moves relative to the solvent front is
known as the Rf value.
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 suspects 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.
24
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 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 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.
25
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 ([Urine EtOH]/[BloodEtOH ]
= 1.3/1) relates the alcohol content in urine to blood alcohol content. This will be used in the final
calculation.
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 L, 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 m 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.
26
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 L.
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.
27
10. GC-ECD ANALYSIS
Precautions
Pesticides are toxic.
Introduction
Pesticides are commonly used in agriculture. Their widespread
Cl Cl availability has led to a number of cases of suicide and attempted
Cl poisoning using pesticides. The determination of pesticides can be
Cl accomplished by a number of methods. In the case of chlorinated
O pesticides a suitable techniq ue is gas chromatography with electron
capture detection (GC-ECD). In this experiment the pesticide must
Cl be extracted from the sample matrix prior to analysis. The most
Cl
common form of extraction is liquid/liquid extraction. However,
solid phase extraction can be used.
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 L 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 m 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.
28
11. GC-MS ANALYSIS
Precautions
BSTFA is flammable and harmful by inhalation.
Introduction
Gas Chromatography (GC) is a widely used technique in forensic
CH3 chemistry. Compounds that are not highly volatile or likely to
decompose at the higher temperature used in GC are usually
H3C Si CH3 derivatised prior to analysis. The most common type of derivative
is a methyl silane. This functional group increases the volatility of
O CH3 the compound. There are a number of different silyating reagents
available. The choice of reagent depends upon the functional
CF3 C N Si CH3 groups to be derivatised. The reagent used here (N-O-
bis(trimethylsilyl) trifluororacetamide, BSTFA) is a multi-purpose
CH3 reagent which is capable of derivatizing a wide range of functional
groups. The mass spectral detector is probably the most important
detector for GC in forensic chemistry. This is partly due to the low limits of detection that the
detector has, but mainly because of the structural information provided by the fragmentation ions
produced.
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.
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 g/mL to 10 g/mL. Start your calibration standard preparation at the derivatization stage.
Your calibration graph should have at least 5 points in it. 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.
29
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
30
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.
Supplies and equipme nt
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.
31
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

(g/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
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,
32
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.
33
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 Examples
n-alkane carbon #s
1 Light Petroleum C4-C8 Petroleum ethers, pocket lighter fuels, rubber
Distillates (LPD) 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 l), Lighter Fluid (250 l), and
Diesel Fuel (250 l). All carpet samples were bagged in nylon with activated charcoal strips
(Albrayco Laboratories, 500 Corporate Row, Cromwell, CT 06416; Ph: 860-635-3369). The
Pyrolyzed sample was heated by propane torch until the carpet started to burn. Accelerant samples
had listed amounts applied to carpet and were ignited by match. Samples were burned to consume
half of the accelerant. Burn time was estimated by burning 250 l of the accelerant only, in a 3"
diameter open dish. All pyrolyzed and accelerant samples were bagged directly after burning. All
samples were heated @ 100 C for 90 min.
Analysis Conditions
column DB-5MS 15 m x 0.25 mm x 0.25 m 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
34
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
l of sample in 10 ml methylene Chloride). The C5 -C20 n-alkane standard is diluted in pentane.
Experimental:
1. Analyze 1.0 l of the C5 -C20 standard with the above temperature program.
2. Take the charcoal strips from nylon bags and place in 1.8 ml vials with appropriate label.
3. In the hood using syringes provided add 250 l 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 l of sample into the GC.
If time permits:
5. Analyze 1.0 l sample of a gasoline sample which has been evaporated to 60% of its original
volume and then diluted. Compare this result to a fresh sample.
6. Run a diesel fuel standard as well.
Discussion
1. What were the complete conditions for sample preparation and instrument operation?
2. What is the range of peaks that are present, light medium or heavy? Is there a broad or narrow
distribution of peaks?
3. Are characteristic class features 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?
35
8. Discuss the use of GC/MS in arson analysis. Explain the advantages of single ion monitoring
36
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 H2 SO4 (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 H2 SO4 (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 % purit y 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
37
38
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.
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
39
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.
40
17. PRESUMPTIVE DRUG SCREENS
PART I Ion Mobility Spectro metry
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 Operators status displa y 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.
41
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.
42
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?
43
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
You will be given example s 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.
44
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.
45
46
Appendix A
COLOUR TEST REAGENTS
Cobalt isothiocyanate Test
Precautions
No specific precautions.
Reagent
Prepare a 2% w/v solution of cobalt isothiocyanate in water.
Method
1. Add reagent to the test substance in a test tube.
Indications
Development of a blue- green colour indicates that cocaine (or surrogates) may be present.
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
47
Appendix A
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.
48
Appendix A
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 L 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.
49
Appendix A
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 (100red-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
(100brown), 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 (100brown), ibuprofen, labetalol, mepivacaine,
methadone, nefopam (brown), tetrahydrozoline
Yellow Amicarbalide (100), clonidine (100orange), dequalinium (100orange), 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),
50
Appendix A
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 (100red), 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, cephalne, 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.
51
Appendix A
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),
ndapamide, mequitazine, methotrexate, nialamide. pericyazine, prajmalium, prolintane, sodium cromoglycate,
thiothixene, xylometazoline
Brown-red Nadolol
Orange Dropropizine (slow), ethylnoradrenaline, hydrastin ine (green), lachesine (green), levamisole (greygreen),
methanthelinium, methixene, methyldopa, methyldopate, methylpiperidyl benzilate (brown green), noradrenaline,
orphenadrine, pipenzolate (green), poldine methylsulphate (greenviolet), propantheline, proquamezine (violet),
solanidine (violetblue), solanine (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, homidium, lidoflazine,
methacycline (orange-violet), paraphenylenediamine, penicillamine, protokylol (brown), tofenacin, tylosin
(yellow-brown), veratrine (orangeviolet -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), -methyltryptamine (orange), metopimazine, monocrotaline, niclosamide,
nitmxotine, norharman (yellow), norrnetanephrine, obidoxime (blue), oleandomycin, oxymetazoline, pecazine
(violet), pentazocine, perazine (violet), phenazone, phenazopyridine, phenformin, phenindamine, phenoxybenzamine
(violet), phenyltoloxamine, pindolol, piperacetazine (redviolet), pipoxolan (brown), prenylamine, proflavine,
promazine (violet), pmmethazine (violet), propranolol, reserpine, ritodrine, thenium, thenyldiamine, thiocarlide
(yellow), tranylcypromine (violet), trifluomeprazine (red-violet)
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 (orangebrown-
yellow), guanoxan, hexobendine, hydromorphone (orange), mepacrine (yellow), mepyramine, methisazone
(yellow), mianserin, morantel, naloxone (brown), oxyclozanide (orange), oxyphenisatin, oxytetracycline
(redorange), penthienate, perphenazine, phenylbutazone, pizotifen (green), prilocaine, primaquine (orange),
propiomazine, pyrantel, pyffobutamine, rolitetracycline (redorange), strychnine, tetracycline (redorange),
thiethylperazine, thiopropazate, triacetyloleandomycin (slow), tridihexethyl, trimeprazine, trimetazidine
52
Appendix A
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, isoprenaline, 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, tolpropamine, 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, trimethobenzamide
Grey-brown Dextropropoxyphene, mephenesin carbamate
Grey Dihydromorphine, diprenorphine, etilefrine (greenbrown), ibogaine (violet), indomethacin, lobeline, lysergide,
oxypertine, propranolol, trazodone (violet)
Blue- grey Alphaprodine, diamorphine, morphine
Black Procyclidine
53
Appendix A
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), -methyltryptamine
(brown), N-methyltryptamine, nefopam (brown), nomifensine (slow), normetanephrine (violet -brown),
oxeladin, oxytetracycline, pentapiperide, pethidine, phenethylamine, phenformin (brown), phentermine, piminodine,
pipenzolate (green blue), piperidolate, pizotifen (red), poldine methylsulphate (greenblue), primaquine,
profadol (red-brown), prolintane (brown), propantheline, prothipendyl, psilocybin, rolitetracycline, spiramycin,
tetracycline, trimethoprim, trimethoxyamphetamine, tryptamine, veratrine, xylometazoline
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
(brownviolet), hydromorphone (red violet), hydroxyephedrine, isoetharine (orange), lidoflazine, lorazepam,
mepacrine, methyldopa (violet), methyidopate (violet), norcodeine (violet), orciprenaline, oxycodone (brown
violet), oxyphenbutazone, phanquone, phenbutrazate (slow), phentolamine, phenyramidol, pindolol (brown),
pramoxine (green), proflavine (orange), salbutamol, salinazid, sodium cromoglycate, solanine (violet),
terbutaline, tetrabenazine, thebacon (violet), tofenacin, triamterene, trimetazidine (fades), vancomycin, viprynium
embonate, zomepirac (100, orange)
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
54
Appendix A
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
55
Appendix A
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.
56
Appendix B
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 (Na2 B4 O7 .10H2 O, 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 M 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
57
Appendix B
Derivatise with BSTFA according to the instructions under amphetamines.

Analysis Conditions
Cocaine
Sample extraction
Derivatisation
Analysis Conditions
Marihuana
Sample extraction
Extract sample using the procedure as detailed under amphetamines.
Derivatisation
Analysis Conditions
Column DB-5, 30 m x 0.32 mm x 0.25 M Detector Temp Flow rate ~1
mL/min
Temp. 100 t0 300 C @ 25 C/min Split ratio Injector temp 250 C
Opiate
Sample extraction
Derivatisation
Analysis Conditions
Temp. 100 t0 300 C @ 25 C/min Split ratio 50:1 Injector temp 250 C
58
Appendix B
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 HA 1
Column: Silica (Spherisorb S5W, 5 m, 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 HB 2
Column: ODS-silica (ODS-Hypersil, 5 m, 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 HC 3
Column: Silica (Spherisorb, 5 m, 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.
59
Appendix B
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
60
Appendix B
Barbiturates
Sample extraction
Analysis Conditions
System HG 4
Column: ODS-silica (ODS-Hypersil, 5 M, 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 HH 5
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.
61
Appendix B
Cocaine
Sample extraction
Analysis Conditions
System HQ 6
Column: ODS-silica (ODS-Hypersil, 5 m 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 HR 7
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.
62
Appendix B
Marihuana
Sample extraction
Analysis Conditions
System HL 8
Column: ODS-silica (Spherisorb-ODS, 5 m, 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.

63
Appendix B
Opiate
Sample extraction
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 m, 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.
64
Appendix B
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
65
Appendix C
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
66
Appendix C
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.
Schematic of Toxi-Gram: Holes at

bottom are for Toxi discs
1 2 3 4 5 6
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.
67
Appendix C
LAB A
Abbreviated Instructions -TOXI
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 L of liquid material to a TOXITUBE 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 TOXIDISC 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 TOXILAB AB Instruction Manual for detailed instructions.
68
Appendix C
LAB B
Abbreviated Instructions -TOXI
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 TOXIDISCS 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 L of liquid material to a TOXITUBE 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 TOXIDISC 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 TOXILAB AB Instruction Manual for detailed instructions.
69
Appendix C
APPENDIX VII
SAMPLE LAB REPORT
Experiment 1
High Performance Liquid Chromatography
Chemistry 325
January 8, 1994
70
Appendix C
INTRODUCTION
In this experiment, high performance liquid chromatography (HPLC) was used to separate a
mixture of three similar alkaloids: caffeine, theobromine, and theophylline. The retention times of
the three compounds were measured, and a calibration model for caffeine was constructed based on
the chromatographic peak heights produced by a series of six caffeine standard solutions. The
derived calibration model was then employed in the prediction of caffeine content in a commercial
soft drink.
PROCEDURE
Instrumentation and Equipment. A modular HPLC setup was used in this experiment,
consisting of (1) a MiltonRoy Model 1100 pump, (2) a 20 L sample injector, (3) an Altex C8
reverse phase HPLC column, (4) an ultraviolet detector operating at 254 nm, and (5) an output
chart recorder. In solution preparation, a Mettler AE160 analytical balance was used to weigh
solid reagents. Class A volumetric glassware was used throughout the experiment.
Solution Preparation. For the study of the retention times of the three alkaloids, separate 1
L solutions of caffeine, theophylline, and theobromine were prepared in deionized water. The
weights of solute used were 0.1011 g, 0.1022 g, and 0.0998 for caffeine, theophylline, and
theobromine, respectively.
In constructing the calibration model for caffeine, six standard solutions were prepared by
serial dilution from a caffeine stock solution. The stock solution was prepared by dissolving
0.5000 g of caffeine in enough deionized water to make 1 L of solution. Employing 5, 10, 15, and
20 mL volumetric pipets, aliquots of 5, 10, 15, 20, 30, and 40 mL were removed from the stock
solution and diluted to 100 mL with deionized water. The unknown for the caffeine determination
was a sample of "Pepsi", a commercial soft drink.
Data Collection and Analysis. A mixture of acetonitrile (20% v/v) in deionized water was
used as the mobile phase. This solution was degassed prior to being used. A flow rate of 1.0
mL/min was employed during the equilibration and during subsequent injections of the samples.
For the study of the retention times of the three alkaloids, three injections of each solution
were made. In constructing the caffeine calibration model, three injections were made of each of
the six standard solutions. Three injections of the "Pepsi" unknown were then made. No dilution
of the unknown was made before injection. The recorded chromatograms were analyzed by
measuring peak heights with a centimeter ruler.
71
Appendix C
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 Peak Height (cm, + 0.05 cm)

Volume
(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 HewlettPackard
Model 42S electronic calculator.
Compound Mean Retention Time (s) Standard Deviation (s) % RSD
Caffeine 180.6 0.6 0.39
72
Appendix C
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)(C stock )/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 # Concent ration (ppm)
1 25.0
2 50.0
3 75.0
4 100.0
5 150.0
6 200.0
73
Appendix C
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 leastsquares calculation was used to compute the slope and intercept of the best calibration
model for the above data. The resident leastsquares functions on the HP 42S calculator were used
for this calculation.
Peak Height = 0.0486 + (0.074220)(Concentration)
To evaluate the quality of the calibration model, the correlation coefficient was computed: The
builtin function of the HP 42S calculator was used.
r = 0.996
On the next page, the calibration curve is plotted. In the plot, the mean peak heights are indicated
by circles, and the leastsquares line is indicated by the solid line.
Computation of Unknown Caffeine Concentration
The mean peak height of the unknown was calculated to be 6.47 cm. This value was used
with the computed slope and intercept to determine the concentration of caffeine in the sample of
"Pepsi".
Cunk = (6.47 + 0.0486)/0.07422 = 87.9 ppm
74
Appendix C
Calibration Curve for Caffeine Determination

16
Mean Peak Height (cm)
14
12
10
8
6
4
2
0
10 30 50 70 90 110 130 150 170 190 210
Caffeine Concentration (ppm)
Questions
1. Two explanations for constituents not showing up in the chromatograms are (1) their possible
coelution 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.
75

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27 March Casts of Footprints

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b) Written statement of purpose.

c) Summary of procedure to be performed condensed from the experiment handout.

6. All notebook entries should be in ink.

7. Each page should be labeled with the date.

Answers to Questions (7)

Next goto http://www.ohiou.edu, go to library, go to ohiolink, go to full text services, go

You are expected to keep the laboratories clean and orderly.

1) J.F. Fallano, Kodak Techbits 1992.

Supplies and equipment

3. Explain the reason derivatization is especially useful for drug metabolites.

6. Derivitization alters the molecular structure. Sometimes this is an advantage in MS analysis,

Element Determination Limit

Supplies and equipment

Pristane (2,6,10,14-tetramethylpentadecane) Mol. Wt. 268.53 CAS. NO. 1921-70-6

PART I Ion Mobility Spectro metry

K0 target = (K0 calibrant * t calibrant ) / t target

1) Record Ionscan instrumental parameters:

3) Place a Teflon filter into the wand.

6) Place the filter holder into the cartridge slide assembly.

9) Within seconds, one of two things will happen:

PARTII Microchemical crystal tests

3. Why is ion mobility a presumptive test?

COLOUR TEST REAGENTS

Cobalt isothiocyanate Test

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

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

Note. Hydrochlorides give a blue colour before the addition of pyrrolidine.

Brown-red Methylchlorophenoxyacetic acid

Pink Trichlorophenoxyacetic acid (brown)

Brown-pink Prazosin (100red-orange)

Black- green Naproxen

Black- violet Methoxychlor

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

Green-brown Methdilazine, norbormide, promazine, thiopropazate

Violet-brown Bamethan, clofibrate, dichlorophen

Black Acetomenaphthone, aloin, aminophenols, amodiaquine, apomorphine, atenolol, benorylate, benzquinamide,

Blue- green Benzoctamine, berberine (brown), edrophonium, hydroxystilbamidine, ketobemidone, methoxyphenamine,

Grey-green Alverine, azapropazone, benzhexol, diamphenethide, diethyltryptamine (yellow), dihydrocodeine, guaiphenesin,

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

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),

Violet-Red Thioridazine (blue-green)

Pink Fenoprofen, metoprolol

Yellow-Orange Orphenadrine, pipradrol

Yellow Acriflavine (red), amiloride, azacyclonol, benzquinamide, benztropine, bromodiphenhydramine, broxaldine,

Blue Clofibrate, embutramide, nicergoline (grey)

Grey-Blue Mebhydrolin, 1-naphthylacetic acid

Brown-Violet Butaperazine, dopamine, tridihexethyl

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

Black-Violet Dextropropoxyphene (green), methapyrilene, thenyldiamine

Orange-Brown Benethamine (brown), clomocycline, nortriptyline

Yellow-Brown Ritodrine, thymoxamine, triacetyloleandomycin, tylosin

Green-Brown Alcuronium, bufotenine, psilocin