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Forensic Chemistry
Laboratory Manual
Revision 3.3
Spring 2001
Laboratory Schedule
27 March
29 March
3 April
5 April
10 April
12 April
17 April
Casts of Footprints
Development of Latent fingerprints
Visualization of Punched Markings on Metal
Firearms Residue on Victims
Marihuana, Amphetamine and Morphine
Cocaine, Barbiturate, Diluent, and Unknown
Thin layer Chromatography
*Rotation Order
Gr
19
Apr
24
Apr
26
Apr
1
May
3
May
8
May
10
May
15
May
17
May
22
May
24
May
29
May
1
2
3
4
5
6
8
16
10
11
18
13
14
16
18
11
8
13
13
8
16
10
11
18
13
14
16
12
11
8
18
13
8
16
10
11
15
13
14
16
17
11
11
18
13
17
16
10
11
10
13
15
16
17
10
11
17
18
15
16
17
11
15
13
14
16
9
15
11
12
13
8
9
17
11
8
13
15
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.
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).
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.
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.
Iodine fuming chamber method: Iodine, TLC tank and lid, blank glass TLC plate, scotch
tape, and glass tray for warming TLC tank with hot water.
Nihydrin method: Ninhydrin solution: ninhydrin (5 g) in methanol (100 mL), nylon brush,
steam iron and distilled water, and cardboard and blotter to protect table top from steam iron.
Silver nitrate method: Silver nitrate (3 g) in distilled water (100 mL). Store in a brown
bottle. Glass tray, nylon brush, and blotters, high intensity ultraviolet lamp and UV
protective goggles.
Cyanoacrylate method: Superglue, sodium hydroxide (10 % aqueous solution), TLC tank, 2
beakers (150 mL), string, scotch tape, paper clips, aluminium weighing cups, cotton gauze
pad.
Procedure
Preparation of fingerprint samples
Prepare the following samples for your own experiments. Print your initials with a felt-tip
marker on the upper right hand corner of each sample. Be careful to handle the items while
wearing gloves. In dry weather, you can improve the fingerprints by wiping your fingers on
sweaty parts of your body or putting moisturising lotion or cream on your hand.
Each group 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.
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)
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
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.
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.
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.
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.
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
13
Procedure
A
1. Mix equal volumes of sulfanilic acid solution and 1-napthol (or naphtyl-ethylenediamine)
solution in a plastic photographic tray.
2. Soak one at a time the dye transfer paper in the mixture until the paper is thoroughly wet. Pick
up the paper by two corners and let the excess solution drain in the tray.
3. Hang the paper on the string with cloth pins to dry.
4. Store the dried paper in a dark and dry place.
5. Just before using, test a sheet of treated paper for nitrite sensitivity by dabbing the four corners
with a nitrite testing swab saturated with 15% acetic acid.
B
1. Place a treated dye transfer paper, gelatin side up, on corrugated cardboard.
2. Place the sample cloth, residue covered side down on the dye transfer paper.
3. Soak a cheesecloth in 15% acetic acid in a 1 L beaker. Pick up a cheesecloth, squeeze out the
excess liquid, and spread it out evenly on the back of the sample fabric.
4. Press the above sandwich from the back of the cheesecloth with a hot iron.
5. Peel off and discard the cheesecloth.
6. Mark the bullet hole. Mark the edges of the fabric if the fabric is smaller than the dye transfer
paper. In actual cases, mark with a pencil important features of the sample fabric: suspected
bullet holes, tears, cuts, rips, button holes, buttons, seams, pockets, etc., for possible future
reference in court.
7. Peel off the sample fabric and save it for the lead test.
8. When the dye transfer paper is dry, mark it on one edge in ink with your initials and case and/or
file number.
LEAD TEST
Supplies and equipment
Spray bottles, test strips containing soluble calcium, strontium, barium, and lead salts.
Solutions
Saturated sodium rhodizonate (or 5,6-dihydroxy-5-cyclohexene-1,2,3,4-tetrone disodium salt) in
buffer (50 mL), the solution should be prepared fresh each day. HC1 (5 mL concentrated HCl and
95 mL distilled water) and pH 2.8 buffer (sodium hydrogen tartrate monohydrate (1.90 g) and
tartaric acid (1.50 g) in distilled water (100 mL).
Procedure
A. Direct application to white sample fabrics
1. Spray the appropriate area with the saturated sodium rhodizonate solution. Pink colour
indicates the presence of lead(II) and other metal ions.
2. Spray the same area with HCl solution. If the pink colour turns to a blue colour, the area has
lead(II).
3. Repeat steps 1, 2, and 3 on test strips containing known metallic elements. Record and
compare the results.
B. Indirect application to coloured sample fabrics
1. Place a piece of filter paper on the area of interest, include any suspected bullet holes. Record
the location of the fabric covered by the filter paper.
2. Spray 15% acetic acid on the filter paper until it is uniformly damp.
3. Cover the dampened filter paper with several layers of dry filter paper and press a hot iron on
the filter paper until the paper is dry.
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
The leaves of marihuana are compound and consist of 5 to 11 separate leaflets. Each leaflet has
characteristic hairs, veins, and serrated edges. The most characteristic feature is the hairs. There
are two types of hairs
1. Cystolith hairs have deposits of calcium carbonate at their base. These hairs are mostly onecelled.
2. Gladular hairs contain and secrete the resin. They are short and may be unicelluar or
multicellular. Larger glandular hairs have a multicellular stalk with heads containing 6 to 8
cells.
Properties
Form
9
-THC
M.Pt. / C
viscous oil
H2 O
Insoluble
Solubility
ethanol
chloroform
1 in 1
Readily soluble
ether
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 disomer 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
Form
Amphetamine
Methamphetamine
M.Pt. / C
liquid
Liquid
H2 O
1 in 50
Slightly soluble
Solubility
ethanol
very soluble
miscible
chloroform
very soluble
miscible
ether
very soluble
miscible
18
Drug
Methamphetamine
Phentermine
Amphetamine
Phenylpropanolamine
Ephedrine/Pseudoephedrine
Rf
0.20
0.40
0.70
0.85
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
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.
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
561-27-3) is a semi- synthetic opiate produced from morphine (a
naturally occurring substance in the opium poppy Papaver
somniferum). The major metabolites are 6-monacteylmorphine
O
then morphine (and glucuronides) and codeine. Morphine is a
NCH3 potent narcotic analgesic, and its primary clinical use is in the
management of moderately severe and severe pain. After heroin,
morphine has the greatest dependence liability of the narcotic
CH3COO
analgesics in common use. Other semi-synthetic analogues of
morphine are also available as painkillers in the US. Heroin is a schedule II drug.
CH3COO
Properties
Form
base
hydrochloride
M.Pt. / C
170
229-233
H2 O
1 in 1700
1 in 1.6
Solubility
ethanol chloroform
ether
1 in 31
1 in 1.5
1 in 100
1 in 12
1 in 1.6
Insoluble
20
Properties
Form
base
hydrochloride
M.Pt. / C
96-98
197 (decomp)
H2 O
1 in 600
1 in 0.5
Solubility
ethanol
chloroform
1 in 7
1 in 0.5
1 in 4
1 in 15
ether
1 in 4
insoluble
21
Barbiturates
Introduction
One of the most common barbiturates is phenobarbital (5-ethyl-5phenylbarbituric acid, C12 H12 N2O3 , MW = 232.2, CAS 50-06-0). It exists as
O
O
colourless crystals or a white powder. Major metabolites are NC6H5
glucopyranosylphenobarbitone and 4- hydroxyphenobarbitone (and
NH
glucuronide). Other common barbiturates are secobarbital, pentobarbital and
C2H5
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.
H
N
Properties
Form
Base
Sodium
M.Pt. / C
174-178
175
H2 O
1 in 1 000
1 in 3
Solubility
ethanol
chloroform
1 in 10
1 in 40
1 in 25
Insoluble
ether
1 in 40
Insoluble
22
HO
CH3O
H
H
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.
Supplies and equipment
A urine sample from a DUI suspect. Blank urine, ethanol and n-propanol. Carry out the alcohol
analysis on the HP5890 GC in Clip 080 using the FID.
Procedure
Construct two calibration graphs using the external standard method (plot area of standard vs
concentration) and the internal standard method (plot area of sample divided by area of standard.vs
conc.) Do this by mixing blank urine (1 mL), n-propanol (200 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
H2
air
DB-624, 0.53 mm x 30 m x 3 m
~40-50 mL/min
~450 mL/min
Temperature
detector temp
injector temp
35 C
250 C
250 C
N2 or He carrier
make up gas
split ratio
50 cm/s
30 mL/min
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.
Supplies and equipment
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
N2 carrier
Detector temp
Injector temp
50 cm/s
300 C
250 C
split ratio
make up gas
50:1
60 mL/min
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
4. Write the reaction mechanism for BSTFA with your drug and using your spectra as a guide,
determine which (if any) sites were modified.
5. Using the paper in your supplementary notes describe 3 different methods for the derivitiztion of
drugs for GC analysis. Which procedure is recommended for cocaine, cannabis, morphine and
amphetamine and why?
6. Derivitization alters the molecular structure. Sometimes this is an advantage in MS analysis,
Why? List some disadvantages to the use of derivatization reagents.
7. Describe how selective ion monitoring and MS/MS can be useful in toxicological analysis.
8. Describe the precautions that must be taken in collecting a urine sample for forensic casework.
Include problems with stability and sample contamination.
Disposal
Chlorinated solvent and other organic solvent waste must be placed in the appropriate container.
Other waste may be disposed of down the drain with copious amounts of water.
30
31
Determination Limit
(g/L)
2.5
0.13
0.25
0.5
0.06
0.25
0.38
0.5
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
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
Supplies and equipment
Procedure
Carpet (2" X 2") samples were all cut from one large piece. Samples consist of a Pyrolyzed carpet,
and carpets spiked with the following accelerants: Gasoline (250 l), Lighter Fluid (250 l), 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
temp
N2 carrier
detector temp
34
35 cm/sec
split flow
make up gas
100 ml/min
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
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
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.
38
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
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
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
44
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)
Red ( no violet on dilution)
Orange (violet on dilution)
Violet
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
Cannabis
Coffee (roasted)
Patchouli Oil
Tea (leaves)
Violet-blue
Violet-brown
Violet
Green-Blue
Colour extracted
by CHCl3 layer
Violet
None
None
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
Sulphuric acid is corrosive
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
Violet-red
Brown-red
Pink
Brown-pink
Orange
Red-orange
Brown-orange
Yellow
Brown-yellow
Green
Blue- green
Brown-green
Grey-green
Black- green
Blue
Ajmaline, alprenolol, arninacrine (100), antazoline, brucine, chlorprothixene, clopenthixol, flupenthixol, mestranol,
oxytetracycline, prajmalium, thiazinamium, thiothixene, tolmetin (100), trifluopemzine, xylazine
Indapamide
Methylchlorophenoxyacetic acid
Trichlorophenoxyacetic acid (brown)
Prazosin (100red-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)
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
Ambutonium, bumetanide, diphenhydramine, fenuron, feprazone (100brown), ibuprofen, labetalol, mepivacaine,
methadone, nefopam (brown), tetrahydrozoline
Amicarbalide (100), clonidine (100orange), dequalinium (100orange), diethylpropion, diloxanide, ethoxzolamide,
fenfluramine (100), flavoxate, gliclazide, metoclopramide, nifenazone (100), piroxicam, propachlor, tropicamide
Amiodarone
Bialamicol, chlorotrianisene, colchicine, dextromoramide (100), diamthazole, hydrastine, mequitazine, naphthols, phenol,
phenothiazine, thiocarlide
Hydrochlorothiazide, hydroflumethiazide, pindolol
Cyclopenthiazide
Azapropazone
Naproxen
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
Green-blue
Violet
Red-violet
Black- violet
Brown
Red-brown
Pink-brown
Orange-brown
Green-brown
Violet-brown
Black-brown
Grey
Black
dipyrone (100), imipramine, mefenamic acid, mefruside, oxypertine, padimate (100), procarbazine (100, 15 s),
propyphenazone (100) (red with water), yohimbine
Amiphenazole (100)
Methocarbamol, mianserin, paracetamol, penthienate methobromide, phenacetin, propiomazine, resorcinol, timolol (100),
trazodone (100) (transient)
Chloroxuron
Methoxychlor
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
Benzthiazide, bisacodyl, carphenazine, chlorpromazine, diclofenac, dothiepin, ethopropazine, etisazole, fenbufen,
fenoprofen, methapyrilene, perphenazine, polythiazide
Metoprolol
Benazolin, diphenadione, maprotiline, methiocarb, piperidolate
Methdilazine, norbormide, promazine, thiopropazate
Bamethan, clofibrate, dichlorophen
Mecoprop
Isopropamide
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
Sulphuric acid is corrosive
Reagent
Dissolve ammonium vanadate (0.5 g) in water (1.5 mL) and dilute to 100 mL with sulphuric acid.
Method
Add a drop of reagent to the sample on a white tile.
Indications
Various colours across the entire visible spectrum are given by a large number of compounds. The
colour given by Liebermann's test should also be taken into account when interpreting the result.
Hydrochlorides give a red colour.
Red
Brown-red
Orange
Red-orange
Green-orange
Brown-orange
Yellow
Orange-yellow
Green-yellow
Green
Yellow-green
Blue- green
Brown-green
Grey-green
Blue
Green-blue
Violet
Ajmaline, azacyclonol, chlorprothixene, diperodon (green), dofamium (brown), flupenthixoi, gelsemine (green),
ndapamide, mequitazine, methotrexate, nialamide. pericyazine, prajmalium, prolintane, sodium cromoglycate,
thiothixene, xylometazoline
Nadolol
Dropropizine (slow), ethylnoradrenaline, 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)
Cotamine (brown)
5-Methyltryptamine
Mexiletine
Azaperone, benztropine, broxaldine, chelidonine (green), conessine, deptropine, desipramine (blue), dihydralazine,
diphenhydramine, diphenidol, diphenylpyraline, dropropizine (orange), halquinol, homidium, lidoflazine,
methacycline (orange-violet), paraphenylenediamine, penicillamine, protokylol (brown), tofenacin, tylosin
(yellow-brown), veratrine (orangeviolet -brown), viprynium
Hexoprenaline
Methoxamine
Acepromazine (red), adiphenine (blue), benorylate, bephenium hydroxynaphthoate, bibenzonium buclosamide (blue
rim), bunamidine, chlorpromazine (violet), clefamide (brown), codeine, coichicine, cyclazocine, cyclomethycaine
(brown), debrisoquine, diaveridine, dibenzepin, diethazine (violet with excess reagent), diethylthiambutene
(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)
Normethadone, opipramol
Benzoctamine, berberine (brown), edrophonium, hydroxystilbamidine, ketobemidone, methoxyphenamine,
phentolamine, profadol (green), viloxazine
Benzydamine, chlorphenesin
Alverine, azapropazone, benzhexol, diamphenethide, diethyltryptamine (yellow), dihydrocodeine, guaiphenesin,
hordenine, levomethadyl acetate, norinorphine, oxyphencyclamine, papaverine, terbutaline
Bamethan (green), clomipramine, deserpidine (green), desferrioxamine (violet), doxapram, droperidol (green),
harmine (green), imipramine (add water), maprotiline, mebhydrolin, metaraminol, phenaglycodol, phenyramidol,
pyridoxine (grey-green), salbutamol (blue rim brown rim), thioridazine (violet), trimipramine (add water),
triphenyltetrazolium (slow), xipamide, xylazine, yohimbine (green)
Chlophedianol, labetalol
Amidephrine, benperidol, bezitmmide (orange), bisacodyl, captodiame, cephaloridine, chloropyrilene (orange),
clomiphene (orange-brown), clomocycline (brown), denatonium, dipyridamole, guanoclor (orangebrownyellow), 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
Blue- violet
Brown-violet
Grey-violet
Black- violet
Brown
Red-brown
Pink-brown
Orange-brown
Yellow-brown
Green-brown
Violet-brown
Grey-brown
Grey
Blue- grey
Black
53
Appendix A
Marquiss Test
Precautions
Sulphuric acid is corrosive
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
Orange-Red
Violet-Red
Brown-Red
Pink
Orange
Red-Orange
Pink-Orange
Yellow-Orange
Brown-Orange
Yellow
Orange-Yellow
Green
Yellow-Green
Blue-Green
Brown-Green
Grey-Green
Blue
Grey-Blue
54
Appendix A
Violet
Red-Violet
Blue-Violet
Brown-Violet
Grey-Violet
Black-Violet
Brown
Red-Brown
Orange-Brown
Yellow-Brown
Green-Brown
Violet-Brown
Grey-Brown
Grey
Blue-Grey
Black
Blue-Black
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
Temp.
Detector Temp
Split ratio
280 C
50:1
Flow rate
Injector temp
~1 mL/min
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
Column
Temp.
Detector Temp
Split ratio
280 C
50:1
Flow rate
Injector temp
~1 mL/min
250 C
Cocaine
Sample extraction
Extract sample using the procedure as detailed under amphetamines, except adjust buffer pH to 9.2.
Derivatisation
Derivatise with BSTFA according to the instructions under amphetamines.
Analysis Conditions
Column
Temp.
Detector Temp
Split ratio
280 C
50:1
Flow rate
Injector temp
~1 mL/min
250 C
Marihuana
Sample extraction
Extract sample using the procedure as detailed under amphetamines.
Derivatisation
Derivatise with BSTFA according to the instructions under amphetamines.
Analysis Conditions
Column
Detector Temp
Flow rate
Temp.
Split ratio
Injector temp
~1
mL/min
250 C
Opiate
Sample extraction
Extract sample using the procedure as detailed under amphetamines, except adjust buffer pH to 9.2.
Derivatisation
Derivatise with BSTFA according to the instructions under amphetamines.
Analysis Conditions
Column
Temp.
Detector Temp
Split ratio
58
280 C
50:1
Flow rate
Injector temp
~1 mL/min
250 C
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
0.63
0.9
8.48
0.99
1.2
0.15
0.2
0.26
1.0
4.39
0.83
0.9
0.82
1.7
0.16
11.08 1.89
1.13
1.0
5.68
1.79
1.3
0.72
0.27
1.3
0.88
2.00
2.24
1.11
0.73
1.8
0.20
1.5
2.48
1.3
16.82 2.17
Adrenaline
Amphetamine
Benzphetamine
Caffeine
Cathine
Chlorphentermine
Diethylpropion
Dimethylamphetamine
DOM
Ephedrine
Fencamfamin
Fenethylline
Fenfluramine
Hordenine
Hydroxyamphetamine
Hydroxyephedrine
Mazindol
Mephentermine
Mescaline
1)
2)
3)
Appendix B
Methoxyamphetamine
Methoxyphenamine
Methylamphetamine
Methylenedioxyamphetamine
Methylephedrine
Methylphenidate
Noradrenaline
Normetanephrine
Oxedrine
Pemoline
Phendimetrazine
Pheneizine
Phenethylamine
Phentermine
Phenylephrine
Phenylpropanolamine
Pipradrol
Prolintane
Pseudoephedrine
Tranylcypromine
Trimethoxyamphetamine
Tyramine
60
1.7
2.0
2.3
1.7
0.2
0.9
1.0
1.2
0.6
1.3
0.9
1.2
2.0
1.2
1.0
1.2
14.95
32.17
10.52
0.10
1.09
0.27
5.91
3.64
19.46
3.87
5.90
0.81
2.07
0.98
1.83
0.36
0.14
0.32
0.37
1.31
0.86
1.64
0.70
0.69
1.26
1.77
0.26
1.48
1.47
Appendix B
Barbiturates
Sample extraction
Extract sample according to the instructions given under amphetamines in GC analysis section.
Reconstitute sample in mobile phase (1 mL).
Analysis Conditions
System 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.
Allobarbitone
Amylobarbitone
Aprobarbitone
Barbitone
Brallobarbitone
Butalbital
Butobarbitone
Cyclobarbitone
Cyclopentobarbitone
Enallylpropymal
Heptabarbitone
Hexethal
Hexobarbitone
Ibomal
Idobutal
Metharbitone
Methohexitone
Methylphenobarbitone
Nealbarbitone
Pentobarbitone
Phenobarbitone
Phenylmethylbarbituric acid
Quinalbarbitone
Secbutobarbitone
Talbutal
Vinbarbitone
4)
5)
System k'
values
HG
HH
2.46
1.33
10.91
7.05
3.42
2.22
1.11
0.63
3.09
1.72
6.17
3.48
5.43
3.42
5.25
2.61
6.00
3.84
8.65
6.96
9.90
4.93
34.28 20.39
7.37
5.67
4.01
2.58
8.12
4.77
2.69
1.99
27.61 20.48
7.27
3.84
10.22
6.19
10.96
8.07
3.09
1.23
1.48
0.94
16.28 11.47
4.89
3.32
7.25
4.67
4.83
2.32
Appendix B
Cocaine
Sample extraction
Extract sample according to the instructions given under amphetamines in GC analysis section.
Reconstitute sample in mobile phase (1 mL).
Analysis Conditions
System 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
2.0
16.25 1.33
0.1
20.06 1.61
0.9t
5.68
0.9
7.19
0.86
1.2
8.97
4.42
0.24
1.9
5.51
2.8
2.68
10.31
2.2
11.24
2.48
2.78
0.6
0.79
0.9
1.09
4.14
16.25 0.86
4.59
0.6
2.4
1.0
1.38
1.9
0.00
1.09
2.1
1.38
Amethocaine
Benzocaine
Benzoylecgonine
Bupivacaine
Butacaine
Butanilicaine
Chloroprocaine
Cinchocaine
Cocaine
Cyclomethycaine
Dimethisoquin
Diperodon
Dyclonine
Lignocaine
Mepivacaine
Oxethazaine
Oxybuprocaine
Piperocaine
Pramoxine
Prilocaine
Procaine
Propoxycaine
Proxymetacaine
t =tailing peak
6)
7)
Appendix B
Marihuana
Sample extraction
Extract sample according to the instructions given under amphetamines in GC analysis section.
Reconstitute sample in mobile phase (1 mL).
Analysis Conditions
System 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).
Cannabichromene
Cannabicyclol
Cannabidiol
Cannabidiolic acid
Cannabigerol
Cannabinol
Cannabivarin
8-Tetrahydrocannabinol
9-Tetrahydrocannabinol
Tetrahydrocannabinolic acid
Tetrahydrocannabivaric acid
Tetrahydrocannabivarin
8)
System
k' values
HL
19.09
14.78
7.47
8.76
8.18
11.77
7.47
14.07
13.35
25.83
14.64
8.18
Appendix B
Opiate
Sample extraction
Extract sample according to the instructions given under amphetamines in GC analysis section.
Reconstitute sample in mobile phase (1 mL).
Analysis Conditions
Systems HA or HC, previously described, may be used or System HS, below.
System HS 9
Column: Amino-propyl bonded silica (Spherisorb S5NH2 , 5 m, 25 cm x 4.6 mm id).
Eluent: Acetonitrile: tetrabutylammonium phosphate (0.005M, pH 7.5) (85:15).
Acetylcodeine
Benzylmorphine
Buprenorphine
Caffeine
Codeine
Dextromethorphan
Dextromoramide
Dextropropoxyphene
Diamorphine
Dihydrocodeine
Dihydromorphine
Diphenoxylate
Dipipanone
Ethoheptazine
Ethylmorphine
Etorphine
Fentanyl
Hydrocodone
Hydromorphone
Ketobemidone
Levallorphan
Levorphanol
Methadone
6-Monoacetylmorphine
Morphine
Nalorphine
Naloxone
Norcodeine
Normethadone
Normorphine
Norpipanone
Noscapine
Oxycodone
Oxymorphone
9)
0.4
0.05
0.2
0.21
4.8t 1.21 1.90
5.6t
0.7
0.09
1.9
0.19
5.7t 2.75
0.2
2.2
1.61
3.3
1.55
0.8
1.11
7.lt
2.17
7.9t
2.8t
1.9t 1.46
4.4t 3.20
2.2
1.03
1.4
0.17
3.lt
3.51
0.53
2.9t 3.92
0.35
0.3
0.15 0.01
6.9t 0.85
6.7t
Appendix B
Papaverine
Pentazocine
Pethidine
Phenazocine
Phenoperidine
Pholcodine
Piritrarnide
Quinine
Strychnine
Thebacon
Thebaine
0.3
1.8
2.8t
1.3
0.8
6.0t
0.6
2.4
1.0t
3.7t
4.6t
65
0.16
0.67
0.55
0.30
0.10
1.63
0.14
0.85
0.94
0.04
2.02
2.43
0.79
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 extractionconcentration, 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 colordeveloping 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.
Points to Note:
Always run samples next to standard discs on the same Toxi-Gram so that direct
comparison can be made
67
Appendix C
Abbreviated Instructions -TOXI
LAB A
1. Briefly shake each TOXI-TUBES A extraction tube. add urine to the 5-mL arrow, cap, and mix
by inversion for 2 min Centrifuge for 2-5 min.
2. Insert the appropriate number of disposable concentration cups into the wells of the OMEGA12 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
Abbreviated Instructions -TOXI
LAB B
1. Using TOXI- TUBES B extraction tubes, add urine to the 4.5- mL arrow, cap and mix by
inversion for 2 min, Centrifuge for 2-5 min.
2. Insert the appropriate number of disposable concentration cups into the wells of theOMEGA-12
extraction solvent concentrator. With a disc-handling pin place one 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
(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
6.58
6.61
Unknown 6.23
% 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
Table I
Exact Concentrations of Standards
Standard #
25.0
50.0
75.0
100.0
150.0
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)
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
74
Appendix C
16
14
12
10
8
6
4
2
0
10
30
50
70
90
110
130
150
Caffeine Concentration (ppm)
170
190
210
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