COLLEGE OF PHARMACY CLINICAL TOXICOLOGY

OUR LADY OF FATIMA UNIVERSITY

COLLEGE OF PHARMACY

LABORATORY MANUAL
IN
CLINICAL TOXICOLOGY

JUNE 2010

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COLLEGE OF PHARMACY CLINICAL TOXICOLOGY

Laboratory Safety Rules and Regulations

1. Laboratory safety information and procedures must be read before using equipment or
conducting an experiment. Instructions should be followed in carrying out the activity or
investigation.
2. Working in the laboratory without the supervision of the instructor is prohibited.
3. Unauthorized activities or investigations are prohibited.
4. The chemical storage area is prohibited to unauthorized personnel.
5. Removing chemicals or equipment from the laboratory is prohibited unless authorized.
6. All accidents, chemical spills and injuries, no matter how trivial they may seem at the time,
must be reported immediately to the instructor.
7. Hair ties should be used by students when necessary to prevent laboratory accidents.
Moreover, students wearing dangling jewelry, acrylic nails, sandals and open-toed shoes
are not allowed inside the laboratory.
8. Location of all safety equipment (e.g. emergency shower area, fire extinguishers and fume
hood) present in the room should be known.
9. Laboratory gloves should be worn throughout the activity when necessary.
10. Precautions should be practiced when using flame-producing devices or heating
equipment. Never leave any fire unattended.
11. Bags and books should remain in an area designated by the instructor.
12. Sitting on the laboratory tables is prohibited.
13. Work areas should be kept clean and neat at all times. Work areas should be cleaned at
the end of the laboratory period.
14. Glassware should be washed with warm water and liquid detergent. They should be
rinsed, dried and returned every after the laboratory period.
15. Hot glassware should not be immersed in cold water.
Handling of Chemicals
1. The reagent bottle labels should be read twice before using. The student should be certain
that the reagent used is the correct one.
2. The reagent bottle should be immediately covered after using it. The reagent should be at
its designated location at all times.
3. Unused chemicals should not be returned to the reagent container. Directions for the
storage or disposal of these chemicals should be followed.
4. Solid chemicals, metals, matches, filter papers, broken glasses and other materials should
be disposed in their proper waste containers, not in the sink.
5. Proper disposal of chemical waste should be practiced at all times. Mixing of chemicals in
the sink should not be practiced.
6. All chemicals in the laboratory should be considered poisonous. Chemicals should not be
handled with bare hands. Chemicals should not be smelled directly.

CHEMICAL WASTE DISPOSAL

1. Only neutral aqueous solutions go down the sink drain.

2. All chemical waste is to be sorted into the appropriate waste container.

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LABORATORY SAFETY RULES AND REGULATIONS

I have read and understood the laboratory safety rules and regulations. I
acknowledge that these rules are necessary to prevent accidents and to ensure my own safety
and of others. Moreover, I will follow any additional instructions given by my instructor. I
understand that I should ask my instructor at any time about the instructions, rules and
regulations if they are not clear to me. I agree to follow and abide to these rules and regulations
in the laboratory.

Conforme:

_________________________ _________________ ________________________

Student Signature Subject Instructor
(Signature over printed name) (Signature over printed name)

_________________________
Date

(To be submitted to the instructor)

LABORATORY SAFETY RULES AND REGULATIONS

Conforme:

_________________________ _________________ ________________________

Student Signature Subject Instructor
(Signature over printed name) (Signature over printed name)

_________________________
Date

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COLLEGE OF PHARMACY CLINICAL TOXICOLOGY

First Aid in the Laboratory

ALL accidents, injuries, and spills should be reported immediately to the instructor.

ALL STUDENTS should know the following:

• Laboratory Safety Techniques

• Procedure in reporting an accident, injury, or spill
• Location of first aid kit, fire alarm, fire extinguisher and emergency shower
• Location of emergency escapes

Basic First Aid Procedures for Laboratories

Situation Safe Response

Burns Minor – include small scalds or burns from hot objects.

✓ Flush the burned area with cool water from the faucet or use
cool wet compresses to the skin.
✓ Cleanse the burned area and apply burn cream from the first
aid kit.

Chemical Burn

✓ Start treatment immediately by placing the burned area under

cool running water for at least 15 minutes.
✓ If the chemical reached the eye, irrigate the injured eye with
cool water for at least 15 minutes. Make sure that the water
will not run into the other eye. Then, cover the eye with a sterile
compress.
✓ Recommend the individual to seek emergency medical
treatment.

Cuts & Bruises ✓ Treat as directed using instructions included in the first aid kit.

Fire ✓ Turn off all flames and gas jets, and then wrap the person on
fire with wet cloth.
✓ Use fire extinguisher to put out the fire, DO NOT use water.

Foreign Matter ✓ Irrigate the injured eye with running water for 15 minutes.
in Eyes ✓ Seek medical help.

Poisoning ✓ Note the suspected poisoning agent.

✓ Consult the instructor for the appropriate antidote.

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✓ Call the nearest poison control center if more help is needed.

Severe bleeding ✓ Apply direct pressure to the source of bleeding.

✓ Seek medical help immediately.

Chemical spills, ✓ Wash the area with running water, use safety shower.
Acid burns, ✓ For acid burns, apply sodium bicarbonate (baking soda).
Base burns ✓ For base burns, apply boric acid.

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USING THE FIRE EXTINGUISHER

1. Pull the Pin at the top of the extinguisher. The pin releases
a locking mechanism that will discharge the extinguisher.

2. Aim at the base of the fire, not the flames. In order to put
out the fire, the fuel must be extinguished.

3. Squeeze the lever slowly. This will release the extinguishing

agent in the extinguisher. If the handle is released, the discharge
will stop.

4. Sweep from side to side. Using a sweeping motion, fire

extinguisher must be moved back and forth until the fire is
completely out. Operate the extinguisher from a safe distance,
several feet away, and then move towards the fire once it starts
to diminish.

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TABLE OF CONTENTS

Experiment No. Title Page No.

1
Ethyl Alcohol 10
2
Methyl Alcohol 13
3
Acetone 16
4
Formaldehyde 18
5
Phenol 21
6
Chloroform 24
7
Carbon Tetrachloride 27
8
Carbon disulfide 30
9
Aniline 33
10
Picric acid 36
11
Naphthalene 38
12
Aspirin 40
13
Salicylic acid 42
14
Acetanilide 44
15
Phenacetin 46
16
Colchicine 49
17
Quinine 52
Phenytoin Sodium Capsules
18 55

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ROUTE OF ENTRY FOR TOXIC SUBSTANCES

Inhalation

The respiratory tract is the most common route of entry for gases, vapors, particles,
and aerosols (smoke, mists and fumes). These materials may be transported into the
lungs and exert localized effects, or be absorbed into the bloodstream. Factors that
influence the absorption of these materials may include the vapor pressure of the material,
solubility, particle size, its concentration in the inhaled air, and the chemical properties of
the material. The vapor pressure is an indicator of how quickly a substance evaporates
into the air and how high the concentration in air can become – higher concentrations in
air cause greater exposure in the lungs and greater absorption in the bloodstream.

Most chemicals have an odor that is perceptible at a certain concentration, referred

to as the odor threshold; however, there is no relationship between odor and toxicity.
There is considerable individual variability in the perception of odor. Olfactory fatigue may
occur when exposed to high concentrations or after prolonged exposure to some
substances. This may cause the odor to seem to diminish or disappear, while the danger
of overexposure remains.

Symptoms of over-exposure may include headaches, increased mucus

production, and eye, nose and throat irritation. Narcotic effects, including confusion,
dizziness, drowsiness, or collapse, may result from exposure to some substances,
particularly many solvents. In the event of exposure, close containers or otherwise
increase ventilation, and move to fresh air. If symptoms persist, seek medical attention.

Volatile hazardous materials should be used in a well-ventilated area, preferably a

fume hood, to reduce the potential of exposure. Occasionally, ventilation may not be
adequate and a fume hood may not be practical, necessitating the use of a respirator. The
Occupational Safety and Health Administration Respiratory Protection Standard regulates
the use of respirators; thus, use of a respirator is subject to prior review by EHS according
to University policy.

Ingestion

The gastrointestinal tract is another possible route of entry for toxic substances.
Although direct ingestion of a laboratory chemical is unlikely, exposure may occur as a
result of ingesting contaminated food or beverages, touching the mouth with contaminated
fingers, or swallowing inhaled particles which have been cleared from the respiratory
system. The possibility of exposure by this route may be reduced by not eating, drinking,
smoking, or storing food in the laboratory, and by washing hands thoroughly after working
with chemicals, even when gloves were worn.

Direct ingestion may occur as a result of the outdated and dangerous practice of
mouth pipetting. In the event of accidental ingestion, immediately go to the Poison Control
Center. Do not induce vomiting unless directed to do so by a health care provider.

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Injection

The final possible route of exposure to chemicals is by accidental injection.

Injection effectively bypasses the protection provided by intact skin and provides direct
access to the bloodstream, thus, to internal organ systems. Injection may occur through
mishaps with syringe needles, when handling animals, or through accidents with pipettes,
broken glassware or other sharp objects that have been contaminated with toxic
substances.

If accidental injection has occurred, wash the area with soap and water and seek
medical attention, if necessary. Cautious use of any sharp object is always important.
Substituting cannulas for syringes and wearing gloves may also reduce the possibility of
injection.

TOXIC SUBSTANCES can act at different systems:

RESPIRATORY SYSTEM

Interference with respiration leads to inadequate ventilation of the lungs and thus
to an inadequate supply of oxygen (hypoxia). When severe, this may be one of the most
serious toxic conditions, because man can live without food and water for days, but he
dies within a few minutes when his oxygen supply is cut off.

CARDIOVASCULAR SYSTEM

Disturbances of the cardiac action are mostly associated with an increase of the
pulse rate, less frequently with slowing. It is often irregular. Changes of the pulse rate may
be due to myocardial damage and may be produced by alterations in the responsiveness
to stimulation of vagus and accelerants.

NERVOUS SYSTEM

The most common injury of nervous system is headache. This may be caused by
nervous strain, such as eye strain, glare, or noise, by hyperemia of the sinuses (frontal
headache), by hyperemia of the meninges, and by increased intracranial pressure. It is
therefore apparent that headache may be produced by many toxic agents, but in most
instances, it is difficult to differentiate these, especially if the symptom may have a multiple
etiology.

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INDUSTRIAL POISONS

Experiment No. 1
ETHYL ALCOHO`L
Objective:
To be able to detect the presence of ethyl alcohol

Materials:
Aspirator Stirring rod
Beaker Test tubes
Erlenmeyer flask Test tube brush
Evaporating dish Test tube holder
Micropipette Water bath
Pipette

Reagents:
1% Potassium dichromate Ethyl alcohol
10% NaOH Lugol's solution
Ammonia molybdate solution KOH pellet
Benzoyl chloride KOH aqueous solution
Carbon disulfide Sodium acetate crystals
Conc. Sulfuric acid

CAUTION:
• Exercise great care in handling KOH as it rapidly destroys tissues. Do not handle it
with bare hands

• CS2 is extremely volatile and flammable compound and also with a disagreeable,
fetid odor.

• Ethanol-water solutions greater than about 50% ethanol by volume are flammable
(in someUrine
Sample: casescontaminated
ethanol will burn
withat as low as a 45% solution) and easily ignited.
Ethanol
Ethanol-water solutions below 50% ethanol by volume may also be flammable if the
solution is vaporized by heating (as in some cooking methods that call for wine to
be added to a hot pan, causing it to flash boil into a vapor, which is then ignited to
"burn off" excessive alcohol).

Procedure:
I. Prepare all the materials needed
II. Perform the different tests for the detection of ethyl alcohol

Detection:

A. Berthelot’s Test
1. Place 1 mL of the sample in a test tube
2. Add a few drops of benzyl chloride, then add an excess of 10% sodium
hydroxide solution until the irritating odor is gone.

Results: _____________________________________________________

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B. Chromic Acid Test

1. Neutralize and distill 100 ml of the sample.
2. To the 10 mL of the distillate, add 2-3 drops of 1% solution of Potassium
dichromate and 4-5 drops of concentrated sulfuric acid.
3. Boil the mixture.
4. Observe for the color and note the odor produced.

Results: ___________________________________________________________

C. Ethyl Acetate
1. Place 5 ml of the distillate in a test tube.
2. To the sample add an equal volume of concentrated sulfuric acid then add a few
crystals of sodium acetate then warm gently.
3. Note for the odor produced.

Results: _____________________________________________________

D. Lieben’s Iodoform Test

1. Place 1 mL of sample in a test tube.
2. Add 1 ml of Lugol’s solution and warm gently.
3. Add enough potassium hydroxide solution until the solution is yellow and allow to
cool.

Results:____________________________________________________

E. Vitali’s Test
1. Place 1 ml of the sample solution in a test tube
2. Add 3 drops of carbon disulfide and a pellet of potassium hydroxide in a small
evaporating dish, when most of the carbon disulfide has evaporated add 1 drop
of ammonium molybdate solution and then acidify with conc. Acid.

Results:__________________________________________________

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Name:____________________________ Score:_________________
Yr and Sec:________________________ Date:__________________

Experiment No. 1
ETHYL ALCOHOL

Questions:
1. Describe the effects of ethanol to one’s health?

2. Name the medical test used to determine whether the person have been exposed to
ethanol?

3. Enumerate means to reduce the risks of exposure to ethanol?

4. What are the manifestations of Ethanol poisoning?

5. How is ethanol poisoning treated?

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COLLEGE OF PHARMACY CLINICAL TOXICOLOGY

Experiment No. 2
ACETONE
Objective:
To be able to detect the presence of Acetone

Materials:
Aspirator Stirring rod
Beaker Test tubes
Condenser Test tube brush
Distilling flask Test tube holder
Micropipette
Pipette

Reagents:
0.5% Sodium nitroprusside Mercuric iodide
Acetic acid Potassium iodide
Acetone KOH solution

CAUTION:
• Acetone is very flammable. Do not use where it may be ignited.
• Exercise great care in handling NaOH, it rapidly destroys tissues

Procedure:
I. Prepare all the materials needed
II. Perform the different tests for the detection of acetone

Detection:
A. Place 1 mL of sample in a test tube. Add a few drops of freshly prepared alkaline solution
of sodium nitroprusside. Observe for the formation of color. Add excess acetic acid and
observe for the color change.

RESULTS: ___________________________________________________________

B. Place the sample in a flask and add Nessler’s reagent (1:1). Distill and observe for the
formation of precipitate.

RESULTS: ___________________________________________________________

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Name:____________________________ Score:_________________
Yr and Sec:________________________ Date:__________________

Experiment No. 2
ACETONE

Questions:
1. Describe the effects of acetone to one’s health?

2. Name the medical test used to determine whether the person have been exposed
to acetone?

3. Enumerate means to reduce the risks of exposure to acetone?

4. What are the manifestations of acetone poisoning?

5. How is acetone poisoning treated?

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Experiment No. 3
FORMALDEHYDE

Objective:
To be able to detect the presence of Formaldehyde

Materials:
Aspirator Stirring rod
Beaker Test tubes
Erlenmeyer flask Test tube brush
Evaporating dish Test tube holder
Micropipette Water bath
Pipette

Reagents:
0.5% Phenyl hydrazine HCl Fehling's reagent
1% Phloroglucinol solution Ferric chloride TS
5% Sodium nitroprusside 15%HCl Formaldehyde
10% NaOH 25% HCl
Nessler's reagent
40% NaOH solution Phloroglucinol
Conc. Ammonia Schiff's reagent
Conc. Sulfuric acid Silver nitrate solution

CAUTION:
• Use care in handling conc. Ammonia, because it is caustic in nature
and has irritating properties of vapor. Cool the container well before
opening and cover the closure with a cloth while opening. Do not
taste it and avoid inhalation of its vapor
• Silver nitrate is caustic and can stain the skin

Procedure:
I. Prepare all the materials needed
II. Perform the different tests for the detection of formaldehyde

Detection:
A. General Aldehyde Reactions
a. Silver Nitrate Test
1. Place 1 mL of sample in a test tube
2. Add a few drops of silver nitrate to the solution to be tested.
3. Heat the solution to boiling.
4. Observe the results
Results:______________________________________________________

b. Fehling’s Test
1. Place 1 mL of sample in a test tube
2. Heat the sample solution with 5 drops of Fehling’s reagent.
3. Observe for the formation of color.
Results:______________________________________________________

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c. Nessler’s Test
1. Place 1 mL of sample in a test tube
2. Add 5 drops of Nessler’s reagent to 2 ml of the solution.
3. Heat the solution to boiling.
4. Observe the results.

Results:______________________________________________________

B. Hehner’s Test as modified by Leonard

1. Place 5 mL of sample in a test tube.
2. Mix with 2 ml of fresh unboiled milk and 7 ml of 25% HCl containing a few
drops of ferric chloride test solution.
3. Boil gently for a minute.
4. Observe the results

Results:______________________________________________________

C. Hexamethylene Tetra-amine Test

1. Place 1 mL of sample in a test tube
2. Treat the sample solution with ammonia and evaporate slowly.
3. Observe the results

Results:______________________________________________________

D. Phloroglucinol Test
1. Place 1 mL of sample in a test tube
2. To the sample solution add an equal quantity of 15% HCl.
3. Sprinkle a pinch of phloroglucinol in the surface of the solution.
4. Observe the results

Results:______________________________________________________

E. Resorcinol Test
1. Place 1 mL of sample in a test tube
2. To the sample solution add a mixture of an equal volume of 5% resorcinol
and 40% sodium hydroxide solution.
3. Heat to boiling.
4. Observe the results

Results:______________________________________________________

F. Rimini’s Phenyl-Hydrazine Test

1. Place 1 mL of sample in a test tube
2. To the sample solution add 10 drops of 0.5% phenyl-hydrazine hydrochloride
solution and then 2 drops of 5% sodium nitroprusside and then finally add 10
drops of 10% sodium hydroxide.
3. Observe the results

Results:______________________________________________________

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Name:____________________________ Score:_________________
Yr and Sec:________________________ Date:__________________

Experiment No. 3
FORMALDEHYDE

Questions:
1. Describe the effects of formalin to one’s health?

2. Name the medical test used to determine whether the person have been exposed to
formalin?

3. Enumerate means to reduce the risks of exposure to formalin?

4. What are the manifestations of Formalin poisoning?

5. How is formalin poisoning treated?

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Experiment No. 4
PHENOL

Objective:
To be able to detect the presence of Phenol

Materials:
Aspirator Stirring rod
Beaker Test tubes
Litmus paper Test tube brush
Micropipette Test tube holder
Pipette

Reagents:
Bromine water Millon’s reagent
Chloroform Phenol
Conc. Sulfuric acid KOH pellet
Conc. HCl NaOCl
Ferric chloride
CAUTION:
• A vapor of Phenol is flammable. It whitens and cauterizes the
skin and mucous membranes
• KOH and Conc. Sulfuric acid are corrosive

Procedure:
I. Prepare all the materials needed
II. Perform the different tests for the detection of phenol

Detection:
A. Bromine Water Test
1. Place 1 mL of sample in a test tube
2. To the solution add an excess of strong bromine water and shake.
Results:________________________________________________________

B. Ferric Chloride Test

1. Place 1 mL of sample in a test tube
2. Add to the solution very dilute ferric chloride drop by drop until there is a color
change. Add to this mixture dilute HCl or sulfuric acid.
3. Observe the change in color. (Difference from salicylic acid)
Results:________________________________________________________

C. Hypochlorite Test
1. Place 1 mL of sample in a test tube
2. Add few drops of ammonium hydroxide, then 2-4 drops of freshly prepared
sodium hypochlorite solution.
3. Warm gently.

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4. Observe the change in color.

Results:________________________________________________________
C. Melzer’s Benzaldehyde Test
1. Place 1 mL of sample in a test tube
2. Add 2 ml of conc. sulfuric acid
3. Heat.
4. Add 10 ml of water in the cooled solution and enough KOH to make it
alkaline.
5. Observe the result.
Results:________________________________________________________

D. Millon’s Test (Plugge’s Reaction)

1. Place 1 mL of sample in a test tube
2. Add to the solution few drops of Millon’s reagent and heat.
Results:________________________________________________________

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Name:____________________________ Score:_________________
Yr and Sec:________________________ Date:__________________

Experiment No. 4
PHENOL
Questions:

1. Describe the effects of phenol to one’s health?

2. Name the medical test used to determine whether the person have been exposed to
phenol?

3. Enumerate means to reduce the risks of exposure to phenol?

4. What are the manifestations of Phenol poisoning?

5. How is phenol poisoning treated?

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COLLEGE OF PHARMACY CLINICAL TOXICOLOGY

Experiment No. 5
CHLOROFORM

Objective:
To be able to detect the presence of Chloroform

Materials:
Aspirator Stirring rod
Beaker Test tubes
Erlenmeyer flask Test tube brush
Evaporating dish Test tube holder
Micropipette Water bath
Pipette

Reagents:
Alpha naphthol Ferrous sulfate solution
Ammonium chloride KOH solution
Ammonium hydroxide KOH solution alc.
Aniline Pyridine
Chloroform NaOH 10%
dil. HCl Resorcinol
Fehling's reagent Silver nitrate
Ferric chloride TS

CAUTION:
• Do not inhale the vapors of chloroform since it is carcinogenic!
Care should be taken not to vaporize chloroform in the presence
of flame, because it can produce harmful gases
• KOH and NaOH are corrosive

Procedure:
I. Prepare all the materials needed
II. Perform the different tests for the detection of chloroform

Detection:

A. Isocyanide Test (Phenyl-carbylamine’s Reaction, Hoffman’s Phenyl-Isocyanide

Reaction)
1. Place 2 mL of the sample solution in a test tube
2. Add 2 drops of aniline and 5 drops of alcoholic or aqueous potassium
hydroxide
3. Warm the mixture until observable change (10 mins)
4. Observe the results

Results:______________________________________________________

B. Schwartz’s Resorcinol Test

1. Mix 2mL of 5% resorcinol solution with 5 drops of sodium hydroxide solution
and finally 2 mL of the sample containing chloroform.

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2. Heat the mixture to boiling (10 mins).

3. Observe the results

Results:______________________________________________________

C. Lustgarten’s Naphthol Test

1. To 2 ml of the sample solution
2. Add a few drops of alpha naphthol solution (Alpha naphthol is made by
dissolving a few grains on a few ml of aqueous potassium hydroxide
solution.)
3. Observe the results
Results:______________________________________________________

D. Fujiwara’s Pyridine Test

1. Mix 2 ml of pyridine with 3 ml of 10% sodium hydroxide solution
2. heat to boiling
3. Add 1 ml of the sample to be tested.
4. Observe the results
Results:______________________________________________________

E. Reduction Test
a. Fehling’s Test
1. Place 1 mL of sample in a test tube
2. Warm the sample with a few drops of the Fehling’s reagent.
Results:______________________________________________________

b. Tollen’s Test
1. Add excess of ammonium hydroxide solution to silver nitrate solution then the
sample containing chloroform.
2. Heat to boiling.
3. Observe the results
Results:______________________________________________________

F. Cyanide Test
1. Place 1 ml of the sample solution in a test tube
2. Add a small amount of ammonium chloride (1 pinch) and 2 ml alcoholic
potassium hydroxide in a test tube.
3. The tube is stoppered with cork containing long glass tubing and heated for
one hour in a water bath.
4. Cool the solution and test the presence of hydrocyanic acid by the Prussian
blue test.
5. Observe the results
Results:______________________________________________________

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Name:____________________________ Score:_________________
Yr and Sec:________________________ Date:__________________

Experiment No. 5
CHLOROFORM

Questions:
1. Describe the effects of chloroform to one’s health?

2. Name the medical test used to determine whether the person have been exposed to
chloroform?

3. Enumerate means to reduce the risks of exposure to chloroform?

4. What are the manifestations of Chloroform poisoning?

5. How is chloroform poisoning treated?

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