SUBJECT FORENSIC SCIENCE

Paper No. and Title PAPER No. 9: Drugs of Abuse

Module No. and Title MODULE No. 25: Colour Tests for Drug Identification

Module Tag FSC_P9_M25

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 TABLE OF CONTENTS

1. Learning Outcomes

2. Introduction

3. Forensic Significance of Drug Identification

4. Colour Tests

5. Summarized Test Indications

6. Limitations of Colour Tests

7. Summary

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 1. Learning Outcomes

After studying this module, you will be able to know about-

 Various preliminary tests for Drug identification

 The significance of preliminary examinations like color tests, crystal tests, etc.
 Limitations of preliminary screening and approaches to overcome them

2. Introduction

Preliminary screening of Drugs are basically done by Colour tests, also sometimes
referred to as chemical spot tests, provide with one of the leading tools for the
presumptive identification of drugs. These colour tests are most practically applied to
pharmaceuticals and scene of crime residues and, to a lesser extent, to biological fluids
such as stomach contents, urine, etc. They are used to place the unidentified into a
specific class of compounds or to eliminate categories or classes of compounds. These
colour tests remain popular for several reasons. They are simple to perform and no
extensive training is required. As such, they appeal in situations where laboratory
facilities may be very limited. They can be performed in the field by police officers or
technicians, require minimal reagents, are inexpensive, and give immediate results that
can be viewed by the bare eye. In many instances, colour tests can also be used as Thin
Layer Chromatography location reagents, applied by spraying or dipping. Colour tests
can provide an indication of compound class far more rapidly than immunoassays and
chromatographic techniques such as Gas Chromatography and High Performance Liquid
Chromatography. Colour tests are only an indication of the presence of a compound or
class of compounds and that all tests must be confirmed by more specific methods. This
is exclusively significant where test results may ultimately result in protective sentences.
Tests are usually carried out either in clear glass test-tubes or on white glazed porcelain
tiles (spotting tiles), which give a uniform background against which colours can be
assessed. A sample known not to contain the compound of interest should be tested at the
same time as the test sample. This enables a comparison of the colours produced by the
sample and by the reagent blank. It is essential to validate all tests and test reagents for
sensitivity and specificity.

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 3. Forensic Significance of Drug Identification

Use of Drugs is not a menace anymore, it has become a rampant. Most cases that arrive
in a forensic laboratory start with the suspicion that a drug is present. A fatality might be
an accident, suicide or murder, but a toxicological examination must be carried out to
assist the investigating officer to decide which of these it might be. Often the
investigating officer will not know whether or not any offence has been committed until
the results of the toxicological analyses are available, so that formulating the correct
questions for him or her to ask is vital if accurate and useful answers are to be given. It
should be remembered that basic tests are not, in any circumstances, intended to replace
pharmacopoeial requirements, but should be used as a rapid and inexpensive means to
substantiate identity and strength of drugs and medicinal products and possibly to detect
poor-quality counterfeit and other substandard products. In the event that suspect
products are detected, these should be tested for compliance against pharmacopoeial
requirements.

4. Some widely used Color Tests

4.1 Chen- Kao Reagent Test

Preparation of Reagent:
 Reagent 1: This is prepared by adding 1 ml of Glacial Acetic Acid in 100 ml
of Water, i.e., 1% (V/V) aqueous Acetic Acid solution.
 Reagent 2: This is prepared by dissolving 1 gm of Copper (II) Sulphate in 100
ml of Water, i.e., 1% (W/V) aqueous CuSO4 solution.
 Reagent 3: This is prepared by dissolving 8 gm of Sodium Hydroxide in 100
ml of Water, i.e., 2N aqueous Sodium Hydroxide solution.

This test is used in to distinguish ephedrine, pseudoephedrine, norephedrine, and

methcathinone from amphetamine and methamphetamine. The latter two do not react
with Chen- Kao reagent.

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 The reagent is prepared in three parts:

Compounds Result in Chen Kao Reagent

Ephedrine Violet
Pseudoephedrine Violet
Norephedrine Bright blue precipitate
Norpseudoephedrine Blue precipitate
Chloropseudoephedrine Bright blue precipitate
N-Methylephedrine Pale blue precipitate
Cathinone Pale blue precipitate
Methcathinone Bright blue precipitate

4.2 Cobalt Thiocyanate Test

This test is a presumptive field test for illegal drugs. The test reagent is a 2% aqueous
solution of Cobalt (II) Thiocyanate which is mixed with a small amount of sample. A
brilliant green- Blue colour is produced by the hydrochloride salts of benzophetamine,
chlordiazepoxide, chlorpromazine, methadone, methylphenidate, and brompheniramine
maleate as well as hydrocodone tartrate. A strong but less green blue colour is produced
by the hydrochloride salts of Cocaine, Diacetylmorphine (Heroin), Ephedrine,
Meperidine, Phencyclidine, Procaine, Propoxyphene and Pseudoephedrine. Alkaloids
present in drugs and other materials are also indicated by the application of this test.

4.3 Dille- Koppayani Test

Preparation of Reagent:

 Part A: This is prepared by dissolving 0.1 g of Cobalt (II) Acetate (dihydrate)

in 100 ml of methanol and subsequently mixed with 0.2 ml of Glacial Acetic
Acid.
 Part B: This is 5% Isopropylamine (V/V) in Methanol.

The Dille–Koppanyi reagent is used as a simple spot-test to presumptively identify

barbiturates. Two drops of Part A are dropped onto the suspected substance followed by
one drop of Part B and any change in colour is observed. The test turns positive for
phenobarbital, pentobarbital, amobarbital and Secobarbital light purple by appearance of
cobalt with the barbiturate nitrogens.

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 4.4 Dragendorff’s Reagent Test
Preparation of Reagent:

1 gm of Bismuth SubNitrate is dissolved in 3 ml. of 10M of Hydrochloric Acid. It is

diluted to 20 ml. 1 gm of Potassium iodide is dissolved in it. If black precipitate of
bismuth tri-iodide separates, it is dissolved in 2M Hydrochloric Acid.

Colour
Red-orange / Brown orange precipitate
Orange Spots
Yellow/ Orange/ Red orange/
Brown orange spot
Orange /Red orange/Brown orange
Compounds
Primary or Secondary or tertiary amine
Alkaloids & benzodiazepines
Amphetamine
Active constituents of Papaver somniferum

4.5 Duquenois Levine Test

The Duquenois Levine reagent was originally developed in the 1930s by Pierre
Duquénois. The reagent is prepared by 5 drops of Acetaldehyde and 0.4 gm. of Vanillin
further dissolved in 20 ml. of 95% Ethanol. This test is recommended for the
identification of Cannabis. When a small amount of suspected residue of the extract is
placed in a test tube and shaken with 2 ml. of a Duquenois Reagent for one minute and
further 2 ml. of concentrated Hydrochloric Acid is added, shaken and allowed to stand for
10 minutes. If a colour develops, 2 ml. of Chloroform is added. The violet colored lower
(Chloroform) layer gives the positive indication of the presence of cannabis.

4.6 Frohde’s Reagent Test

Preparation of reagent:
50 mgs of Molybdic Acid or Sodium Molybdate is dissolved in 10 ml of hot concentrated
Sulphuric Acid. The resulting solution should be colourless.

Appropriate amount of the suspected material or exhibit is taken on a spot plate and
added to it few drop of Frohde’s reagent. The appearance of brown colour indicates the
presence of Mescaline. The conversion of Brown colour to purple indicates the presence
of Methylenedioxyamphetamine (MDA).

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 Amphetamine gives brown yellow color, whereas presence of opium alkaloids is
indicated by violet colour changing to green and finally blue. In case of Ergot alkaloids a
colour change from deep green to red, grey and finally blue is observed.

4.7 Forrest Reagent Test

Preparation of Reagent:

Equal volumes of a 0.2% solution of Potassium Dichromate, 30% (v/v) solution of

Sulphuric Acid, and 20% solution of Perchloric Acid and a 50% solution of Nitric Acid
are mixed.

Phenothiazines gives Red / Violet red / Brown red / Orange / Pink orange / Red Orange /
Brown, whereas, Imipramine and related compounds gives Blue colour.

4.8 Fisher Morris Test

Preparation of Reagents:

 Reagent A: Concentrated Formic Acid.

 Reagent B: 5% aqueous Sodium Nitrite solution.

5 drops of A and 3 drops of B are added to sample. After 2 minutes, 15 drops of

Chloroform are added and colours of layers are observed. Heroin, Diphene, hydramine,
Diazepam can be detected by this test.

4.9 Marquis Test

Preparation of Reagent:

1 volume of Formalin is added with 9 volumes of concentrated Sulphuric Acid.

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MODULE No. 25: Colour Tests for Drug Identification
 Yellow / Orange Benzodiazepines
Brown Orange Amphetamine
purple red colour is produced which Opium Alkaloids
changes to violet and finally blue
Brown Ergot Alkaloids

4.10 Mandelin’s Test

Preparation of Reagent:

1% solution of Ammonium Vanadate is added in concentrated Sulphuric Acid

This test is performed for the detection of Ergot Alkaloids. To a portion of the dried
residue of the extract, one drop of reagent is added. A purple brown colour develops
indicating the presence of ergot alkaloids

4.11 Van- Urk Reagent Test

Preparation of Reagent:

1 gm. of p-amino benzaldehyde is dissolved in 100 ml. ethanol and adding 10 ml. of
Hydrochloric Acid.

To the dried residue of the extract, one drop of Van Urk Reagent is added. A green colour
changing to blue is observed.

4.12 Husemann’s Test

To a little dried extract in a porcelain basin, 2-3 drops of concentrated Sulphuric Acid is
added and heated on a water bath for 30 minutes over a small flame for a few minutes
until white fumes appear. A reddish or reddish-brown or black colour appears. It is
cooled. One drop of concentrated Nitric Acid and a crystal of Potassium Nitrate are
added. A reddish violet colour appears which immediately changes to blood red and then
to reddish yellow and finally fades away indicating the presence of Opium Alkaloids.

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MODULE No. 25: Colour Tests for Drug Identification
 4.13 Zwikker’s Test
The residue of extract is taken up in Chloroform. To 1 ml. of Chloroform extract, 2-3
drops of 0.5 ml. of 5% pyridine in Chloroform is added and shaken. The colour of
Chloroform layer becomes purple. Then 1 drop of Glacial Acetic Acid is added. If the
colour of Chloroform layer changes from purple to weak blue, the presence of non-
Thiobarbiturates is indicated. If Chloroform layer becomes green after adding pyridine in
Chloroform, the presence of Thiobarbiturates is indicated. This green colour changes to
light green on adding Acetic Acid.

4.14 Mecke’s test

The Mecke reagent is used as a simple spot-test to presumptively identify alkaloids as
well as other compounds. It is composed of a mixture of Selenious Acid and concentrated
Sulphuric Acid, which is dripped onto the substance being tested.

4.15 Simon’s Reagent Test

Simon’s reagent is Sodium Nitroprusside in a basic buffer. It works by detecting
‘secondary amines’, such as the ‘meth’ in Methamphetamine and in
methylenedioxymethamphetamine (MDMA). If a substance tests positive for an ecstasy-
like substance by turning to purple/black with Marquis or Mecke, or to blue/black with
Mandelin, then a color-change reaction with Simon’s indicates the presence of MDMA or
MDE.

4.16 Mayer’s Reagent Test

Preparation of Reagent:

Potassium Mercury Iodide prepared by dissolving 1.357 gm. of Mercuric Chloride and 5
gms of Potassium Iodide in 100 ml. of water

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MODULE No. 25: Colour Tests for Drug Identification
 This test is performed for the detection of Nicotine. The dried residue of extract is
acidified with Acetic Acid followed by addition of 2 drops of reagent. A white or
yellowish precipitate is obtained.

4.17 FPN Test

Preparation of Reagent:

5 ml. of Ferric Chloride solution, 45 ml. of 20% (w/v) solution of Perchloric Acid and
50 ml. of 50% (v/v) solution of Nitric Acid is added to make a final solution.

Phenothiazines give Orange red / Violet red / Brown red / Orange / Red orange / pink
orange /Blue/ Violet / Red Brown colour.

4.18 McNally Test

To the residue of extract of biological materials a few drops of Acetone and 1-2 ml. of
water are added. 2 drops of 0.5% Copper Sulphate solution in 10% Acetic Acid is added
followed by a pinch of solid Sodium Nitrite. It is shaken and heated gradually to boiling
and maintained in boiling condition for a few minutes. A red colour is formed if
Salicylates and Salicylic Acid are present.

4.19 Urotropine Test

This test is performed to identify opium alkaloids. To a little dried extract in a porcelain
basin, a few drops of aqueous solution of Urotropine is added and warmed slightly. A
purple colour changing to blue and then green is observed.

4.20 Scott’s Test

Preparation of Reagent:

Reagent is prepared by dissolving 2 gms of Cobalt Thiocyanate in water and diluted with
96% Glycerine in water in 1:1 proportion.

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification
 The residue of the extract is taken in a test tube and reagent is added. The mixture is
shaken and a blue colour develops at once which indicates the presence of cocaine.
Methaqualone also gives positive reaction. If a blue colour develops, one drop of
concentrated Hydrochloric Acid is added. The blue colour disappears and a clear pink
colour appears. If a blue colour does not disappear, one drop of concentrated
Hydrochloric Acid is added. Then a few drops of Chloroform is added and shaken. The
Chloroform layer becomes intense blue.

4.21 Fast Blue- B Test

A small amount of residue of the extract is placed in a test tube and a very small amount
solid Fast blue B reagent (solid Fast blue B: anhydrous Sodium sulphate in 2.5: 100 ratio)
is added. 1 ml. of Chloroform is then added and shaken. It is kept for two minutes. The
Chloroform layer becomes purple red in colour indicating the presence of Cannabis.

4.22 Gerrard’s Test

1-2 ml. of 2% Mercuric Chloride solution in 50% of alcohol is added to a portion of
residue of the extract. A red colour develops immediately. Hyoscyamine produces a
yellow colour which becomes red on burning, while hyocine does not produce any
change of colour.

4.23 Vitali’s Test

A portion of residue of the extract is treated with a few drops of fuming Nitric Acid in a
porcelain basin. It is evaporated to dryness on a water bath. The residue is cooled and
moistened with a few drops of freshly prepared alcoholic Caustic Potash solution when a
violet colour is produced, which soon changes to red and finally disappears. The colour
may be made to reappear by adding more alcoholic Caustic Potash solution. This test is
performed for the detection of Atropine, Datura and ergot alkaloid.

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MODULE No. 25: Colour Tests for Drug Identification
 5. Summarized Test Indications
Substance/functional group
 Alcohols
 Alkaloids and nitrogenous
bases
 Amides (aliphatic)
 Aldehydes (aliphatic)
 Amphetamines
 Antidepressants
 Barbiturates
 Benzodiazepines
 Cannabis
 Carbamates (non-aromatic)
 Cocaine
 Chlorinated phenols
 Chlorinated hydrocarbon
insecticides
 Cyanide
 Cyanide groups
 Dithiocarbamates
 Halogenated hydrocarbons
 Imides
FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse
MODULE No. 25: Colour Tests for Drug Identification
Useful tests/reagents
Potassium Dichromate
Dragendorff’s reagent
Nessler’s reagent
Schiff’s reagent
Marquis test, Mecke test, Frohde’s reagent and
Mandelin’s test Sodium Nitroprusside–Acetone
Marquis test
Dille–Koppanyi reagent Koppanyi–Zwikker
reagent
Mercurous Nitrate
Vanillin reagent
Zwikker reagent
Formaldehyde–sulfuric acid
Duquenois reagent
Furfuraldehyde
Cobalt thiocyanate
p-Dimethylaminobenzaldehyde
Mandelin’s test
Scott’s test
Nitric Acid (fuming)
Nitric–sulfuric acid
Ferrous sulfate (B)
Sodium picrate
Sodium picrate
Sodium Nitroprusside Ergot alkaloids p-
Dimethylaminobenzaldehyde
Fujiwara test
Koppanyi–Zwikker test
  Ketones Sodium Nitroprusside
 Methadone Tetrabromophenolphthalein ethyl ester Cobalt
thiocyanate
Mandelin’s test
Marquis test
 Mono-substituted pyridine Cyanogen bromide
ring
 Nitrates and nitrites Ferrous sulfate
 Opiates Marquis, Mecke, Frohde’s, Mandelin’s tests
 Oxidizing agents Diphenylamine
 Phencyclidine Tetrabromophenolphthalein ethyl ester
 Phenols Ferric chloride Folin–Ciocalteu reagent
Millon’s reagent p-Dimethylaminobenzaldehyde
 Phenothiazines Formaldehyde–sulfuric acid
Forrest reagent
FPN reagent
Ferric chloride
 Phenylpyrazolines Nitrous acid
 Primary aromatic amines Diazotization Coniferyl alcohol
 Primary and secondary Dragendorff’s reagent
amines Simon’s test
 Propoxyphene Cobalt thiocyanate
Frohde’s reagent
Liebermann’s test
Tetrabromophenolphthalein ethyl ester
 Quaternary Ammonium Tetrabromophenolphthalein ethyl ester
compounds
 Quaternary amines Dragendorff’s reagent
 Quinines Thalleioquin Cobalt thiocyanate
 Quinones Methanolic Potassium hydroxide
 Salicylates Ferric chloride
Trinder’s reagent
 Steroids Antimony Pentachloride
Naphthol sulfuric acid
Sulfuric acid

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MODULE No. 25: Colour Tests for Drug Identification
  Sulfonamides
 Sulfur-containing
 Tertiary amines
Copper sulfate
Koppanyi–Zwikker reagent
Mercurous Nitrate
Nitrous acid
Palladium chloride
Sodium Nitroprusside
Dragendorff’s reagent
Tetrabromophenolphthalein ethyl ester

6. Limitations of Colour Tests

Colours exhibited by these tests cannot be described with any accuracy. They may vary
in intensity or tint with the concentration of compounds in the test samples and the
presence of extraneous material. In addition, their assessment is always a subjective one,
even in people with normal colour vision. For example, the Marquis test is stated to give
a black or blue-black colour with MDMA and a green-black with lysergide. Even in the
pure form these colours may not be readily distinguishable; add this to the fact that most
drugs of abuse are more routinely encountered diluted with a variety of other substances,
and one can begin to appreciate the difficulties that can arise in the real world with colour
tests. Some of the complexes formed are unstable, so that the colour changes or fades
with time. Salts may give different colours from those of the corresponding acid or base.
In general, free acids or bases that have been isolated from the test material by an
extraction process give better colours than their salts. The colour of a salt may be
modified by the nature of the other ions present.

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MODULE No. 25: Colour Tests for Drug Identification
 7. Summary

 Colour Tests provides a relatively rapid and inexpensive indication of the presence or
absence of a substance and an indication of the identity of a substance.

 The emphasis for colour tests is on ‘indication’. Such tests do not provide
confirmation of identity.

 Colour tests provide toxicologists and drug analysts with one of the first tools for the
presumptive identification of drugs and poisons.

 They are used to place the unknown into a specific class of compounds or to
eliminate categories or classes of compounds.

 Colour tests are particularly important in clinical toxicology, especially where a

patient is being treated in accident and emergency and clinical symptoms may
indicate some form of poisoning.

 Colour tests can provide an indication of compound class far more rapidly than
immunoassays and chromatographic techniques such as GC and HPLC.

 Colour tests are widely used by police authorities and customs to detect drugs of
abuse.

 Colour tests range from those that rely on reactions with certain functional groups, to
those that are almost specific for a given group.

FORENSIC SCIENCE PAPER No. 9: Drugs of Abuse

MODULE No. 25: Colour Tests for Drug Identification