Identification of Active Pharmaceutical Ingredients by Color Test.

SANJIV MENON
Laboratory of Instrumental Analysis
Department of Pharmaceutical Analysis and Medicinal Chemistry
Faculty of Pharmacy, Universitas Padjadjaran.

ABSTRACT
There are certain ways to identify functional groups for alkaloid, nitrogen base compounds,
sulfonamide and barbiturate and antibiotics in drug molecules using chemical reagents or by
color test. There would be specific reaction for functional groups that could be observed by
the formation of color and precipitation. The common methods use for alkaloid
identifications are using wagner test, concentrated acid and acetone water. Whereas for
nitrogen base compounds, it can be tested by using Lieberman test as well as Chen-Kao test
therefore for hexamine which belongs to can be nitrogen determine thru red litmus paper.
Lastly is identification technique for sulfonamide is such as Koppayi-Zwikker test and by
involving vanillin-H2SO4 and for barbiturates, the test is done by using acetone water. This
test is done in order to find out which drug belongs to which group and how it is being
identified. For the identification alkaloid reagents used are papaverine hydrochloride.
Moreover for nitrogen base compounds,ephedrine HCL and hexamine are used as reagents .
For sulfonamide, sulfamethoxazole whereas for barbiturate, luminal and barbital being used
as the reagents. Concentrated sulphuric acid is used in most of test as catalyst and for speed
up the reaction.
Keywords : alkaloid, barbiturate, ephedrine, H2SO4, nitrogen base compound, sulfonamide,
test.

INTRODUCTION
Alkaloids
Alkaloids are a group of diverse biomolecules.They are all secondary compounds and are
derived from amino acids or from the transamination process. From the biological point of
view, the alkaloid is any biologically active and heterocyclic chemical compound which
contains nitrogen and may have some pharmacological activity and, in many cases,
medicinal, or ecological use. They are a special group of chemicals that are active at different
cellular levels of organisms. Alkaloid, any of a class of naturally occurring organic nitrogencontaining bases. Alkaloids have diverse and important physiological effects on humans and
other animals. Well-known alkaloids include morphine, strychnine,quinine, ephedrine,
and nicotine. Alkaloids are derived from L-lysine, L-ornithine, L-tyrosine, L-tryptophan, Lhistidine, L-phenylalanine, nicotinic acid, anthranilic acid, or acetate. The terpenoid, steroid,
and purine alkaloids are also important. Alkaloids also occur in the animal kingdom.
Differently from plants, the source of these molecules in an animal's body can be endogenous
or exogenousThe chemical structures of alkaloids are extremely variable. Generally, an
alkaloid contains at least one nitrogen atom in an amine-type structurei.e., one derived
from ammonia by replacing hydrogen atoms with hydrogen-carbon groups called
hydrocarbons. This or another nitrogen atom can be active as a base in acid-basereactions.
The name alkaloid (alkali-like) was originally applied to the substances because, like the
inorganic alkalis, they react with acids to form salts. Most alkaloids have one or more of their
nitrogen atoms as part of a ring of atoms, frequently called a cyclic system. Alkaloid names
generally end in the suffix -ine, a reference to their chemical classification as amines. In their
pure form most alkaloids are colourless, nonvolatile, crystalline solids. They also tend to have
a bitter taste. A precise definition of the term alkaloid (alkali-like) is somewhat difficult
because there is no clear-cut boundary between alkaloids and naturally occurring complex
amines. Typical alkaloids are derived from plant sources, they are basic, they contain one or
more nitrogen atoms (usually in a heterocyclic ring) and they usually have a marked
physiological action on man or other animals. The name proto-alkaloid or amino-alkaloid
is sometimes applied to compounds such as hordenine, ephedrine and colchicine which lack
one or more of the properties of typical alkaloids. Other alkaloids, not conforming with the
general definition, are those synthetic compounds not found in plants but very closely related
to the natural alkaloids (e.g. homatropine). In practice, those substances present in plants and
giving the standard qualitative tests outlined below are termed alkaloids, and frequently in
plant surveys this evidence alone is used to classify a particular plant as alkaloid-containing.
Barbiturates are a type of depressant drug that cause relaxation and sleepiness. A barbituate
overdose occurs when someone accidentally or intentionally takes more than the normal or
recommended amount of this medication. This is life threatening.At relatively low doses,
barbiturates may cause you to seem like you are drunk or intoxicated.Barbiturates are
addictive, producing physical dependence and a withdrawal syndrome that can be lifethreatening. Whereas tolerance to the mood-altering effects of barbiturates develops rapidly
with repeated use, tolerance to the lethal effects develops more slowly, and the risk of severe
toxicity increases with continued use. They are synthetic drugs derived from barbituric acid ,

which is a synthetic condensation product of malonic acid and urea. They differ mainly in the
substitution pattern at position-5 with some also including an N-methyl at N-1. The most well
known derivative, phenobarbitone , has been used medicinally since 1912, mainly in the
treatment of epilepsy.Over 2,500 barbiturates have reportedly been synthesized with more
than 50 of these presently marketed for clinical use throughout the world. Barbiturates such
as phenobarbital were long used as anxiolytics and hypnotics, but today have been largely
replaced bybenzodiazepines for these purposes because of less potential for lethal overdoses.
[7][8][9] However, barbiturates are still used as anticonvulsants, as para-operative sedatives
(ex. sodium thiopental), and analgesics for cluster headaches/ migraines (ex. fioricet).

Sulphonamides is A drug may be classified by the chemical type of the active ingredient or by
the way it is used to treat a particular condition. Each drug can be classified into one or more
drug classes.Sulphonamides (sulfa drugs) are one of a group of drugs derived from
sulphanilamide that prevents the growth of bacteria.Sulphonamides compete with paminobenzoic acid (PABA) for the enzyme dihydropteroate synthetase, which is important in
the formation of folic acid that is required by the bacteria. Folic acid is required for the
synthesis of precursors of DNA and RNA both in bacteria and in mammals. Mammals obtain
their folic acid in their diet but bacteria need to synthesize it. Sulphonamides inhibit the
growth of bacteria but do not kill them i.e. their action is bacteriostatic.Many sulphonamides
are rapidly excreted and very soluble in urine so they are used to treat infections of the
urinary tract. Sulfanilamide which was the first compound used has H's at R1 and R4. To date
about 15,000 sulfonamide derivatives, analogues, and related compounds have been
synthesized. This has lead to the discovery of many useful drugs which are effective for
diuretics, antimalerial and leprosy agents, and antithyroid agents. The basic structure of
sulfonamide cannot be modified if it is to be an effective competitive "mimic" for paminobenzoic acid. Essential structural features are the benzene ring with two substituents
para to each other; an amino group in the fourth position; and the singly substituted 1sulfonamido group. Sulfonamides, as antimetabolites, compete with para-aminobenzoic acid
(PABA) for incorporation into folic acid. The action of sulfonamides illustrates the principle
of selective toxicity where some difference between mammal cells and bacterial cells is
exploited. All cells require folic acid for growth. Folic acid (as a vitamin is in food) diffuses
or is transported into human body

METHODS
Alkaloids
A.1 Alkaloid that contains quinolone structure
A1.1 Quinine Hydrochloride
I.
II.

III.

IV.
V.

A few milligram of quinine HCL is dissolved in 3 mL of water in a test tube. Add a

few drops of H2SO4. Observe the fluorescence under UV lamp 366 nm.
A few drops of bromine water is added to a saturated solution of quinine HCL, a
yellow precipitate is formed, which gradually disappears with formation of rose-red
coloration, changing to cherry-red. The colour disappear after a time, but can be
reproduced by adding more bromine water, and the reaction is more delicate and
prompt if the quinine solution be previously gently warmed.
Thalleioquin test: a few milligram of quinine HCL is dissolved in 3 mL of water in a
test tube. Add 2-3 drops of bromine water. Agitate the mixture. Add 1-2 drops of
ammonia water (NH3 liquid). A green color of thalleioquim (ammoniacal solution)
indicates positive result.
A few milligram of quinine HCL is dissolved in 3 mL of Hg2Cl2 solution. Observe
the crystal formed under microscope.
Erythroquine reaction: a few milligram is dissolved in 3 mL of water in a test tube.
Add 2-3 drops of bromine water. Agitate the mixture. Add 1-2 drops of potassium
hexacynoferrate and NH4OH.

A.2 Alkaloid that contains Benzylisoquinoline

A2.1 Papaverine Hydrochloride
I.

II.

III.

IV.

A few milligram of papaverine hydrochloride is dissolved in 3 mL of water. Add 2-3

drops of potassium permanganate solution. Add 2-3 drops of Marquis reagent
(formaldehyde in H2SO4 1:9). A green color will be formed and further develops to
blue.
A few milligrams of papaverine hydrochloride is dissolved in 3mL of water. Add 2-3
drops of concentrated H2SO4. Heat the mixture gently. A violet color indicates
positive result.
A few milligrams of papaverine hydrochloride is reacted with 3 mL of acetic
anhydride, add cautiously 3 drops of H2SO4 and heat on a water bath for 3-4
minutes, a yellow colour with a green fluorescence is produced.
A few milligrams of papaverine hydrochloride is dissolved in 3 mL of Hg2Cl2
solution. Observe the crystal formed under microscope.

Nitrogen Base Compound

B.1 Ephedrine
I.
II.

III.

IV.

Liebermann test: A few milligrams of ephedrine Hcl is added with 3 drops of

Liebermann reagent (1g of sodium nitrite in 10 mL of H2SO4)
Chen-Kao test: Dissolved a few milligrams of ephedrine Hcl in 3 mL of water and add
0.1 mL of copper (ii) sulfate. Followed by 2 mL of sodium hydroxide, a violet colour
produced. Add 1 mL of ether and shake, a purple colour is produced in the ethereal
layer and a blur colour in the aqueous layer.
Dissolved a few milligrams of ephedrine Hcl in 3 mL of water. Add a few drops of
sodium hydroxide and 4 mL of potassium ferricyanide TS, and heat. An odour of
benzaldehyde is perceptible.
A few milligrams of ephedrine Hcl is dissolved in 3 mL of Hg2Cl2 solution. Observe
the crystal formed under microscope.

B.2 Hexamine
I.
II.

In a test tube, mix an equivalent weight of hexamine and salicylic acid. Add 1 mL of
H2SO4. Heat the mixture gently.
In a test tube, mix few milligrams of hexamine with dilute H2SO4 amd a drop of
formaldehyde. Place a piece of cotton and a wet red litmus paper on the upper part of
the tube. Check the change of litmus color during reaction.

Sulfonamide
A.1 Sulfamethoxazole
I.
II.
III.

IV.

A few milligrams of sulfamethoxazole is dissolved in 3 mL of dilute Hcl. Add 2-3

drops of p-diaminobenzaldehyde solution.
A few milligrams of sulfamethoxazole is dissolved in 3 mL of NaOH. Add 3 drops of
15% CuSO4 pentahydrate solution.
Koppayi-Zwikker test: A few milligrams of sulfamethoxazole is dissolved in 2 mL of
ethanol. Add 1 drop of Koppayi-Zwikker reagent (1% CO(NO3)2 in ethanol) and 1
drop of pyrolidine.
Add vanillin-H2SO4 to a few milligrams of sulfamethoxazole.

Barbiturate
B.1 Luminal (phenobarbital)
I.

II.
III.

Parri test: 5mg of luminal is dissolved in 3 mL of methanol. Add 0.1 mL of Parri

reagent (contains 1g Co(NO3)2 and 1g of CaCl2 in 10 mL of methanol). Shake the
mixture and add 0.1 mL of NaOH solution. A violet-blue color and precipitate are
formed.
A few milligrams of luminal is put onto an object glass. Add 2-3 drops of a mixture of
acetone-water. Observe the crystals and microscope.
Add a few drops of vanillin reagent [1g of vanillin in 20mL of H2SO4] to luminal,
dark color change to violet.

B.2 Barbital

I.

II.
III.

Parri test: 5 mg of barbital is dissolved in 3 mL of methanol. Add 0.1 mL of parri

reagent (contains 1g Co(NO3)2 and 1 g of CaCl2 in 10 Ml of methanol). Shake the
mixture and add 0.1 mL of NaOH solution. A violet-blue color and precipitate are
formed.
A few milligrams of barbital is put onto an object glass. Add 2-3 drops of a mixture of
acetone-water. Observe the crystals under microscope.
Add a few drops of vanillin reagent [1g of vanillin in 20Ml of H2SO4] to barbital,
dark colour changed to violet.

Antibiotics
A.1 Amoxicillin
I.
II.
III.

A few milligrams of amoxicillin is put on a metal spatula. Burn the sample. Specific
odor is formed.
A few milligrams of amoxicillin is put on a porcelain spot plate. Add 1-2 drops of
concentrate H2SO4. Observe the fluorescence under UV lamp.
A few milligrams of amoxicillin is put onto an object glass. Add 2-3 drops of a
mixture of acetone-water. Observe the crystal under microscope.

A.2 Erythromycin
I.
II.

A few milligrams of erythromycin is put on a porcelain spot plate. Add 1-2 drops of
concentrate H2SO4. Observe the fluorescence under UV lamp.
A few milligrams of erythromycin is put onto an object glass. Add 2-3 drops of a
mixture of acetone-water. Observe the crystals under microsope.

A.3 Chloramphenicol
I.
II.

III.

A few milligrams of chloramphenicol is put on a metal spatula. Burn the sample.

Specific odor is formed.
In a test tube, a few milligrams of chloramphenicol is dissolved in 2 mL of freshly
prepared NaOH solution. Add 1 mL of pyridine. Heat the mixture gently in a
waterbath at 100C for 2 minutes. A positive result is indicated by red color in pyridine
layer.
A few milligrams of chloramphenicol is put onto an object glass. Add 2-3 drops of
mixture of acetone-water. Observe the crystals under microscope.

A.4 Tetracycline
I.
II.

Liebermann test: A few milligrams of tetracycline is added with 3 drops pf

Liebermann reagent (1g of sodium nitrite in 10mL of H2SO4).
Marquis test: Add 2-3 drops of Marquis reagent (formaldehyde in H2SO4 1:9) to a
few milligrams of tetracycline.

Mandelin test: Add 2-3 drops pf mandolin reagent (ammonium vanadate in H2SO4) to a few
milligrams of tetracycline. A violet-red-orange color is changed.

RESULTS
Functional
Group
1.Alkaloid
1.1Quinoline
Structure

Reagents

Procedures

Result

Water+
H2SO4

1. A few milligrams of
quinine HCl is dissolve in
3mL of water in a test
tube.
2. Add a few drop of
H2SO4.
3. Observe the fluorescence
under UV lamp 366nm.

Blue colour solution

being observed under UV
lamp 366nm.

Acetone
water

1. A few milligram of
quinine HCl is dissolved
in 3mL of acetone water.
2. Observe the crystal
formed under the
microscope.

Crystal formed

Potassium
Permanganat
e+
Marquis
reagent
(formaldehy
de in H2SO4
1:9)

1. A few milligrams of
papverin HCl is dissolve
in 3mL of water in a test
tube.
2. Add 1 drop of
formaldehyde and 9 drops
of H2SO4 into the test
tube.

Negative result:
The yellow colour
formed

Quinine
Hydrochlorid
e

1.2Benzyliso
quinoline

Papaverin
Hydrochlorid
e

 Concentrated 1. A few milligrams of

papaverin HCl is dissolve
H2SO4
in 3mL of water in a test
tube.
2. Add 2-3 drops of
concentrated H2SO4
3. Heat the mixture gentally.

Negative result:
The colour did not
change

Acetic
anhydride+
H2SO4

A yellow colour with a

green fluorescene is
produced.

Acetone
water

1. A few milligrams of
papverin HCl is reacted
with 3mL of acetic
anhydride in a test tube.
2. Add cautiously 3 drops of
H2SO4
3. Heat on a water bath for
3-4 minutes

1. A few milligram of papverin

HCl is dissolved in 3mL
of acetone water.
2. Observe the crystal formed
under the microscope.

A cystal formed

2.Nitrogen
base
compound
2.1
Ephedrine

Lieberman
Liberman Test
A yellow colour formed
reagent(1g of 1. A few milligram of
ephedrine HCl is add with
sodium
3 drops of Liberman
nitrite in 10
reagent (1g of sodium
mL of
nitrite in 10 mL of
H2SO4)
H2SO4)

Water+
Copper(II)
Sulphate+
Sodium
Hydroxide

1. Dissolve s few milligrams

of ephedrine HCl in 3mL
of water
2. Add 2 drops of copper(II)
sulphate
3. Add 2 mL of Sodium
Hydroxide

A violet colour formed

Water+
sodium
hydroxide+
potassium
ferricyanide
TS

1. 1.Dissolve s few
milligrams of ephedrine
HCl in 3mL of water..
2. Add 3 drops of sodium
hydroxide.
3. Add 4 mL of potassium
ferricyanide TS
4. Heat the solution

An odour of almond is
produced

 Acetone
water

2.2 Hexamin Salicylic

e
acid
( Hexame
thylene
tetramine
)

1. A few milligram of
ephedrine HCl is
dissolved in 3mL of
acetone water.
2. Observe the crystal
formed under the
microscope.

A Crystal formed

1. Mix an equivalent weight

of hexamine and salicylic
acid in a test tube
2. Add 1mL of H2SO4
3. Heat the solution gentally

Formaldehyd 1. Mix a few milligram of

Negative result :
hexamine with dilute
The colour of red litmus
e
H2SO4 in a test tube
paper did not change
2. Add a drop of
formaldehyde
3. Place a piece of cotton
and a wet red litmus
paper on upper part of test
tube

3Sulfonamid
e

3.1
Sulfamethox
azole

Dilute HCl+
pdiaminobenz
aldehyde

1. A few miligrams of
sulfamethoxazale is
dissolved in 3 mL of
dilute HCl
2. Add 2-3 drops of pdiaminobenzaldehyde
solution

A yellow colour formed

 NaOH+
CuSO4
pentahydrate
solution

1. A few miligrams of
sulfamexazole is dissolve
in 3mL of NaOH.
2. Add 3 drops of 15%
CuSO4 pentahydrate
solution

A green-yellowish colour
formed

VanillinH2SO4

1. Add vanillin-H2SO4 to a
few miligrams of
sulfomexazole

Yellow colour formed

4.Barbiturate

Acetone
water

1. A few milligram of
luminal is put onto an
object glass.
2. Add 2-3 drops of a
mixture of acetone water.
3. Observe crystal under
microscope

A crystal formed

Vanilin
reagent(1g
vanillin in
20mL of
H2SO4)

1. Add a few drops of

vanillin reagent (1g
vanillin in 20mL of
H2SO4) to luminal

Negative result:
A yellow-brownish
colour formed

4.1 Lumi
nal
(phenoba
rbital)

4.2Barbital

5.Antibiotic
5.1
Amoxicilin

Acetone
water

1. A few milligram of
barbital is put onto an
object glass.
2. Add 2-3 drops of a
mixture of acetone water.
3. Observe crystal under
microscope

A crystal formed

Vanilin
reagent(1g
vanillin in
20mL of
H2SO4)

1. Add a few drops of

vanillin reagent (1g
vanillin in 20mL of
H2SO4) to luminal

Negative result:
Yellow colour formed

amoxicilin

1. A few miligrams of
amoxicillin is put on a
metal spatula.
2. Burn the sample

5.2
Erythromyci
n

3. Actone water 1. A few milligram of

erythromycin is put onto
an object glass.
2. Add 2-3 drops of a
mixture of acetone water.
3. Observe crystal under
microscope

A crystal formed

Concentrated 1. A few miligrams of

erythromycin is put on a
H2SO4
porcelain spot plate.
2. Add 1-2 drops of
concentrated H2SO4
3. Observe under UV lamo

A green solution being

observed

5.3
Chloramphe
nicol

Acetone
water

1. A few milligram of
erythromycin is put onto
an object glass.
2. Add 2-3 drops of a
mixture of acetone water.
3. Observe crystal under
microscope

A crystal formed

Chloramphe
nicol

1. A few miligrams of
amoxicillin is put on a
metal spatula.
2. Burn the sample

Bad smell

NaOH+
pyridine

1. In a test tube, a few

miligrams of
chloramphenicol is
dissolved in 2mL of
freshly prepared NaOH
2. Add 1 mL of pyridine.
3. Heat the mixture in a
waterbath at 100C for 2
minutes

A red clour formed at

pyridine layer

Acetone
water

1. A few milligram of
erythromycin is put onto
an object glass.
2. Add 2-3 drops of a
mixture of acetone water.
3. Observe crystal under
microscope

A crystal formed

5.4
Tetracycline

Lieberman
Lieberman test
reagent(1g of 1. A few miligrams of
tetracycline is added with
sodium
3 drops of Liberman
nitrite in
reagent(1g of sodium
10mL of
nitrite in 10mL of
H2SO4)
H2SO4)

Black-chocolate colour
formed

Marquis
reagent(For
maldehyde
in
H2SO41:9)

Marquis Test
1. Add 2-3 drops of Marquis
reagent(formaldehyde in
H2SO4 1:9) to a few
miligrams of tetracycline.

Chocolate-yellow colour
is formed

Mandolin
reagent(amm
onium
vanadate in
H2SO4)

1. Add 2-3 drops of

mandolin reagent
(ammonium vanadate in
H2SO4) to a few
miligrams of tetracycline)

Dark colour near orange

colour is formed

DISCUSSION
The purpose to carry out the experiment is to identify the alkaloid, nitrogen base
compounds, sulphonamide, barbiturate and antibiotic in drug molecule by using chemical
reagent.The principle for this experiment is the functional group (such as alkaloid, nitrogen
base compound, sulphonamide, barbiturate and antibiotic) provides specific chemical
reaction that could be observed by the formation of colour or precipitate in the experiment.
The first experiment of Quinine HCl is observation of Quinine Hydrochloride under UV
lamp.The reagent used water and sulphuric acid. 3mL of water into the test tube to dissolve
the Quinine Hydrochloride.A few drops of the sulphuric acid is added into the test tube.The
sulphuric acid will exchange position with the HCl in the Quinine HCL become Quinine
H2SO4 which will formed the blue fluorescence under the UV lamp. The blue fluorescence
will disappear if adding in a few drop of HCl.
The second experiment is to test the alkaloid with sulphuric acid. The reagent used are water
and concentrated sulphuric acid. A few milligram of the papaverin HCl is dissolved in 3mL of
water.2-3 drops of concentrated H2SO4 is added in the colourless solution. After that the
mixture is heated gentally with Bunsen burner.This is done so the concentrated sulphuric acid
can react with Benzylisoquonine. This experiment also give the negative result that the
colourless solution did not change . this is because of a few reason which the reagent and
sample is contaminated with the impurities or the reagent and the sample is expired.
The third experiment is testing of the papverin HCl with the acetic anhydride and sulphuric
acid. The reagent are acetic anhydride and sulphuric acid. A few miligrams of the papaverin
HCl is reacted with 3ml acetic anhydride, Then 2-3 drops of sulphuric acid is added. The
sulphuric acid act as the catalyst in the experiment. The mixture is heat gentally in water
bath .A yellow colour with green fluorescene was produced. The acetic anhydride is added to
dehydrated the hydrogen ion of papavein HCL.
The fourth experiment is the crystal test. The reagent used are acetone water. The Hg2Cl2
and the acetone water will cause the forming of the crystal of the sample. The crystal of
sample can be seem by microscope.

Next up is the experiment of Ephedrine HCl. Ephedrine HCl is one of the Nitrogen base
compound. The first experiment of the ephedrine HCl is Lieberman test. The reagent for this
test is the Lieberman reagent which contain 1g sodium nitrite in 10mL sulphuric acid. A few
miligrams of ephedrine HCl is added with 3 drop of Lieberman reagent.A yellow colour
precipitate formed.
The second experiment is the Chen-Kao test.The reagents are water, copper(II)sulphate,
sodium Hydroxide. A few miligrams of ephedrine HCl was dissolved in 3mL of water. Then
the copper(II)sulphate which is blue in colour is added in the test tube, followed by 2mL of
sodium hydroxide .There was no reaction between copper sulphate and Hydrochloric acid but
in the presence of sodium hydroxide copper hydroxide formed is precipitated which is bluish
in color and dissolves in hydrochloric acid due to neutralisation of sodium hydroxide.A violet
colour is produced
The third test is to test the odour or smell of benzaldehyde. The reagent for this experiment
are water, sodium hydroxide and potassium ferricyanide TS. A few miligrams of ephendrine
HCl is dissolved in 3mL of water.A few drop of NaOH is added in the text tube. 4 mL of .
potassium ferricyanide TS to react with ependrine to form benzaldehyde which produce the
almond adour. . The purpose to add the NaOH is to neutralize the HCl of ephendrine
The fourth experiment is the crystal test. The reagent is acetone water, supposed the
reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2.
Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause
the forming of the crystal of the sample. The crystal of sample can be seem by microscope.
Hexamine is also one of the nitrogen base compound.The first experiment carry out to test
the reaction between hexamine and salicylic acid.
The second test is to test the basicity of hexamine with the litmus paper. The reagent for this
experiment is dilute sulphuric acid, formaldehyde. Mix a few milligram of hexamine is mixed
with dilute H2SO4 and a drop of formaldehyde.A piece of cotton was placed with a wet red
litmus paper on the upper part of test tube and then the solution was heated. The reaction of
the sample produces NH3 gas which is base that will turns the re litmus paper to blue colour.
.The first experiment pf sulfamethoxazole is to test the reaction of sulfamethoxazole during
acid form. The reagent that used in this experiment is dilute HCl and p-diaminobenzaldehyde.
A few miligrams of sulfamethoxazole was dissolved in 3 mL of dilute HCl.2-3 drops pdiaminobenzaldehyde is adeed in the test tube of The HCl will react with the NH2 in
sulfamethoxazole to form NH3+ which will later react with p-diaminobenzaldehyde to
produce yellow precipitate solution.
The second experiment is to test the reaction of sulfamethoxazole in base condition.The
reagent that used in this experiment is NaOH, 15% CuSO4 pentahydrate. A few miligrams of
sulfamethoxazole is dissolved in 3mL of NaOH. 3 drops of NaOH, 15% CuSO4 pentahydrate
is added in the test tube. The NaOH added to sulfamethaxazole is to react with the NH which

is acid in sulfamethoxazole to form N- ions which will react with 15% CuSO4 pentahydrate
to form the green-yellow precipitate solution.
The third experiment is to test the reaction of vanillin with the sulfamethoxazole. The
reagent used in this experiment is vanillin-H2SO4. The vanillinH2SO4 was added to a few
milligram of sulfamethoxazole. The sulphuric acid will react with NH2 to form NH3+ that
will react with vanillin to form yellow colour solution.

Luminal (phenobarbital) is one chemical that form from barbiturate. The first experiment
that form barbiturate is is the crystal test. The reagent is acetone water The crystal of sample
can be seem by microscope.
The second experiment is vanillin test. The reagent used in this experiment is the vanillin
reagent which contain 1g vanillin in 20mL of sulphuric acid.A few drops of vanillin reagent
is added to luminal. The sulphuric acid will react with NH2 to form NH3+ that will react with
vanillin
The first experiment which is been used for is the crystal test as the parri test was not carried
out. The reagent is acetone water, supposed the reagent is Hg2Cl2,but because the lab
shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is more effective than acetone water.
The Hg2Cl2 and the acetone water will cause the forming of the crystal of the sample. The
crystal of sample can be seem by microscope.
The third experiment is vanillin test. The reagent used in this experiment is the vanillin
reagent which contain 1g vanillin in 20mL of sulphuric acid. A few drops of vanillin reagent
is added to barbital. The H2SO4 will react with NH2 to form NH3+ that will react with
vanillin.

For antibiotics there are four sample was used to test. Firstly is Amoxicilin which is one of
the antibiotic. For the first experiment a few milligram of the amoxicillin is put on a metal
spatula. Then, the sample was burn. A specific odour is produced.
The second experiment is the testing of the amoxicillin under UV Lamp. The reagent that
used in this experiment is concentrated sulphuric acid . A few miligrams of amoxicillin is put
on a porcelain spot plate.1-2 drops of concentrated sulphuric acid is added to the porcelain
spot plate. The blue fluorescene is formed under the UV lamp. This is because the
concentrated H2SO4 replace the NH in the amoxicillin structure.
The third experiment is the crystal test. The reagent is acetone water, supposed the reagent
is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is
more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming
of the crystal of the sample. The crystal of sample can be seem by microscope.

For Erythromycin The first experiment is to test it under UV Lamp.The reagent that used in
this experiment is concentrated sulphuric acid. A few milligrams Erythromycin of is put on a
porcelain spot plate.1-2 drops of concentrated H2SO4 is added to the porcelain spot plate.
The green fluorescene is formed under the UV lamp. This is because the concentrated H2SO4
replace the NH in the amoxicillin structure.
The second experiment is the crystal test. The reagent is acetone water, supposed the
reagent is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2.
Hg2Cl2 is more effective than acetone water. The Hg2Cl2 and the acetone water will cause
the forming of the crystal of the sample. The crystal of sample can be seem by microscope.
Chloramphenicol is one of the antibiotic. The first experiment is to test the odour of the
amoxicillin. A few milligram Chloramphenicol of the is put on a metal spatula. Then, burn
the sample. A specific odour which is a bad spell is produced.
The second experiment is to test the reaction of chloramphenicol in pyridine layer. The
reagent that used in this experiment is NaOH and the pyridine. A few miligrams of
chloramphenicol is dissolved in the 2mL of freshly prepared NaOH. 1mL of the pyridine is
added in the test tube. The mixture is heated in the waterbath at 100C for 2 minutes. The red
colour formed in pyridine layer is obtained. The NaOH added to sulfamethaxazole is to react
with the NH which is acid in sulfamethoxazole to form N- ions which will react with pyridine
layer to formed red colour.
The third experiment is the crystal test. The reagent is acetone water, supposed the reagent
is Hg2Cl2,but because the lab shortage of Hg2Cl2, so we replace the Hg2Cl2. Hg2Cl2 is
more effective than acetone water. The Hg2Cl2 and the acetone water will cause the forming
of the crystal of the sample. The crystal of sample can be seem by microscope.
Tetracycline is also one of the antibiotic.The first experiment is Lieberman test.The reagent
is the Lieberman reagent which contain 1g sodium nitrite in 10mL H2SO4. A few miligrams
of tetracycline is add with 3 drop of Lieberman reagent. The dark-brown colour precipitate
formed.
The second test is Marquis test. The reagent of this experiment have marquis reagent which
contain formaldehyde in H2SO4 with ratio of 1:9.The experiment start with 2-3 drops of
marquis reagent is added to a few milligrams of Tetracycline. A brown-yellow precipitate is
formed.
The third test in this experiment is mandolin test. The reagent that used in this experiment
is mandolin reagent which contain ammonium vanadate in sulphuric acid to a few miligrams
of tetracycline.A dark colour near orange colour form because the ammonium vanadate react
with th NH3+ ion that formed from the reaction of H2SO4 and NH2.

CONCLUSION

By using chemical reagents the identification of alkaloid nitrogen base compound

sulphonamide barbiturate and antibiotics can be done

REFERENCES
1.Alkaloids - Secrets of Life.Tadeusz Aniszewski.Available online 1 October 2007. Alkaloid
Chemistry, Biological Significance, Applications and Ecological Role2007, Pages 159
2.http://chemwiki.ucdavis.edu/Biological_Chemistry/Drug_Activity/Sulfa_Drugs
3. Laboratory and Scientific Section United nationS office on drUgS and crime Vienna
Recommended Methods for the Identification and Analysis of Barbiturates and
Benzodiazepines under International Control