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Chapter
33
Chapter
Limit Tests
Drugs are aimed to be free from impurities, which may arise from various
sources. Every pharmaceutical substance may contain an impurity, which may
vary in proportion. To control these impurities, Indian Pharmacopoeia has fixed
a limit. To detect the impurity, some tests have been designed, which are known
as limit tests. A limit test is based on quantitative analysis.
DEFINITION Limit tests are quantitative or semiquantitative tests designed to
identify and control small quantities of impurities, which are likely to be present
in the substance.
It involves simple comparison of opalescence, turbidity or colour produced
by the test sample with the standard. The extent of opalescence, colour, etc.,
depends on the quantity of the impurities present.
CHARACTERISTICS OF A LIMIT TEST The factors that have to be considered while
designing a limit test include the following:
1. Specificity: The test must give specific reaction with the impurities present
in the drugs.
2. Sensitivity: The sensitivity of various tests depends on a number of variable
factors that are unlikely to be changed and are favourable towards produc-
ing reproducible results.
Some sources of impurity are listed below:
1. Raw material
2. Material used in manufacturing
3. Solvent, especially water—the cheapest source, and reagents
4. Metal of the plant in which the manufacturing process has been carried out
5. Improper storage
NESSLER’S CYLINDER This is a specially designed cylinder for limit tests. It is made
up of clear, leadfree glass. To test a sample Nessler’s cylinders are used in pairs, one
for sample and other for reference standard. For testing, a pair should be made of
same glass, having same diameter and identical mark of height, i.e. 50 mL.
128
Practical Pharmaceutical Analytical Chemistry
3.1 Limit Test for Acid Radical Impurities
The impurities in the medicinal compounds may come from differ-
ent sources. The most common acid radical impurities are chlorides and
sulphates, which may arise from water, the cheapest solvent used in the
manufacturing process.
Experiment 1 To perform the limit test of chloride ions in the given sample.
Procedure
The two solutions used in the test are prepared as follows:
Test Standard
Dissolve a specified quantity of the sample in
minimum quantity of water or treat as otherwise
directed in the monograph.
Take 1 mL of 0.01 N hydrochloric acid or 1 mL of
0.05845% w/v solution of sodium chloride.
Add 10 mL of dilute nitric acid and dilute it
further to 50 mL with water. Add 1 mL of
5% w/v silver nitrate solution.
Add 10 mL of dilute nitric acid and dilute it
further to 50 mL with water. Add 1 mL of
5% w/v silver nitrate solution.
Stir immediately with a glass rod and set aside
for 5 min.
Stir immediately with a glass rod and set aside for
5 min.
Compare the opalescence of the two solutions after 5 min. The opalescence of
the test solution should not be greater than that of the standard.
Reaction involved
AgNO + Cl AgCl
3
Silver nitrate
Chloride ion
HNO
Silver chlo
3
⎯→⎯⎯⎯⎯ ↓
rride
+NO
3
THEORY This test is based on the precipitation of chloride as silver chloride by
reacting chloride ions with silver nitrate in the presence of nitric acid. The opal-
escence produced is compared with standard opalescence containing a known
amount of chloride ions. The acidity of the solution is kept equivalent to 10 mL
of dilute nitric acid.
Some important modifications are as follows:
1. For the limit test of alkali hydroxides and carbonates, additional acid is
required to neutralize them and to leave the required 10 mL excess.
2. Insoluble substances such as light kaolin and magnesium trisilicate are boiled
with a mixture of water and dilute nitric acid. The solution is filtered, and
the test is applied to the filtrate.
129
Limit Tests
3. Coloured substances are specially treated before the test. For example,
potassium permanganate is decolourized with ethanol, filtered from the
precipitated magnesium dioxide and the test is applied to the filtrate.
Observation
Test Standard Inference
Opalescence Opalescence produced The given sample
1. not produced, or 1. passes the limit test.
2. produced but less than standard or 2. passes the limit test.
3. produced but more than standard. 3. fails the limit test.
Result The given sample…………………the limit test for chloride.
Experiment 2 To perform the limit test of sulphate ions in the given sample.
Procedure
The two solutions used in the test are prepared as follows:
Test Standard
Dissolve a specified quantity of the sample in
minimum quantity of water or treat as otherwise
directed in the monograph.
Take 1.25 mL of 0.01 N sulphuric acid or 1 mL
of 0.1089% w/v solution of potassium sulphate.
Add 2 mL of dilute hydrochloric acid and dilute
it further to 45 mL with water. Add 5 mL of
barium sulphate solution (25% w/v).
Add 2 mL of dilute hydrochloric acid and dilute
it further to 45 mL with water. Add 5 mL of
barium sulphate solution (25% w/v).
Stir immediately with a glass rod and set aside
for 5 min.
Stir immediately with a glass rod and set aside
for 5 min.
Compare the turbidities of the two solutions after 5 min. The turbidity of the test
solution should not be greater than that of the standard.
Reaction involved
BaCl + SO BaSO
24
HCl
4
Barium chloride
Sulphate ion Barium s
⎯→⎯⎯⎯⎯ ↓
uulphate
+2Cl
THEORY This test is based on the precipitation of sulphate as barium sulphate.
Barium sulphate reagent, which contains barium chloride, alcohol and a small
130
Practical Pharmaceutical Analytical Chemistry
amount of potassium sulphate, is used. Alcohol prevents supersaturation and
potassium sulphate increases the sensitivity of the test by providing ions to
the solution. The turbidity produced is compared with the standard turbidity
containing a known amount of sulphate ions. The acidity of the solution is kept
equivalent to 2
mL of dilute hydrochloric acid.
Observation
Test Standard Inference
Turbidity Turbidity produced The given sample
1. not produced, or 1. passes the limit test.
2. produced but less than standard or 2. passes the limit test.
3. produced but more than standard. 3. fails the limit test.
Result The given sample…………………the limit test for sulphate.
3.2 Limit test for Metallic Impurities
The impurities in the medicinal compounds, which may affect the physiological
functions, are of special concern. Arsenic and lead contamination is a common
impurity due to atmospheric pollution.
Experiment 3 To perform the limit test of iron ions in the given sample.
Procedure
The two solutions used in the test are prepared as follows:
Test Standard
Dissolve a specified quantity of the sample in
35 mL of water or treat as otherwise directed
in the monograph.
Take 2 mL of standard iron solution containing a
known amount of 0.04 mg of iron with 40 mL of
water.
Add 2 mL of 20% w/v aqueous citric acid. Add 2 mL of 20% w/v aqueous citric acid.
Further add two drops of thioglycollic acid. Further add two drops of thioglycollic acid.
Make the solution alkaline with iron free
ammonia.
Make the solution alkaline with iron free
ammonia.
Dilute it to 50 mL with water. Dilute it to 50 mL with water.
Stir immediately with a glass rod and set aside for
5 min.
Stir immediately with a glass rod and set aside for
5 min.
131
Limit Tests
Compare the colour of the two solutions after 5 min. The intensity of colour of
the test solution should not be greater than that of the standard.
Reaction involved
T
HEORY This test is based on the formation of purple colour when iron reacts
with thioglycollic acid in a solution buffered with ammonium citrate. The
purple colour is due to the formation of ferrous thioglycollate, a coordina-
tion compound. Thioglycollic acid also causes the reduction of ferric ions to
ferrous ions, if any. Citric acid is added to prevent the precipitation of iron by
ammonia. Ammonia is added to maintain the alkaline pH in which thiogly-
collate appears as a purple colour. The intensity of colour produced is com-
pared with the standard colour containing a known amount of ferrous ions.
Observation
Test Standard Inference
Colour Colour produced The given sample
1. not produced or 1. passes the limit test.
2. produced but less than
standard or
2. passes the limit test.
3. produced but more than
standard.
3. fails the limit test.
Result The given sample…………………the limit test for iron.
Experiment 4 To perform the limit test of lead ions in the given sample.
Procedure
Dilute lead solution: It is a solution of lead nitrate (0.016 g) containing nitric
acid (0.5
mL) per litre. Each millilitre contains the equivalent of 0.01 mg of lead.
The weights of the sample required for the preparation of the primary and
auxiliary solutions may be found by refering to the appropriate appendix of the
British Pharmacopoeia. The two solutions are prepared as follows:
2Fe
+++
2CH
2
–SH-COOH
2Fe
++
HCOO-CH
2
-S-S-CH
2
-COOH
2H
+
Fe
++
2CH
2
SH-COOH
Ferric
Ferrous
Ferrous
Dil. ammonia
Citric acid
Thioglycollic acid
Thioglycollic acid
(Ferrous thioglycolate complex)
CH
2
–SH
CO-O
O-CO
HS-CH
2
e
2H
+
132
Practical Pharmaceutical Analytical Chemistry
Primary solution Auxiliary solution
Dissolve a specified quantity of the sample in
hot water containing the specified volume of
acetic acid or treat as otherwise directed in the
monograph.
Dissolve a specified quantity of the sample in
hot water containing the specified volume of
acetic acid or treat as otherwise directed in the
monograph.
Boil the solution to remove carbon dioxide. Boil the solution to remove carbon dioxide. Add
the specified volume of dilute lead solution.
Further, add ammonia solution PbT and 1 mL of
10% w/v potassium cyanide solution PbT.
Further, add ammonia solution PbT and 1 mL of
10% w/v potassium cyanide solution PbT.
If turbid, filter the solutions and dilute it to 50 mL
with water followed by addition of two drops of
sodium sulphide solution PbT.
If turbid, filter the solutions and dilute it to 50 mL
with water followed by addition of two drops of
sodium sulphide solution PbT.
Stir immediately with a glass rod and set aside for
5 min.
Stir immediately with a glass rod and set aside for
5 min.
Compare the colour of the two solutions after 5 min. The colour of the primary
solution should not be darker than that of the auxiliary solution.
Reaction involved
Pb + Na S PbS + 2Na
Lead
Sodium sulphate
Lead sulphide
2
+
⎯→⎯⎯⎯
THEORY This test is based on the formation of colloidal form of lead
sulphide by reaction of lead salts with sodium sulphide in a slightly alkaline
solution buffered by a fairly high concentration of ammonium acetate. The
brown colour is due to the formation of colloidal lead sulphide, which is
then compared with that obtained from a known amount of lead. Potassium
cyanide is added to prevent the precipitation of iron and copper as sulphides
by converting them to complex cyanides, which are unaffected by sodium
sulphide. The colour of the primary solution should not be darker than that
of the auxiliary solution.
Some important modifications are as follows:
1. For the limit test of acidic substance like acetic acid, citric acid, lactic acid,
nitric acid, etc., there is no need for the addition of acetic acid.
2. Substances like benzoic acid, boric acid, methyluracil, sodium salicylate,
etc., which are insoluble in acetic acid, get dissolved by the addition of
ammonia solution.
133
Limit Tests
3. Sulpha drugs are insoluble in both acetic acid and ammonia. With sodium
hydroxide they form water-soluble sodium salts; hence instead of acetic
acid and ammonia, sodium hydroxide is preferred.
4. In some places, dithizone is used in place of sodium sulphide for the limit
test of lead. The test is based on the reaction between lead and dithizone
(diphenylthiocarbazone) to give lead-dithizone complex.
ead
Dithizone
Lead-dithizone complex
N
C
6
H
5
NC
6
H
5
H
N
S2
Pb
NN
N
NN
NN
H
S
S
C
6
H
5
C
6
H
5
C
6
H
5
C
6
H
5
Observation
Primary Auxiliary Inference
Brown colour Brown colour Produced The given sample
1. not produced, or 1. passes the limit test.
2. produced but less than
auxiliary or
2. passes the limit test.
3. produced but more than
auxiliary.
3. fails the limit test.
Result The given sample…………………the limit test for lead.
Experiment 5 To perform the limit test of arsenic ions in the given sample.
Procedure
The limit test of arsenic is performed in a special type of apparatus. It consists of
a wide-mouthed bottle of 120
mL capacity, fitted with a rubber bung through
which a glass tube passes. The glass tube has a length of 200
mm and an inter-
nal diameter of 6.5
mm. It is tapered at one end, i.e. its one end has a diameter
of 2
mm and the other end has a diameter of about 1 mm. A hole is blown
in the tube’s side with 2
mm diameter. Two rubber bungs with a hole bored
centrally (diameter 6.5
mm) are fitted with a rubber band for holding them
tightly together. Place a mercuric chloride paper flat on the top of the rubber
bung in such a fashion that the borings of two bungs meet to form a true tube
interrupted by the diaphragm of mercuric chloride paper. The assembly should
be such that the entire evolved gas must pass through the paper.
134
Practical Pharmaceutical Analytical Chemistry
Test Standard
Dissolve a specified quantity of the sample in
50 mL of water or treat as otherwise directed in
the monograph.
Take 1 mL of dilute arsenic solution AsT.
Add 10 mL of stannated hydrochloric acid AsT
and transfer to the bottle.
Add 10 mL of stannated hydrochloric acid AsT
and transfer to the bottle.
Add 1 g of potassium iodide AsT and 10 g
of zinc AsT.
Add 1 g of potassium iodide AsT and 10 g of zinc
AsT.
Place the glass tube in position and set aside the
bottle for 40 min.
Place the glass tube in position and set aside the
bottle for 40 min.
Remove the mercuric chloride paper and compare the yellow stain, if any, that
appears on the paper. The colour of the test stain should not be darker than that
of the standard stain.
Reaction involved
As AsO
3+
33
Trivalent
in acidic medium
Arsenic acid
H⎯→⎯⎯⎯⎯⎯⎯⎯
As H AsO
5+
34
Pentavalent
in acidic medium
Arsenic acid
⎯→⎯⎯⎯⎯⎯⎯⎯
H AsO H AsO
34 33
Arsenic acid
SnCl
Arsenious acid
2
⎯→⎯⎯⎯⎯
H AsO 2AsH + 3H O
33
Zn+HCl
32
Arsenious acid
reduction
Arsine
W
⎯→⎯⎯⎯⎯⎯
aater
Mercuric chloride
Hydrochloric acid
Arsine
2AsH
3
HgCl
2
2HCl
Yellow colour
+
+
Hg
AsH
2
AsH
2
THEORY This test is based on the reduction of arsenic compounds to gaseous
arsenious hydride or arsine with zinc and hydrochloric acid. When arsine comes
in contact with mercuric chloride it produces a yellow stain. To perform this
test, the tube is lightly packed with cotton wool previously moistened with lead
acetate to trap any hydrogen sulphide generated, which would interfere in the
test by staining the mercuric chloride paper.
Hydrogen gas is generated by the action of hydrochloric acid on zinc. Stan-
nous chloride ensures a steady evolution of hydrogen by this reaction. It also
helps in reduction of pentavalent arsenic to its trivalent state. Potassium iodide
forms hydroiodic acid, a reducing agent also causing the same effect. Arsenic
135
Limit Tests
gets converted into arsine by reaction with hydrogen gas. This entire reaction
requires 40
min for completion. The reaction is temperature dependent and best
suited temperature is 40ºC.
The test is performed simultaneously in two sets of apparatus and the stain, if
any, produced in the test is compared with that produced by the standard.
Observation
Test Standard Inference
Yellow stain Yellow stain produced The given sample
1. not produced, or 1. passes the limit test.
2. produced but less than standard or 2. passes the limit test.
3. produced but more than standard. 3. fails the limit test.
Result The given sample…………………the limit test for arsenic.
136
Practical Pharmaceutical Analytical Chemistry
Judge yourself
1. Define the following:
(i) Turbidity (ii) Opalescence
2. Give reasons for the use the of following in the limit test of arsenic.
(i) Zinc with hydrochloric acid (ii) Potassium iodide
3. Write the reaction involved in the limit test of the following
(i) Iron (ii) Sulphate (iii) Lead
4. Give reasons for the following:
(i) Nitric acid is added in the limit test of chloride.
(ii) Silver nitrate is added in the limit test of chloride.
(iii) Additional acid is required in the limit test of chloride for alkali
hydroxides.
(iv) Hydrochloric acid is added in the limit test of sulphate.
(v) Potassium sulphate is added in the limit test of sulphate.
(vi) Thioglycollic acid is added in the limit test of iron.
(vii) Citric acid is added in the limit test of iron.
(viii) Ammonia is added in the limit test of iron.