Asian J. Pharm. Res. 2017; Vol. 7: Issue 1 [AJPRes.

ISSN- 2231–5683 (Print) www.asianpharmaonline.org

ISSN- 2231–5691 (Online) www.asianjpr.com

REVIEW ARTICLE

Pharmaceutical Importance and Significance of Limit Tests

Habeeb Ahmed1*, Mohammed Ehtesham1, Nuha Rasheed2 , Abdul Saleem Mohammad3*
1
Department of Pharma. D, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally (M), Behind Mount
Opera, Yadadri (Dist)-508284, Telangana, India.
2
Department of Pharmaceutics, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally (M), Behind Mount
Opera, Yadadri (Dist)-508284, Telangana, India.
3
Department of Pharmaceutical Analysis and Quality Assurance, Nizam Institute of Pharmacy, Deshmukhi (V),
Pochampally (M), Behind Mount Opera, Yadadri (Dist)-508284, Telangana, India.
*Corresponding Author E-mail: [email protected]

ABSTRACT:
The present subject is on the review of various limit test and titrations which are commonly conducted in the
Chemistry lab. Limit tests are a category of impurity tests in which a solution of an unknown sample is
compared to a standard that contains the impurity at the product limit. Generally this definition applies to limit
tests that are in widespread use in various pharmacopoeial compendia, such as United States Pharmacopoeia
(USP), European Pharmacopoeia (EP), Japanese Pharmacopoeia (JP) and Food Chemicals Codex (FCC). These
tests are used to make a simple pass/fail evaluation of the sample. Typically this category of test relies on the
comparison of an unknown sample to a standard solution. Often these tests rely on a visual evaluation by the
technician, though some may rely on instrumental analysis of the sample.

KEY WORDS: Limit tests, Arsenic, end point, titration, equivalent point, and indicator

INTRODUCTION: which appears as solid particles (Opalescence) in the

In Chemistry, Limit means a value or amount that is solution. Limit test of sulphate is based on the reaction
likely to be present in a substance and test means to of soluble sulphate with barium chloride in presence of
examine or to investigate. Thus, limit test is nothing but alcohol and potassium sulphate to form barium sulphate,
to identify the impurities in the substance and compare it which appears as solid particles (turbidity) in the
with standard. In general, limit test is defined as solution. Here alcohol is added to prevent super
quantitative or semi quantitative test designed to identify saturation. Limit test of heavy metals is based on the
and control small quantities of impurity which is likely reaction of metallic impurities with hydrogen sulfide in
to be present in the substance. Limit test is generally acidic medium to form colored solution. Metals that
carried out to determine the inorganic impurities present response to this test are lead, mercury, bismuth, arsenic,
in compound. Limit test of chloride is based on the antimony, tin, cadmium, silver, copper, and
reaction of soluble chloride with silver nitrate in molybdenum. Limit test of lead is based on the reaction
presence of dilute nitric acid to form silver chloride, of lead and diphenyl thiocabazone (dithizone) in alkaline
solution to form lead dithizone complex which is read in
color. Limit test of Iron is based on the reaction of iron
Received on 08.12.2016 Accepted on 08.01.2017 in ammonical solution with thioglycollic acid to form
© Asian Pharma Press All Right Reserved iron thioglycolate which is pink-reddish purple in color.
Asian J. Pharm. Res. 2017; 7(1): 30-34. Limit test of Arsenic is based on the reaction of arsenic
DOI: 10.5958/2231-5691.2017.00006.5

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Asian J. Pharm. Res. 2017; Vol. 7: Issue 1 [AJPRes.]

gas with hydrogen ion to form yellow stain on mercuric Procedure:

chloride paper in presence of reducing agents like TABLE :2
potassium iodide. It is also called as Gutzeit test and Test sample Standard compound
requires special apparatus. [1-3] Specific weight of compound is Take 1ml of 0.1089 % W/V
dissolved in water or solution is solution of potassium sulphate in
prepared as directed in the Nessler cylinder
pharmacopoeia and transferred in
VARIOUS LIMIT TEST Nessler cylinder
Limit test for Chlorides Add 2ml of dilute hydrochloric acid Add 2ml of dilute hydrochloric
acid
Principle: Dilute to 45 ml in Nessler cylinder Dilute to 45 ml in Nessler
Limit test of chloride is based on the reaction of soluble cylinder
chloride with silver nitrate in presence of dilute nitric Add 5ml of barium sulphate reagentAdd 5ml of barium sulphate
acid to form silver chloride, which appears as solid reagent
particles (Opalescence) in the solution. Keep aside for 5 min Keep aside for 5 min
Observe the Turbidity Observe the Turbidity

Barium sulphate reagent contains barium chloride,

sulphate free alcohol and small amount of potassium
sulphate.
Procedure:
TABLE :1 Observation:
Test sample Standard compound The turbidity produce in sample solution should not be
Specific weight of compound is Take 1ml of 0.05845 % W/V
dissolved in water or solution is solution of sodium chloride in
greater than standard solution. If turbidity produces in
prepared as directed in the Nessler cylinder sample solution is less than the standard solution, the
pharmacopoeia and transferred in sample will pass the limit test of sulphate and vice versa.
Nessler cylinder
Add 1ml of nitric acid Add 1ml of nitric acid
Reasons:
Dilute to 50ml in Nessler cylinder Dilute to 50ml in Nessler cylinder
Add 1ml of AgNO3 solution Add 1ml of AgNO3 solution
Hydrochloric acid helps to make solution acidic.
Keep aside for 5 min Keep aside for 5 min Potassium sulphate is used to increase the sensitivity of
Observe the Opalescence/Turbidity Observe the the test by giving ionic concentration in the reagent
Opalescence/Turbidity Alcohol helps to prevent super saturation. [5-7]
Observation: Limit test for Iron
The opalescence produce in sample solution should not Principle:
be greater than standard solution. If opalescence Limit test of Iron is based on the reaction of iron in
produces in sample solution is less than the standard ammonical solution with thioglycollic acid in presence
solution, the sample will pass the limit test of chloride of citric acid to form iron thioglycolate which is pale
and visa versa. pink to deep reddish purple in color.

Reasons:
Nitric acid is added in the limit test of chloride to make
solution acidic and helps silver chloride precipitate to
make solution turbid at the end of process.[3-5]
Procedure:
Limit test for Sulphates TABLE :3
Principle: Test sample Standard compound
Limit test of sulphate is based on the reaction of soluble Sample is dissolved in specific
amount of water and then volume is
2 ml of standard solution of
iron diluted with water upto
sulphate with barium chloride in presence of dilute made up to 40 ml 40ml
hydrochloric acid to form barium sulphate which appears Add 2 ml of 20 % w/v of citric acid Add 2 ml of 20 % w/v of
as solid particles (turbidity) in the solution. (iron free) citric acid (iron free)
Add 2 drops of thioglycollic acid Add 2 drops of thioglycollic
acid
Add ammonia to make the solution Add ammonia to make the
alkaline and adjust the volume to solution alkaline and adjust
50 ml the volume to 50 ml
Keep aside for 5 min Keep aside for 5 min
Color developed is viewed Color developed is viewed
vertically and compared with vertically and compared
standard solution with standard solution
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Asian J. Pharm. Res. 2017; Vol. 7: Issue 1 [AJPRes.]

Earlier aamoniumthiocyanate reagent was used for the sample will pass the limit test of heavy metals and vice
limit test of iron. Since thioglycolic acid is more versa. [7-9]
sensitive reagent, it has replaced ammonium thiocyanate
in the test. Method II: Use for the substance which do not give clear
colorless solution under the specific condition.
Observation:
The purple color produce in sample solution should not TABLE :5
be greater than standard solution. If purple color Test sample Standard compound
produces in sample solution is less than the standard Weigh specific quantity of test Take 2 ml of standard lead
substance, moisten with sulphuric acid solution and dilute to 25 ml
solution, the sample will pass the limit test of iron and and ignite on a low flame till with water
vice versa. completely charred
Add few drops of nitric acid and heat
to 500 °C
Reasons: Allow to cool and add 4 ml of
Citric acid helps precipitation of iron by ammonia by hydrochloric acid and evaporate to
forming a complex with it. dryness
Moisten the residue with 10 ml of
hydrochloric acid and digest for two
Thioglycolic acid helps to oxidize iron (II) to iron (III).
minutes
Ammonia to make solution alkaline. [7-9] Neutralize with ammonia solution and
make just acid with acetic acid
Limit test for Heavy Metals Adjust the pH between 3 to 4 and filter Adjust the pH between 3 to 4
Principle: if necessary by adding dilute acetic acid
‘Sp’ or dilute ammonia
Limit test of heavy metals is based on the reaction of solution ‘Sp’
metallic impurities with hydrogen sulfide in acidic Dilute with water to 35 ml Dilute with water to 35 ml
medium to form brownish colour solution. Metals that Add freshly prepared 10 ml of Add freshly prepared 10 ml of
response to this test are lead, mercury, bismuth, arsenic, hydrogen sulphide solution hydrogen sulphide solution
antimony, tin, cadmium, silver, copper, and Dilute with water to 50 ml Dilute with water to 50 ml
Allow to stand for five minutes Allow to stand for five minutes
molybdenum. The metallic impurities in substances are
View downwards over a white surface View downwards over a white
expressed as parts of lead per million parts of the surface
substance. The usual limit as per Indian Pharmacopoeia
is 20 ppm Observation:
The color produce in sample solution should not be
Procedure: greater than standard solution. If color produces in
The Indian Pharmacopoeia has adopted three methods sample solution is less than the standard solution, the
for the limit test of heavy metals. sample will pass the limit test of heavy metals and vice
versa.
Method I: Use for the substance which gives clear
colorless solution under the specific condition. Method III: Use for the substance which gives clear
colorless solution in sodium hydroxide solution.
TABLE :4
Test sample Standard compound TABLE :6
Solution is prepared as per the Take 2 ml of standard lead Test sample Standard compound
monograph and 25 ml is transferred solution and dilute to 25 ml with Solution is prepared as per the Take 2 ml of standard lead solution
in Nessler’s cylinder water monograph and 25 ml is
Adjust the pH between 3 to 4 by Adjust the pH between 3 to 4 by transferred in Nessler’s cylinder or
adding dilute acetic acid ‘Sp’ or adding dilute acetic acid ‘Sp’ or weigh specific amount of
dilute ammonia solution ‘Sp’ dilute ammonia solution ‘Sp’ substance and dissolve in 20 ml of
Dilute with water to 35 ml Dilute with water to 35 ml water and add 5 ml of dilute
Add freshly prepared 10 ml of Add freshly prepared 10 ml of sodium hydroxide solution
hydrogen sulphide solution hydrogen sulphide solution Make up the volume to 50 ml with Add 5 ml of dilute sodium
Dilute with water to 50 ml Dilute with water to 50 ml water hydroxide solution and make up the
Allow to stand for five minutes Allow to stand for five minutes volume to 50 ml with water
View downwards over a white View downwards over a white Add 5 drops of sodium sulphide Add 5 drops of sodium sulphide
surface surface solution solution
Mix and set aside for 5 min Mix and set aside for 5 min
Observation: View downwards over a white View downwards over a white
The color produce in sample solution should not be surface surface
greater than standard solution. If color produces in
sample solution is less than the standard solution, the
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Asian J. Pharm. Res. 2017; Vol. 7: Issue 1 [AJPRes.]

Observation: interference and influence of other impurities have been

The color produce in sample solution should not be eliminated.
greater than standard solution. If color produces in
sample solution is less than the standard solution, the Phenol red is used as indicator to develop the color at the
sample will pass the limit test of heavy metals and vice end of process.
versa.[9-10]
Lead present as an impurities in the substance, gets
Limit test for Lead separated bye extracting an alkaline solution with a
Lead is a most undesirable impurity in medical dithizone extraction solution.
compounds and comes through use of sulphuric acid,
lead lined apparatus and glass bottles use for storage ofLimit test for Arsenic
chemicals. Principle:
Limit test of Arsenic is based on the reaction of arsenic
Principle: gas with hydrogen ion to form yellow stain on mercuric
Limit test of lead is based on the reaction of lead and chloride paper in presence of reducing agents like
diphenylthiocabazone (dithizone) in alkaline solution to potassium iodide. It is also called as Gutzeit test and
form lead dithizone complex which is read in color. requires special apparatus.

Dithizone is green in color in chloroform and lead- Arsenic, present as arsenic acid in the sample is reduced
dithizone complex is violet in color, so the resulting to arsenious acid by reducing agents like potassium
color at the end of process is red. iodide, stannous acid, zinc, hydrochloric acid, etc.
Arsenious acid is further reduced to arsine (gas) by
Procedure: hydrogen and reacts with mercuric chloride paper to give
TABLE :6 a yellow stain.
Test sample Standard compound
A known quantity of sample A standard lead solution is prepared
solution is transferred in a equivalent to the amount of lead
separating funnel permitted in the sample under H3AsO4 + H2SnO2 → H3AsO3 + H2SnO3
examination Arsenic acid Arsenious acid
Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide Add 2 ml of potassium cyanide and H3AsO3 + 3H2 → AsH3 +3H2O
and 2 ml of hydroxylamine 2 ml of hydroxylamine
hydrochloride hydrochloride
Arsenious acid Arsine
Add 2 drops of phenol red Add 2 drops of phenol red
Make solution alkaline by adding Make solution alkaline by adding The depth of yellow stain on mercuric chloride paper
ammonia solution. ammonia solution. will depend upon the quality of arsenic present in the
Extract with 5 ml of dithizone until Extract with 5 ml of dithizone until sample.
it becomes green it becomes green
Combine dithizone extracts are Combine dithizone extracts are
shaken for 30 mins with 30 ml of shaken for 30 mins with 30 ml of Procedure:
nitric acid and the chloroform nitric acid and the chloroform layer Test solution:
layer is discarded is discarded The test solution is prepared by dissolving specific
To the acid solution add 5 ml of To the acid solution add 5 ml of amount in water and stannated HCl (arsenic free) and
standard dithizone solution standard dithizone solution
Add 4 ml of ammonium cyanide Add 4 ml of ammonium cyanide
kept in a wide mouthed bottle.
Shake for 30 mins Shake for 30 mins
Observe the color Observe the color To this solution 1 gm of KI, 5 ml of stannous chloride
acid solution and 10 gm of zinc is added (all this
Observation: reagents must be arsenic free).
The intensity of the color of complex, is depends on the
amount of lead in the solution. The color produce in Keep the solution aside for 40 min and stain obtained on
sample solution should not be greater than standard mercuric chloride paper is compared with standard
solution. If color produces in sample solution is less than solution.
the standard solution, the sample will pass the limit test
of lead and vice versa. Standard solution:
A known quantity of dilute arsenic solution is kept in
Reasons: wide mouthed bottle and rest procedure is followed as
Ammonium citrate, potassium cyanide, hydroxylamine described in test solution (Gutzeit apparatus shown in
hydrochloride is used to make pH optimum so Fig.1).

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Asian J. Pharm. Res. 2017; Vol. 7: Issue 1 [AJPRes.]

Fig.1 Gutzeit apparatus.

A ： approximately 60 ml generator bottle with 40 ml indicating line.

B ： glass tube with 6.5 mm inner diameter
C and D ： a ground joint glass tube with 6.5 mm inner diameter and
18 mm outer diameter at the joint. Inner joint and the outer joint form a
concentric circle.
E ： rubber stopper
F ： narrow part of the glass tube B. Glass wool is inserted up to this
part.
G ： rubber board (Lead acetate cotton plug)
H ： clamp

Reasons:
Stannous chloride is used for complete evolution of
arsine Zinc, potassium iodide and stannous chloride is
used as a reducing agent Hydrochloric acid is used to
make the solution acidic Lead acetate pledger or papers
are used to trap any hydrogen sulphide which may be
evolved along with arsine.[2-5]

REFFERENCE
1. A.H. Beckett and J.B. Stenlake, Practical pharmaceutical
chemistry, Part-I. The Athtone press, University of London,
London.
2. P. Gundu Rao, Inorganic Pharmaceutical Chemistry; Vallabh
Prakashan, Delhi.
3. Advanced Inorganic Chemistry by Satya Prakash, G.D. Tuli
4. Jolly-Modern Inorganic Chemistry
5. Pharmaceutical Inorganic Chemistry Textbook by Alagarsamy.
6. L.M. Atherden, Bentley and Driver’s Textbook of Pharmaceutical
Chemistry Oxford University Press, London.
7. Indian Pharmacopoeia 1996, 2006.
8. J.H Block, E. Roche, T.O Soine and C.O. Wilson, Inorganic
Medical and Pharmaceutical Chemistry Lea & Febiger
Philadelphia PA.
9. Pharmaceutical Inorganic Chemistry by S. Chand, R.D. Madan,
Anita Madan
10. Pharmaceutical Inorganic Chemistry by Soma Shekar Rao

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