Limit test is defined as quantitative or semi quantitative test designed to

identify and control small quantities of impurity which is likely to be present in

the substance.

Importance of Limit test

•To trace out the harmful amount of impurities during preparation of drugs.
•To find out the avoidable or unavoidable amount of impurities during
formulation of drugs.
•To identify the impurities present in the substance and compare it with
standard to limit an error.

A. Test for Chloride:

Principle: The limit test for chlorine has been based on simple reaction
between silver nitrate and soluble chlorides to obtain silver chloride which is
insoluble on dilute nitric acid. The silver chloride produced in present of dilute
nitric acid makes test solution turbid. When turbidity from sample has less than
standard turbidity then sample will pass limit test and vice versa.
HNO3
NaCl + AgNO3 → AgCl + NaNO3
Standard solution: Take 1ml of N/100 NaCl in Nessler cylinder & add 1 ml
of HNO3 and make up to 50ml with water with addition of 1ml of AgNO3 then
Stir and allow to stands for 5 minutes.

Test solution: Dissolve specified quantity of the substance in water or

prepare solution specified in pharmacopeia and transfer to Nessler cylinder.
Add 1ml of dil. HNO3 and make solution up to 50ml with addition of water. Add
1ml of N/10 AgNO3 then stir and allow stand for 5 minutes. Compare the
opalescence with the standard.

B. Test for Sulfate:

Principle: Limit test of sulphate is based on the reaction of soluble sulphate
with barium chloride in presence of dilute hydrochloric acid to form barium
sulphate. The barium chloride test solution on I.P. is replaced by barium
sulphate Reagent which is composed of barium chloride, sulphate free alcohol
and solution of potassium sulphate.

HCl
K 2 SO4 + BaCl2 → BaSO4 + KCl
Standard solution: Take 1ml of N/100 K 2 SO4 and 2ml dilute HCl in a
Nessler cylinder then dilute to 45ml with distilled water and add 5ml of BaCl2
reagent. Stir and allow to stands for 5 minutes.

Test with substance: Dissolve specified quantity of the substance in water

or prepare solution specified in pharmacopeia and transfer to a Nessler cylinder
and add 2ml of dil HCl and dilute to 45 ml with distilled water with addition of
5ml of the BaCl2 reagent. Stir and allow to stands for 5 minutes. Compare the
turbidity with the standard.

C. Test for Iron:

Principle: Limit test of Iron is based on reaction of iron in ammonical solution
with thioglycollic acid in presence of citric acid to form iron thioglycolate which
is pale pink to deep reddish purple in color. Citric acid does not allow to forms
precipitation of iron by ammonia by forming of complex with it.

Initially thioglycollic acid is formed by a reaction between Potassium hydrogen

sulphide (KSH) and Monochloroacetic acid (CH2 Cl.COOH).
KSH + CH2 Cl.COOH KCl + HS.CH2COOH (Thioglycollic acid)
Citric Acid
Thioglycollic acid + Fe2+ → Ferrous Thioglycolate

Standard solution: Take 2ml of 20% w/v citric acid and add on 2ml of
iron solution. Then add 2 drops of thioglycolic acid and make the solution
alkaline with ammonia solution then make up to 50 ml with distilled water.

Test solution: Dissolve specified quantity of the substance in water or

prepare solution specified in pharmacopeia and transfer to a Nessler cylinder.
Then proceeds the same procedure as on standard solution preparation.
Compare the two solutions.

D. Test for Lead:

Preparation of Thioacetamide Reagent: Take 15ml NaOH add 5ml
water and 20ml glycerol. Add 0.2 ml of 4% Thioacetamide solution
(CH3 CSNH2 ) to above solution and heat for 20min in a water bath and cool.

Standard Solution: Take 10ml of Pb solution and add 2ml acetate buffer
(PH 3.5) with addition of 1.2ml of Thioacetamide reagent and make up to 50ml
with distilled water then allow to stands for 5minutes.
Test Solution: Dissolve specified quantity of the substance in water or
prepare solution specified in pharmacopeia and transfer to a Nessler cylinder.
Then proceeds the same procedure as on standard solution preparation.
Compare the two solutions.

E. Test for Arsenic:

Principle: Limit test of Arsenic is based on reaction on which Arsenic acid in
present of stannous chloride change to Arsenious acid and finally nasent
hydrogen reduce arsenious acid to arsine. Arsine reacts with mercuric chloride
paper giving yellow stain. It is also called as Gutzeit test.

SnCl2
H3 AsO4 (Arsenic acid) → H3 AsO3 (Arsenious Acid)
H3 AsO3 + 3H2 → AsH3 (Arsine) + 3H2 O

Procedure: The glass tube is first of all tightly packed with cotton wool which
is moistened with solution of lead acetate and dried. The upper end of tube has
been then inserted into narrow end of one of the bugs and two bugs are secured
by using rubber band after keeping mercuric chloride paper on between them as
like as sandwich.
The test solution is prepared by dissolving specific amount in water and
stannated HCl (arsenic free) and kept in wide mouthed bottle. To this solution
1 gm of KI, 5 ml of stannous chloride acid solution and 10 gm of zinc is added.
The solution is keep around for 40 minute and stain obtained on mercuric
chloride paper is compared with standard solution.