Pharmaceutical Inorganic chemistry

UNIT-I (Part-III)
Limit Test

Presented By
Prof. (Dr.) Aditi Kaushik
Laureate Institute of Pharmacy
Kangra, H.P
❑ Limit tests:-
Definition: Limit tests are quantitative or semi quantitative test designed to
identify and control small quantities of impurities which are likely to be
present in the substances.
• Tests being used to identify the impurity.
• Tests being used to control the impurity.
 ❖ Factors affecting limit tests:
• Specificity of the tests

• Sensitivity

• Control of personal errors (Analyst errors)

a) Test in which there is no visible reaction

b) Comparison methods

c) Quantitative determination
❖ Limit test for Chloride:
• The test is used to limit the amount of Chloride as an impurity in inorganic
substances.

Principle:

• Limit test of chloride is based on the reaction of soluble chloride with silver nitrate
in presence of dilute nitric acid to form silver chloride, which appears as solid
particles (Opalescence) in the solution.

Cl- + AgNO3 --------------------------AgCl + NO3-

Soluble chloride

present as impurity
• The silver chloride produced in the presence of dilute Nitric acid makes
the test solution turbid, the extent of turbidity depending upon the amount
of Chloride present in the substance is compared with the standard
opalescence produced by the addition of Silver nitrate to a standard
solution having a known amount of chloride and the same volume of
dilute nitric acid as used in the test solution.
 Test sample Standard compound

Specific weight of compound Take 1 ml of 0.05845 % W/V

is dissolved in water or solution is prepared as solution of sodium chloride in Nessler cylinder
directed in the pharmacopoeia and transferred in
Nessler cylinder
Add 10 ml of nitric acid Add 10 ml of nitric acid

Dilute to 50 ml with Distilled water in Nessler Dilute to 50 ml with Distilled water in Nessler
cylinder with cylinder with

Add 1 ml of AgNO3 solution Add 1 ml of AgNO3 solution

Keep aside for 5 min Keep aside for 5 min

Observe the Opalescence/Turbidity Observe the Opalescence/Turbidity

• Observation: The opalescence produce in sample solution should not be
greater than standard solution. If opalescence produces in sample solution
is less than the standard solution, the sample will pass the limit test of
chloride and visa versa.

• 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 as Dilute HNO3 is insoluble in AgCl.
• Pharmacopoeia does not prescribe any numerical value of limit test for
chlorides, sulphate and iron because limit test is based on the simple
comparison of opalescence or colour between the test and standard solution
prescribed according to pharmacopoeia.

• In this type of limit test, the extent of turbidity or opalescence or colour

produced in influenced by the presence of other impurities present in the
substance and also by variation in time and method of performance of test.

• Thus the pharmacopoeia does not prescribe any numerical value of the limit
test.
• The limit test involve simple comparisons of opalescence, turbidity, or colour
with standard.

• These are semi-qualitative reactions in which extent of impurities present can

be estimated by comparing visible reaction response of the test and standard.

• By this way, extent of reaction is readily determined by direct comparison of

test solution with standard. So pharmacopoeia prefers comparison methods.
❖ Limit test for sulphate:
• The Sulfate Limit Test is designed to determine the allowable limit of sulfate contained in a sample.

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 which appears as solid particles (turbidity) in the
solution.

• Then comparison of turbidity is done with a standard turbidity obtained from a known amount of Sulphate
and same volume of dilute Hydrochloric acid have been added to both solutions.

• The barium chloride test solution in the IP has been replaced by Barium sulphate reagent which is having
barium chloride, sulphate free alcohol (Avoid supersaturation) and a solution of potassium sulphate.
Potassium sulphate has been added to increase the sensitivity of the test.
 Test sample Standard compound

Specific weight of compound Take 1 ml of 0.1089 % W/V solution of potassium

is dissolved in water or solution is prepared as sulphate in Nessler cylinder
directed in the pharmacopoeia and transferred in
Nessler cylinder
Add 2 ml of dilute hydrochloric acid Add 2 ml of dilute hydrochloric acid

Dilute to 45 ml with Distilled water in Nessler Dilute to 50 ml with Distilled water in Nessler
cylinder. cylinder.

Add 5 ml of barium sulphate reagent Add 5 ml of barium sulphate reagent

Keep aside for 5 min Keep aside for 5 min

Observe the Opalescence/Turbidity Observe the Opalescence/Turbidity

• Barium sulphate reagent contains barium chloride, sulphate free alcohol and small amount of potassium
sulphate.

• Observation:

• The turbidity produce in sample solution should not be greater than standard solution. If turbidity
produces in sample solution is less than the standard solution, the sample will pass the limit test of
sulphate and vice versa.

• Reasons:

• Hydrochloric acid helps to make solution acidic.

• Potassium sulphate is used to increase the sensitivity of the test by giving ionic

• concentration in the reagent.

• Alcohol helps to prevent super saturation and so produces a more uniform opalescence.
❖ Limit test for Arsenic:
• Arsenic is a well known undesirable and harmful impurity which is
present in medicinal substances.

• All pharmacopoeias prescribe a limit test for it.

• Pharmacopoeial method is based on the Gutzeit test.

• All the special reagents used in the limit test for Arsenic are marked and
distinguished by letter ‘As T’, which means that they all should be
Arsenic free and should themselves conform to the test for Arsenic.
Principle:
• Limit test of Arsenic is based on the reaction of arsenic gas with hydrogen ion to form
yellow stain on mercuric chloride paper in presence of reducing agents like potassium
iodide. It is also called as Gutzeit test and requires special apparatus.
• Arsenic, prese3nt as arsenic acid (H3AsO4) in the sample is reduced to arsenious acid
(H3AsO3) by reducing agents like potassium iodide, stannous acid, zinc, hydrochloric
acid, etc. Arsenious acid is further reduced to arsine (gas) (AsH3) by hydrogen and reacts
with mercuric chloride paper to give a yellow stain.
Substance + dil HCl -------------------------H3AsO4
(contains Arsenic impurity) Arsenic acid
• H3AsO4 + H2SnO2 -------------------→ H3AsO3 + H2SnO3
Arsenic acid Stannous acid Arsenious acid
H3AsO3 + 6[H] ---------------------→ AsH + 3H2O
Arsenious acid nascent hydrogen Arsine gas
The depth of yellow stain on mercuric chloride paper will depend upon the quantity of
arsenic present in the sample.
• When the sample is dissolved in acid, the Arsenic present in the sample gets
converted to Arsenic acid.

• By action of reducing agents like Potassium iodide, stannous acid etc.,

Arsenic acid gets reduced to arsenious acid.

• The nascent hydrogen formed during the reaction, further reduces

Arsenious acid to Arsine gas, which reacts with mercuric chloride paper,
giving a yellow stain.
• Apparatus:
• It is having a wide mouthed glass bottle of 120 mL capacity having mouth of about 2.5 cm in
diameter. This bottle is fitted with a rubber bung through which passes a glass tube, 20 cm long.
• External diameter=0.8 cm
• Internal diameter=0.65 cm
• The tube is constricted at its lower end extremity to about 1 mm diameter and there is blown a hole,
not less than 2 mm in diameter, in the side of the tube near the constricted part.
• The upper end of the glass tube is fitted with two rubber bungs(25 mm x 25 mm), each having a
hole bored centrally and exactly 6.5 mm in diameter.
• One of the bungs has been fitted to the upper end of the tube, while the second bung has to be fitted
upon the first bung in such a way that the mercuric chloride paper gets exactly sandwiched between
the central perforation of the two.
• The bungs are kept in close contact by using rubber band or spring clip in such a manner that the
gas evolved from the bottle must have to pass through the 0.65 mm internal circle of mercuric
chloride paper.
• During the test, the evolved gases have been passing through the side hole, the lower hole serving
as an exit for water which condenses in the constricted part of the tube.
• An important feature has been the standardization of the area of Mercuric chloride paper which is
exposed to the reaction of arsine gas.
 Test sample Standard compound

The test solution is prepared by dissolving specific A known quantity of dilute arsenic solution in water
amount in water and stannated HCl (arsenic free) and and stannated HCl (arsenic free) is kept in wide
kept in a wide mouthed bottle. mouthed bottle.
1 g of KI 1 g of KI

5 ml of stannous chloride acid solution 5 ml of stannous chloride acid solution

10 g of granulated zinc is added (all this reagents 10 g of granulated zinc is added (all this reagents
must be arsenic free). Close the cork immediately must be arsenic free). Close the cork immediately
than place bottle in water bath at 40 C for 40 min than place bottle in water bath at 40 C for 40 min

Compare the yellow stain on MgCl2 paper Compare the yellow stain on MgCl2 paper
• Stain obtained on mercuric chloride paper is compared with standard solution. Standard
stain must be freshly prepared as it fades on keeping.

• Inference: If the stain produced by the test is not deeper than the standard stain, then
sample complies with the limit test for Arsenic.

Reasons:

• Stannous chloride is used for complete evolution of arsine.

• Zinc, potassium iodide and stannous chloride is used as a reducing agent.

• Hydrochloride acid is used to make the solution acidic

• Lead acetate paper are used to trap any hydrogen sulphide which may be evolved
along with arsine.
• Use of stannated Hydrochloric acid:
• If pure zinc and HCl are used, the steady evolution of gas does not occur. This
produces improper stain (e.g slow evolution produces short but intense stain
while rapid evolution of gas produces long but diffused stain.)
• So, to get steady evolution of gas, stannated hydrochloric acid is used.
• Use of Lead Acetate solution:
• H2S gas may be formed during the experiment as zinc contains sulphides as
impurities. It gives black stain to HgCl2 paper and so will interfere the test.
• Hence, gases evolved are passed through cotton wool plug moistened with
lead acetate, where H2S gas is trapped as PbS.
• Use of Potassium iodide:
• KI is converted to HI which brings about reduction of unreacted pentavalent
arsenic to trivalent Arsenic. Thus, reproducible results can be obtained. If it is
not used then some pentavalent Arsenic may remain unreacted.
❖ Limit test for IRON:
• Limit test of Iron is based on the reaction of iron in ammonical solution with
thioglycollic acid in presence of citric acid to form iron thioglycolate (Ferrous
thioglycolate complex) which produces pale pink to deep reddish purple color
in alkaline media.
• Thioglycolic acid is used as reducing agent.
• The color of the Ferrous thioglycolate complex fades in the presence of air
due to oxidation.

• Also, the color is destroyed in presence of oxidizing agents and strong

alkalis.

• The purple color is developed only in alkaline media. So ammonia

solution is used.

• But ammonia reacts with iron, forms precipitate of ferrous hydroxide.

• Thus citric acid is used which prevents the precipitate of iron with
Ammonia by forming a complex with iron as iron citrate.
Procedure:
Test sample Standard compound
Sample is dissolved in specific amount 2 ml of standard solution of iron diluted (0.173
of water and then volume is made up to 40 ml with water upto 40 ml (0.173gm ferric ammonium
sulphate +1.5 ml HCl Volume make upto 1000ml
with distelled water)

Add 2 ml of 20 % w/v of citric acid Add 2 ml of 20 % w/v of citric acid

(iron free) (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 solution
alkaline and adjust the volume upto 50 ml with alkaline and adjust the volume upto 50 ml with
distilled water. distilled water.
Keep aside for 5 min Keep aside for 5 min

Color developed is viewed vertically Color developed is viewed vertically

and compared with standard solution and compared with standard solution

Note: All the reagents used in the limit test for Iron should themselves be iron free.
• Observation:

• The purple color produce in sample solution should not be greater than standard solution. If
purple color produces in sample solution is less than the standard solution, the sample will pass
the limit test of iron and vice versa.

Reasons:

• Citric acid forms complex with metal cation and helps precipitation of iron by ammonia by
forming a complex with it.

• Thioglycolic acid helps to oxidize iron (II) to iron (III).

• Ammonia is added to make solution alkaline. The pale pink color is visible only in the alkaline
media. The color is not visible in acidic media as ferrous thioglycolate complex decomposes in
high acidic media.
❖ Limit test for heavy metals
• The limit test for heavy metals is designed to determine the content of metallic impurities
that are coloured by hydrogen sulphide or sodium sulphide under the condition of the test
should not exceed the heavy metal limits given under the individual monograph.

• The heavy metals (metallic impurities) may be iron, copper, lead, nickel, cobalt, bismuth,
antimony etc.. The limit for heavy metals is indicated in the individual monograph in
term of ppm of lead i.e. the parts of lead per million parts of the substance being
examined

• In substances the proportion of any such impurity (Heavy metals) has been expressed as the
quantity of lead required to produce a color of equal depth as in a standard comparison
solution having a definite quantity of lead nitrate.
• Principle: It based upon the reaction between metallic impurities with
hydrogen sulphide in acidic medium it formed the brownish coloured
solution.

• Metal that response the limit test lead, mercury, bismuth, silver, copper etc

+ Acidic media +
2 M + H2S---------------------M2S + H
Metal impurity

• Example:
+ Acidic media +
Copper 2 Cu + H2S --------------- Cu2S + H
• The quantity is stated as the heavy metal limit and is expressed as parts of
lead (by weight) per million parts of the test substance.

• The limit test for heavy metals has been based upon the reaction of the
metal ion with hydrogen sulphide, under the prescribed conditions of the
test causing the formation of metal sulphides.

• These remain distributed in colloidal state, and give rise to a brownish

coloration.
+ +

Acidic media
• 2 M + H2S---------------------M 2S + H
Metal Hydrogen sulphide
impurity
• I.P limit for heavy metals in 20 ppm.

• The test solution is compared with a standard prepared using a lead solution (as the heavy
metal).The metallic impurities in substance are expressed as parts of lead per million parts of
substance.

• IP has adopted 3 methods for this:

• Method I: The method is applicable for the samples which give clear colourless solutions under
specified conditions of test.

• Method II: The method is applicable for the samples which DO NOT give clear colourless
solutions under specified conditions of test.

• Method III: Used for substances which give clear colourless solutions in sodium hydroxide
medium.
Test solution Standard solution
Specified amount of sample with 2ml standard lead solution
25 ml water dissolve. (20ppm) 25 ml water dissolve
Adjust the pH 3 to 4 Adjust the pH 3 to 4
Dilute up to 35 ml water and mix Dilute up to 35 ml water and mix
10 ml H2S solution add again 10 ml H2S solution add again
dilute upto 50 ml dilute upto 50 ml
Mix the solution darkness observed Mix the solution darkness observed
white background white background
❖ Limit test for lead:
• Lead is a most undesirable impurity in medical compounds and comes
through use of sulphuric acid, lead lined apparatus and glass bottles use for
storage of chemicals.

• Principle:

• Limit test of lead is based on the reaction of lead and diphenyl thiocabazone
(dithizone) in alkaline solution to form lead dithizone complex which is red in
color.
• Dithizone in chloroform, is able to extract lead from alkaline aqueous
solutions as a lead complex (Red in colour)
• The original dithizone is having a green colour in chloroform while the
lead-dithizone is having a violet color. So, resulting color at the end of the
process is red.
• The intensity of the color of complex is dependant upon the amount of lead in

• the solution.

• The color of the lead-dithizone complex in chloroform has been compared with a

• standard volume of lead solution, treated in the same manner.

• In this method, the lead present as an impurity in the substances, gets separated by

• extracting an alkaline solution with a dithizone extraction solution.

• The interference and influence of the other metal ions has been eliminated by

adjusting the optimum pH for the extraction by employing Ammonium citrate/

potassium cyanide.
Method:

• Sample solution is transferred to a separating funnel.

• To it 6 ml of ammonium citrate, 2 ml potassium cyanide and 2 ml of hydroxalamine HCl are

added.

• 2 drops of phenol red

• Solution is made alkaline by adding ammonia solution.

• This is then extracted with 5 ml portions of dithizone solution until it becomes green.

• The combined dithizone extracts are shaken for 30 min with 30 ml of nitric acid and chloroform
layer is discarded.

• To the acid solution 5 ml of standard dithizone solution is added and 4 ml ammonium cyanide and
solution is shaken for 30 sec.

• Similarly prepare standard.

Observation:
• The intensity of the color of complex, is depends on the amount of lead in
the solution. The color produced in sample solution should not be greater
than standard solution. If color produces in sample solution is less than the
standard solution, the sample will pass the limit test of lead and vice versa.
Reasons:
• Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is
used to make pH optimum so interference and influence of other
impurities have been eliminated.
• Phenol red is used as indicator to develop the color at the end of process
Lead present as an impurities in the substance, gets separated by extracting
an alkaline solution with a dithizone extraction solution.
TEST SOLUTION STANDARD SOLUTION
A known quantity of sample solution is A standard lead solution is prepared equivalent to
transferred in a separating funnel the amount of lead permitted in the sample under
examination
Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide and 2 ml of Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride hydroxylamine hydrochloride
Add 2 drops of phenol red Add 2 drops of phenol red
Make solution alkaline by adding ammonia solution. Make solution alkaline by adding ammonia solution.
Extract with 5 ml of dithizone until it becomes green Extract with 5 ml of dithizone until it becomes green
Combine dithizone extracts are shaken for 30 mins Combine dithizone extracts are shaken for 30 mins
with 30 ml of nitric acid and the chloroform layer is with 30 ml of nitric acid and the chloroform layer is
discarded discarded

To the acid solution add 5 ml of standard dithizone To the acid solution add 5 ml of standard dithizone
solution solution
Add 4 ml of ammonium cyanide Add 4 ml of ammonium cyanide
Shake for 30 mins Shake for 30 mins
Observe the colour Observe the colour
• Aq. Ammonia is added in limit test of lead:

• Pb+ S ------------ PbS

• In limit test of lead, PbS is produced by addition of standard H2S , to the

solution containing lead.

• pH 3-4 is necessary for the precipitation of PbS. So aq. Ammonia /acetic acid
is added to maintain that pH.
❖ Modified limit test for Chlorides
• Depending upon the nature of the substance, some modifications have to be
adopted for the preparation of the solution.
Principle:
The limit test for chloride based on the reaction between soluble chloride
impurities present in the substance and silver nitrate solution to give white
precipitates of silver chloride.
These white precipitates are insoluble in dilute nitric acid and hence give
turbidity or opalescence to the test solution.
The extent of the turbidity produced depends upon the amount of the chloride
present in the substance which is compared with a standard opalescence produce
by addition of silver nitrate to a standard solution having known amount of
chloride and the same volume of the dilute nitric acid as the use in the test
solution.
If the turbidity developed in the sample is less than the standard turbidity, the
sample passes the limit test for chloride and vice-versa.
As potassium permanganate gives purple color aqueous solution that interferes in
the comparison of opalescence or turbidity, therefore the aqueous solution must
first be decolorized. Potassium permanganate is oxidizing agent while ethanol is
reducing agent.
• When potassium permanganate solution is treated with ethanol in presence of
heat the redox reaction will take place, i.e. potassium permanganate gets
reduced to manganese dioxide (precipitates). The filtrate of the reaction is
colorless that is subjected to proceed for limit test for chloride.

Chemical Reaction:
Redox
reaction
2 KMnO4 + 3 C2H5OH-----------------2MnO2 + 2 KOH + 2 CH3CHO + 2 H2O

Coloured compound------------------- Colourless compound

Basic Reaction

Cl- + AgNO3 --------------------------AgCl + NO3-

• Preparation of Test solution (KMnO4) : Dissolve 1.5 gm KMnO4 in 50 ml distilled water
(purple colour) heat on water bath slowly added 6ml of (95%) ethanol and cool the solution
then dilute with 60 ml distilled water and filter it------ gives ( colourless solution)
TEST SOLUTION STANDARD SOLUTION

Specified amount Transfer the prepared test Take 10 ml chloride standard solution (25
solution in Nessler’s cylinder ppm chloride) and add 5 ml water in a
Nessler’s cylinder.
Add 10 ml of dilute nitric acid and dilute to Add 10 ml of dilute nitric acid and dilute to
50 ml with distilled water 50 ml with distilled water

Add 1ml of 0.1 M silver nitrate solution and Add 1ml of 0.1 M silver nitrate solution and
stir immediately with glass rod and allow stir immediately with glass rod and allow
standing for 5 minutes protected from light. standing for 5 minutes protected from light.

• Compare the turbidity or opalescence produced in test solution with respect to

standard solution and report the result and conclusion.
• Observation and conclusion will be of two types:

• If the intensity of turbidity or opalescence appears to be more in test solution

than the standard solution then conclusion is impurities of chloride in given
sample is over the limit as per IP’96. Hence, sample do not passes the limit
test for chlorides.

• If the intensity turbidity or opalescence appears to be less or equal in test

solution than the standard solution then conclusion is impurities of chloride
in given sample is under the limit as per IP’96. Hence, sample passes the
limit test for chloride
❖ Modified limit test for Sulphate
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 which appears as solid particles (turbidity) in
the solution.

• Then comparison of turbidity is done with a standard turbidity obtained from a known amount of
Sulphate and same volume of dilute Hydrochloric acid have been added to both solutions.

• The barium chloride test solution in the IP has been replaced by Barium sulphate reagent which is
having barium chloride, sulphate free alcohol and a solution of potassium sulphate. Potassium
sulphate has been added to increase the sensitivity of the test.
• As potassium permanganate gives purple color aqueous solution that interferes
in the comparison of opalescence or turbidity, therefore the aqueous solution
must first be decolorized. Potassium permanganate is oxidizing agent while
ethanol is reducing agent.

• When potassium permanganate solution is treated with ethanol in presence of

heat the redox reaction will take place, i.e. potassium permanganate gets
reduced to manganese dioxide (precipitates). The filtrate of the reaction is
colorless that is subjected to proceed for limit test for chloride.

Chemical Reaction:

2 KMnO4 + 3 C2H5OH-----------------2MnO2 + 2 KOH + 2 CH3CHO + 2 H2O

Coloured compound------------------- Colourless compound

• Preparation of Test solution (KMnO4) : Dissolve 1.5 gm KMnO4 in 50 ml distilled water
(purple colour) heat on water bath slowly added 6ml of (95%) ethanol and cool the solution
then dilute up to 60 ml with distilled water and filter it------ gives ( colourless solution)
TEST SOLUTION STANDARD SOLUTION

Specified amount Transfer the prepared test Take 1ml of 0.1089% w/v K2SO4.
solution (40ml) in Nessler’s cylinder

Add 2ml of dilute HCl/ acetic acid and Add 2ml of dilute HCl/ acetic acid and
dilute to 50 ml with distilled water dilute to 50 ml with distilled water.

Add 5ml of BaSO4 reagent and stir Add 5ml of BaSO4 reagent and stir
immediately with glass rod and allow immediately with glass rod and allow
standing for 5 minutes protected from light. standing for 5 minutes protected from light.

• Compare the turbidity or opalescence produced in test solution with respect to

standard solution and report the result and conclusion.
• Observation and conclusion will be of two types:

• If the intensity of turbidity or opalescence appears to be more in test

solution than the standard solution then conclusion is impurities of sulphate
in given sample is over the limit as per IP’96. Hence, sample do not passes
the limit test for sulphate.

• If the intensity turbidity or opalescence appears to be less or equal in test

solution than the standard solution then conclusion is impurities of sulphate
in given sample is under the limit as per IP’96. Hence, sample passes the
limit test for sulphate.
Thank
You !