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UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
2
INDEX
S.NO. TOPIC PAGE NO.
1 Impurity:-
 Introduction
 Sources
 Types
 Effects
1-3
2 Limit Test:-
 Introduction
 Importance
4
3 Limit Test for Chloride:-
Principle,Reaction,Procedure,Observation
5-6
4 Limit Test for Sulphate:-
Principle,Reaction,Procedure,Observation
7-8
5 Limit Test for Iron:-
Principle,Reaction,Procedure,Observation, Reason
9-10
6 Limit Test for Heavy metal:-
Principle,Reaction,Procedure,Observation, Reason
11-12
7 Limit Test for Arsenic:-
Principle,Reaction,Procedure,Observation, Reason
13-15
1
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Unit-1
Pharmaceutical Chemistry
 Impurities in Pharmaceuticals: - Unwanted substances
 Impurity: - Any component of the API that is not the chemical entity defined
as the API is called as an impurity.
 Impurities in pharmaceutical are the unwanted chemical that remain with the
API or develop during formulation.
 Impurities are defined as the presence of undesired & unexpected material
during any procedure & that may affect the final product.
 The presence of these unwanted chemicals even in small amounts may
influence the efficacy & safety of the Pharmaceutical product.
 Sources of Impurity: -
During manufacturing the different types & amount of impurity present in the
chemical or pharmaceutical product (API) depend upon several factor like those
listed below: -
 Raw material used in mfg.
 Process used in mfg.
 Plant material used in mfg.
 Solvent used in mfg.
 Adulteration
 Improper storage area
 Atmospheric contamination
 Reagents used in mfg.
 Raw material used in mfg.: -When any medicine is manufactured, the starting
material used in mfg. is called raw material. If there are any impurities in raw
material, then the final product will also have impurities. Example: - If arsenic,
heavy metal, lead is present in raw material then this will also be found in the
final product which may cause harmful effect to human being.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
2
 Process used in mfg.: - There are a number of drug & chemicals which are
manufactured by different methods. Sometimes during mfg. impurities are also
incorporated & this will appear in final product.
If the mfg. process is long, then the number of
impurities will also increase in the final product.
 Plant material used in mfg.: - During the mfg. process, various chemicals are
mixed in vessels, these vessels are made up iron, copper, steel, aluminum.
Sometimes during mixing reaction takes place between chemical & vessels
which produce impurities in final product.
So, it very necessary to use a proper vessel which does not react
with any chemicals.
 Solvent used in mfg.: - Water is the cheapest solvent & most commonly used
in mfg. chemicals. But water may act as a source of impurities if proper
precaution is not taken. If a company is using tap water, then tap water contains
loss of inorganic impurities.
So, if a company is using distilled water then the number of
impurities will reduce.
 Adulteration: - It is a process in which high quality chemical are mixed with
low quality chemical. Because of this impurity found in the final product.
Example: Potassium bromide is a costly chemical is mixed with sodium
bromide which is cheaper product.
 Improper Storage area: - Many substances may undergo changes if proper
storage area is not provided. Because of these impurities get involved in the
final product.
 Atmospheric Contamination: - If the atmosphere is very polluted, then it may
affect the purity of the product & impurities get involved in the final product.
 Reagents used in mfg. process: -Many reagents are used in the process of mfg.
a product. If the products are not washed properly to remove excess reagents,
then these reagents are liable to be carried as impurity to final preparation.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
3
 Types of Impurities:-
There are following three types:
1. Organic Impurities: - Starting material, By-product, Reagents, Degradation
products.
2. Inorganic Impurities: - Reagents, Heavy metal, Inorganic salt etc.
3. Residue Solvents:-
 Class 1:- Solvents to be avoided
 Class 2:- Solvents to be limited
 Class 3:- Solvents with low toxic potential.
 Effect of Impurity: -
The impurities present in the substances may give
following effects:
 Impurities may have toxic effects & can be injurious when present in higher
amount.
 Impurities may bring changes in the physical properties of final product. Like:
Colour, Odor, Taste etc.
 Impurities may bring change in the chemical properties of final product.
 Impurities may reduce the shelf life of the drugs.
 Impurities may cause technical difficulties in the formulation while mfg. it.
 Impurities may cause incompatibility with the other substance
Incompatibility: - Is defined as a change resulting & an undesired product is
formed, which may affect the safety, efficacy, appearances, and stability of the
pharmaceutical product.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
4
Limit test
Introduction: - Limit Test
A value Examine
or, an amount or, Investigate
 Limit: - A value or an amount that is likely to be present in a substance.
 Test: - To examine or investigate
 Definition: -Limit test is quantitative & semi-quantitative test designed to
identify small amount of impurities which are present in the substance.
 Limit test is generally carried out to determine the inorganic impurity present in
the substance.
 Limit test- Determine the impurities.
 Limit tests Bing used to identify & control the impurities.
 The limit test performed in Nessler Cylinder.
Fig: 01. Nessler’s Cylinder
Importance of Limit test: -
 Limit test are used to identify impurities.
 Limit test are very important for differentiating into avoidable & non-avoidable
impurities.
 Limit test are very important for check the purity of substance.
 To find out harmful amount of impurities.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
5
Limit test for Chloride
PRINCIPLE: - Limit test of chloride principle 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 particle (turbidity or opalescence) in the solution.
Reaction: -
Clˉ + AgNO3 Dilute HNO3 AgCl + NO3ˉ
(Soluble) (Silver nitrate) (Silver chloride ppt)
PROCEDURE: -
 Take two 50 ml Nessler’s cylinders.
 Label one as “Test” and the other as “Standard”.
S.no Test solution Standard solution
1 Dissolve the specified quantity of
the substance in distilled water and
transfer it to the Nessler’s cylinder.
The solution prepared as directed
pharmacopoeia.
Place 1 ml of 0.05845% w/v
solution of NaCl in a Nessler’s
cylinder.
2 Add 10 ml of dilute. HNO3 Add 10 ml of dilute. HNO3
3 Dilute to 50 ml with water in
Nessler’s cylinder
Dilute to 50 ml with water in
Nessler’s cylinder
4 Add 1 ml of silver nitrate solution. Add 1 ml of silver nitrate solution.
5 Stir immediately with a glass rod
and allow standing for 5 minutes.
Stir immediately with a glass rod
and allow standing for 5 minutes.
6 Observe the opalescence
(turbidity) developed and compare
it with that of the standard.
Observe the opalescence (turbidity)
developed and compare it with that
of the sample.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
6
Observation: - The turbidity produce in test solution should not be greater than
standard solution. If opalescence (turbidity) produced in the test solution is less
than the standard solution, the sample will pass the limit test of chloride.
Fig: 02. The turbidity show in test & standard solution
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
7
Limit test for sulphate
Principle: -
Limit test for sulphate principle is based on the reaction of
soluble sulphate(SO4
-
) with barium chloride(BaCl2) in presence of dilute
hydrochloric acid (Dil.HCl) to form barium sulphate(BaSO4) which
appears as solid particle (turbidity or opalescence) in the solution.
Reaction: -
SO4
2-
+ BaCl2 dil. HCl BaSO4 + 2Cl-
(Soluble sulphate) (Barium chloride) (Barium Sulphate as turbidity)
Procedure: -
 Take a two Nessler cylinder. Properly washed & dry.
 Mark on Two Nessler cylinder as Standard solution& Test solution.
 Solution Prepare according to Indian Pharmacopoeia.
Test Solution Standard solution
Specific weight of compound is
dissolved in water is prepared as
directly in the pharmacopoeia and
transferred in Nessler cylinder.
Take 1 ml of 0.1089% w/v solution
of potassium sulphate(K2SO4) in
Nessler cylinder
Add 2 ml of Dil. HCl. Add 2 ml of Dil. HCl.
Dilute to 45 ml in Nessler cylinder. Dilute to 45 ml in Nessler cylinder.
Add 5ml of barium sulphate
(BaSO4) reagent.
Add 5ml of barium sulphate
(BaSO4) reagent.
Keep aside for 5 min. Keep aside for 5 min.
Observe the turbidity. Observe the turbidity.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
8
Note: Barium sulphate (BaSO4) reagent contains barium chloride (BaCl2) &
Sulphate free alcohol.
Observation: - The turbidity produce in test sample solution should not
be greater than standard solution. If turbidity produce in test sample
solution is less than the standard solution, the sample will pass.
Fig: 03. The turbidity show in test & standard solution
Reason: -
 HCl helps to make solution acidic.
 Alcohol helps to prevent super saturation & so produces a more uniform opalescence.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
9
Limit test for Iron
Principle: -
Limit test for iron principle is based on the reaction of iron in
ammonical solution with thioglycolic acid in presence of dilute citric acid
to form iron thioglycolate (ferrous thioglycolate complex) which produces
pale pink to deep reddish purple colour in alkaline medium.
 Thioglycolic acid is used as reducing agents.
Reaction: -
or,
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
10
Procedure: -
 Take a two Nessler cylinder. Properly washed & dry.
 Mark on Two Nessler cylinder as Standard solution& Test solution.
 Solution Prepare according to Indian Pharmacopoeia.
Test Solution Standard solution
Sample is dissolved in specific amount
of water and then volume is made up to
40ml.
2ml of standard solution of iron diluted
with water up to 40ml.
Add 2ml of 20% w/v of citric acid(iron
free)
Add 2ml of 20% w/v of citric acid(iron
free)
Add 2 drops of thioglycolic acid Add 2 drops of thioglycolic acid
Add ammonia to make the solution
alkaline and adjust the volume to 50ml.
Add ammonia to make the solution
alkaline and adjust the volume to 50ml.
Keep aside for 5 min. Keep aside for 5 min.
Color developed is viewed vertically
and compared with standard solution.
Color developed is viewed vertically
and compared with standard solution.
Observation: - The purple color produce in test sample solution should not be
greater than standard solution. If purple color produce in test sample solution is
less than the standard solution, the sample will pass.
Test solution Standard solution
Fig: 04. The color developed in test & standard solution
Reason: -
 Citric acid forms complex with metal cation and helps precipitation of iron by ammonia
by forming a complex with it.
 Ammonia to make solution alkaline.
 Thioglycolic acid used as reducing agent.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
11
Limit test for Heavy metals
Principle: -
Limit test for heavy metal principle is based on the reaction of
metallic impurities with hydrogen sulphide (H2S)/ nitrogen sulphide
(N2S) in presence of acidic medium to form heavy metal sulphide which
produces brownish color solution.
Reaction: -
Heavy metal + hydrogen sulphide (H2S) Heavy metal sulphide
/ Nitrogen sulphide (N2S) (brownish color)
Procedure: -
The IP has adopted 4 methods for the limit test of heavy
metals.
Method I: - The method is applicable for the samples which give clear
colorless solutions under specified conditions of test.
 Method II: - The method is applicable for the samples which give do not give
clear colorless solutions under specified conditions of test.
 Method III: - The method is used for substances which give clear colorless
solution in sodium hydroxide medium.
 Take a two Nessler cylinder. Properly washed & dry.
 Mark on Two Nessler cylinder as Standard solution& Test solution.
 Solution Prepare according to Indian Pharmacopoeia.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
12
Test Solution Standard solution
Solution prepared as per the
pharmacopoeia and 25ml is transferred
in Nessler’s cylinder.
Take 2ml of standard lead solution and
dilute to 25ml with water.
Adjust the pH between 3 to 4 by adding
dilute acetic acid or dilute ammonia
solution.
Adjust the pH between 3 to 4 by adding
dilute acetic acid or dilute ammonia
solution.
Dilute with water to 50ml. Dilute with water to 50ml.
Add freshly prepared 10ml of hydrogen
sulphide solution. Dilute with water up
to 50ml.
Add freshly prepared 10ml of hydrogen
sulphide solution. Dilute with water up
to 50ml.
Allow to stand for 5min. Allow to stand for 5min.
Color developed is viewed downwards
over a white surface and compared with
standard solution.
Color developed is viewed downwards
over a white surface and compared with
standard solution.
Observation: - The color produce in test sample solution should not be greater
than standard solution. If color produce in test sample solution is less than the
standard solution, the sample will pass.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
13
Limit test for Arsenic
 Limit test for arsenic prescribe as all pharmacopoeia.
 It is also known as Gutzeit test.
 Limit test for arsenic performed in Gutzeit apparatus.
 Arsenic is undesirable and harmful impurity which is present in pharmaceutical
product.
PRINCIPLE: -
Limit test of arsenic principle is based on the reaction of arsenic gas
with hydrogen ion to form yellow stain on mercuric chloride paper is presence
of reducing agents like potassium iodide.
Arsenic, present as arsenic acid in the sample is reduced to arsenious acid by
reducing agent like potassium iodide, stannous acid, zinc etc. Arsenious acid
further reduced to arsine gas by hydrogen & react with mercuric chloride paper
to give yellow stain.
Reaction: -
As3+
H3AsO4 (Arsenic acid)
H3AsO4 H3AsO3 (Arsenious acid)
H3AsO3 + 3H2 AsH3 (gas) + 3H2O
AsH3 (gas) + HgCl2 Hg (AsH2)2(Yellow stain) + 2 HCl
(Mercuric chloride paper)
Note- The depth of yellow stain on mercuric chloride paper will depend upon the
quality of arsenic present in the sample.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
14
Apparatus:-
Limit test for arsenic is performed by an apparatus known as Gutzeit apparatus. It
is also called arsenic apparatus.
Fig: 05. Gutzeit Apparatus
 A wide-mouthed glass bottle capable of holding about 120 ml is fitted with a
rubber bung through which passes a glass tube.
 Glass tube total length of 200 mm and an internal diameter of exactly 6.5 mm
(external diameter about 8 mm).
 Rubber stopper.
 Narrow part of the glass tube. Glass wool is inserted up to this part.
 Lead acetate cotton plug or wool.
UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST
15
Procedure: -
Test solution Standard solution
The test solution is prepared by
dissolving specific amount in water and
stannated HCl (arsenic free) and kept in a
wide mouthed bottle.
A known quantity of dilute arsenic
solution in water and stannated HCl
(arsenic free) is kept in wide mouthed
bottle.
The add 1gm of KI , 5ml of stannous
chloride & 10gm of Zinc is added( all
should be arsenic free)
The add 1gm of KI , 5ml of stannous
chloride & 10gm of Zinc is added( all
should be arsenic free)
Keep the solution a side for 40 min. Keep the solution a side for 40 min.
Compare the stain obtained on the
mercuric chloride paper with that in the
apparatus containing the test solution.
Compare the stain obtained on the
mercuric chloride paper with that in the
apparatus containing the test solution.
Observation: - The yellow stain produce in test sample solution should not be
greater than standard solution. If yellow stain produce in test sample solution is
less than the standard solution, the sample will pass.
Reason: -
 Stannous chloride is used for complete evolution of arsine.
 Zinc, KI & Stannous chloride is used as reducing agent.
 HCl 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.

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Impurity & Limit test.pdf

  • 1. 1 Subscribe my YouTube channel @thepharmacywithadsir
  • 2. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 2 INDEX S.NO. TOPIC PAGE NO. 1 Impurity:-  Introduction  Sources  Types  Effects 1-3 2 Limit Test:-  Introduction  Importance 4 3 Limit Test for Chloride:- Principle,Reaction,Procedure,Observation 5-6 4 Limit Test for Sulphate:- Principle,Reaction,Procedure,Observation 7-8 5 Limit Test for Iron:- Principle,Reaction,Procedure,Observation, Reason 9-10 6 Limit Test for Heavy metal:- Principle,Reaction,Procedure,Observation, Reason 11-12 7 Limit Test for Arsenic:- Principle,Reaction,Procedure,Observation, Reason 13-15
  • 3. 1 Subscribe my YouTube channel @thepharmacywithadsir Unit-1 Pharmaceutical Chemistry  Impurities in Pharmaceuticals: - Unwanted substances  Impurity: - Any component of the API that is not the chemical entity defined as the API is called as an impurity.  Impurities in pharmaceutical are the unwanted chemical that remain with the API or develop during formulation.  Impurities are defined as the presence of undesired & unexpected material during any procedure & that may affect the final product.  The presence of these unwanted chemicals even in small amounts may influence the efficacy & safety of the Pharmaceutical product.  Sources of Impurity: - During manufacturing the different types & amount of impurity present in the chemical or pharmaceutical product (API) depend upon several factor like those listed below: -  Raw material used in mfg.  Process used in mfg.  Plant material used in mfg.  Solvent used in mfg.  Adulteration  Improper storage area  Atmospheric contamination  Reagents used in mfg.  Raw material used in mfg.: -When any medicine is manufactured, the starting material used in mfg. is called raw material. If there are any impurities in raw material, then the final product will also have impurities. Example: - If arsenic, heavy metal, lead is present in raw material then this will also be found in the final product which may cause harmful effect to human being.
  • 4. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 2  Process used in mfg.: - There are a number of drug & chemicals which are manufactured by different methods. Sometimes during mfg. impurities are also incorporated & this will appear in final product. If the mfg. process is long, then the number of impurities will also increase in the final product.  Plant material used in mfg.: - During the mfg. process, various chemicals are mixed in vessels, these vessels are made up iron, copper, steel, aluminum. Sometimes during mixing reaction takes place between chemical & vessels which produce impurities in final product. So, it very necessary to use a proper vessel which does not react with any chemicals.  Solvent used in mfg.: - Water is the cheapest solvent & most commonly used in mfg. chemicals. But water may act as a source of impurities if proper precaution is not taken. If a company is using tap water, then tap water contains loss of inorganic impurities. So, if a company is using distilled water then the number of impurities will reduce.  Adulteration: - It is a process in which high quality chemical are mixed with low quality chemical. Because of this impurity found in the final product. Example: Potassium bromide is a costly chemical is mixed with sodium bromide which is cheaper product.  Improper Storage area: - Many substances may undergo changes if proper storage area is not provided. Because of these impurities get involved in the final product.  Atmospheric Contamination: - If the atmosphere is very polluted, then it may affect the purity of the product & impurities get involved in the final product.  Reagents used in mfg. process: -Many reagents are used in the process of mfg. a product. If the products are not washed properly to remove excess reagents, then these reagents are liable to be carried as impurity to final preparation.
  • 5. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 3  Types of Impurities:- There are following three types: 1. Organic Impurities: - Starting material, By-product, Reagents, Degradation products. 2. Inorganic Impurities: - Reagents, Heavy metal, Inorganic salt etc. 3. Residue Solvents:-  Class 1:- Solvents to be avoided  Class 2:- Solvents to be limited  Class 3:- Solvents with low toxic potential.  Effect of Impurity: - The impurities present in the substances may give following effects:  Impurities may have toxic effects & can be injurious when present in higher amount.  Impurities may bring changes in the physical properties of final product. Like: Colour, Odor, Taste etc.  Impurities may bring change in the chemical properties of final product.  Impurities may reduce the shelf life of the drugs.  Impurities may cause technical difficulties in the formulation while mfg. it.  Impurities may cause incompatibility with the other substance Incompatibility: - Is defined as a change resulting & an undesired product is formed, which may affect the safety, efficacy, appearances, and stability of the pharmaceutical product.
  • 6. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 4 Limit test Introduction: - Limit Test A value Examine or, an amount or, Investigate  Limit: - A value or an amount that is likely to be present in a substance.  Test: - To examine or investigate  Definition: -Limit test is quantitative & semi-quantitative test designed to identify small amount of impurities which are present in the substance.  Limit test is generally carried out to determine the inorganic impurity present in the substance.  Limit test- Determine the impurities.  Limit tests Bing used to identify & control the impurities.  The limit test performed in Nessler Cylinder. Fig: 01. Nessler’s Cylinder Importance of Limit test: -  Limit test are used to identify impurities.  Limit test are very important for differentiating into avoidable & non-avoidable impurities.  Limit test are very important for check the purity of substance.  To find out harmful amount of impurities.
  • 7. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 5 Limit test for Chloride PRINCIPLE: - Limit test of chloride principle 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 particle (turbidity or opalescence) in the solution. Reaction: - Clˉ + AgNO3 Dilute HNO3 AgCl + NO3ˉ (Soluble) (Silver nitrate) (Silver chloride ppt) PROCEDURE: -  Take two 50 ml Nessler’s cylinders.  Label one as “Test” and the other as “Standard”. S.no Test solution Standard solution 1 Dissolve the specified quantity of the substance in distilled water and transfer it to the Nessler’s cylinder. The solution prepared as directed pharmacopoeia. Place 1 ml of 0.05845% w/v solution of NaCl in a Nessler’s cylinder. 2 Add 10 ml of dilute. HNO3 Add 10 ml of dilute. HNO3 3 Dilute to 50 ml with water in Nessler’s cylinder Dilute to 50 ml with water in Nessler’s cylinder 4 Add 1 ml of silver nitrate solution. Add 1 ml of silver nitrate solution. 5 Stir immediately with a glass rod and allow standing for 5 minutes. Stir immediately with a glass rod and allow standing for 5 minutes. 6 Observe the opalescence (turbidity) developed and compare it with that of the standard. Observe the opalescence (turbidity) developed and compare it with that of the sample.
  • 8. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 6 Observation: - The turbidity produce in test solution should not be greater than standard solution. If opalescence (turbidity) produced in the test solution is less than the standard solution, the sample will pass the limit test of chloride. Fig: 02. The turbidity show in test & standard solution
  • 9. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 7 Limit test for sulphate Principle: - Limit test for sulphate principle is based on the reaction of soluble sulphate(SO4 - ) with barium chloride(BaCl2) in presence of dilute hydrochloric acid (Dil.HCl) to form barium sulphate(BaSO4) which appears as solid particle (turbidity or opalescence) in the solution. Reaction: - SO4 2- + BaCl2 dil. HCl BaSO4 + 2Cl- (Soluble sulphate) (Barium chloride) (Barium Sulphate as turbidity) Procedure: -  Take a two Nessler cylinder. Properly washed & dry.  Mark on Two Nessler cylinder as Standard solution& Test solution.  Solution Prepare according to Indian Pharmacopoeia. Test Solution Standard solution Specific weight of compound is dissolved in water is prepared as directly in the pharmacopoeia and transferred in Nessler cylinder. Take 1 ml of 0.1089% w/v solution of potassium sulphate(K2SO4) in Nessler cylinder Add 2 ml of Dil. HCl. Add 2 ml of Dil. HCl. Dilute to 45 ml in Nessler cylinder. Dilute to 45 ml in Nessler cylinder. Add 5ml of barium sulphate (BaSO4) reagent. Add 5ml of barium sulphate (BaSO4) reagent. Keep aside for 5 min. Keep aside for 5 min. Observe the turbidity. Observe the turbidity.
  • 10. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 8 Note: Barium sulphate (BaSO4) reagent contains barium chloride (BaCl2) & Sulphate free alcohol. Observation: - The turbidity produce in test sample solution should not be greater than standard solution. If turbidity produce in test sample solution is less than the standard solution, the sample will pass. Fig: 03. The turbidity show in test & standard solution Reason: -  HCl helps to make solution acidic.  Alcohol helps to prevent super saturation & so produces a more uniform opalescence.
  • 11. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 9 Limit test for Iron Principle: - Limit test for iron principle is based on the reaction of iron in ammonical solution with thioglycolic acid in presence of dilute citric acid to form iron thioglycolate (ferrous thioglycolate complex) which produces pale pink to deep reddish purple colour in alkaline medium.  Thioglycolic acid is used as reducing agents. Reaction: - or,
  • 12. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 10 Procedure: -  Take a two Nessler cylinder. Properly washed & dry.  Mark on Two Nessler cylinder as Standard solution& Test solution.  Solution Prepare according to Indian Pharmacopoeia. Test Solution Standard solution Sample is dissolved in specific amount of water and then volume is made up to 40ml. 2ml of standard solution of iron diluted with water up to 40ml. Add 2ml of 20% w/v of citric acid(iron free) Add 2ml of 20% w/v of citric acid(iron free) Add 2 drops of thioglycolic acid Add 2 drops of thioglycolic acid Add ammonia to make the solution alkaline and adjust the volume to 50ml. Add ammonia to make the solution alkaline and adjust the volume to 50ml. Keep aside for 5 min. Keep aside for 5 min. Color developed is viewed vertically and compared with standard solution. Color developed is viewed vertically and compared with standard solution. Observation: - The purple color produce in test sample solution should not be greater than standard solution. If purple color produce in test sample solution is less than the standard solution, the sample will pass. Test solution Standard solution Fig: 04. The color developed in test & standard solution Reason: -  Citric acid forms complex with metal cation and helps precipitation of iron by ammonia by forming a complex with it.  Ammonia to make solution alkaline.  Thioglycolic acid used as reducing agent.
  • 13. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 11 Limit test for Heavy metals Principle: - Limit test for heavy metal principle is based on the reaction of metallic impurities with hydrogen sulphide (H2S)/ nitrogen sulphide (N2S) in presence of acidic medium to form heavy metal sulphide which produces brownish color solution. Reaction: - Heavy metal + hydrogen sulphide (H2S) Heavy metal sulphide / Nitrogen sulphide (N2S) (brownish color) Procedure: - The IP has adopted 4 methods for the limit test of heavy metals. Method I: - The method is applicable for the samples which give clear colorless solutions under specified conditions of test.  Method II: - The method is applicable for the samples which give do not give clear colorless solutions under specified conditions of test.  Method III: - The method is used for substances which give clear colorless solution in sodium hydroxide medium.  Take a two Nessler cylinder. Properly washed & dry.  Mark on Two Nessler cylinder as Standard solution& Test solution.  Solution Prepare according to Indian Pharmacopoeia.
  • 14. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 12 Test Solution Standard solution Solution prepared as per the pharmacopoeia and 25ml is transferred in Nessler’s cylinder. Take 2ml of standard lead solution and dilute to 25ml with water. Adjust the pH between 3 to 4 by adding dilute acetic acid or dilute ammonia solution. Adjust the pH between 3 to 4 by adding dilute acetic acid or dilute ammonia solution. Dilute with water to 50ml. Dilute with water to 50ml. Add freshly prepared 10ml of hydrogen sulphide solution. Dilute with water up to 50ml. Add freshly prepared 10ml of hydrogen sulphide solution. Dilute with water up to 50ml. Allow to stand for 5min. Allow to stand for 5min. Color developed is viewed downwards over a white surface and compared with standard solution. Color developed is viewed downwards over a white surface and compared with standard solution. Observation: - The color produce in test sample solution should not be greater than standard solution. If color produce in test sample solution is less than the standard solution, the sample will pass.
  • 15. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 13 Limit test for Arsenic  Limit test for arsenic prescribe as all pharmacopoeia.  It is also known as Gutzeit test.  Limit test for arsenic performed in Gutzeit apparatus.  Arsenic is undesirable and harmful impurity which is present in pharmaceutical product. PRINCIPLE: - Limit test of arsenic principle is based on the reaction of arsenic gas with hydrogen ion to form yellow stain on mercuric chloride paper is presence of reducing agents like potassium iodide. Arsenic, present as arsenic acid in the sample is reduced to arsenious acid by reducing agent like potassium iodide, stannous acid, zinc etc. Arsenious acid further reduced to arsine gas by hydrogen & react with mercuric chloride paper to give yellow stain. Reaction: - As3+ H3AsO4 (Arsenic acid) H3AsO4 H3AsO3 (Arsenious acid) H3AsO3 + 3H2 AsH3 (gas) + 3H2O AsH3 (gas) + HgCl2 Hg (AsH2)2(Yellow stain) + 2 HCl (Mercuric chloride paper) Note- The depth of yellow stain on mercuric chloride paper will depend upon the quality of arsenic present in the sample.
  • 16. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 14 Apparatus:- Limit test for arsenic is performed by an apparatus known as Gutzeit apparatus. It is also called arsenic apparatus. Fig: 05. Gutzeit Apparatus  A wide-mouthed glass bottle capable of holding about 120 ml is fitted with a rubber bung through which passes a glass tube.  Glass tube total length of 200 mm and an internal diameter of exactly 6.5 mm (external diameter about 8 mm).  Rubber stopper.  Narrow part of the glass tube. Glass wool is inserted up to this part.  Lead acetate cotton plug or wool.
  • 17. UNIT-1 PHARMACEUTICAL CHEMISTRY IMPURITY & LIMIT TEST 15 Procedure: - Test solution Standard solution The test solution is prepared by dissolving specific amount in water and stannated HCl (arsenic free) and kept in a wide mouthed bottle. A known quantity of dilute arsenic solution in water and stannated HCl (arsenic free) is kept in wide mouthed bottle. The add 1gm of KI , 5ml of stannous chloride & 10gm of Zinc is added( all should be arsenic free) The add 1gm of KI , 5ml of stannous chloride & 10gm of Zinc is added( all should be arsenic free) Keep the solution a side for 40 min. Keep the solution a side for 40 min. Compare the stain obtained on the mercuric chloride paper with that in the apparatus containing the test solution. Compare the stain obtained on the mercuric chloride paper with that in the apparatus containing the test solution. Observation: - The yellow stain produce in test sample solution should not be greater than standard solution. If yellow stain produce in test sample solution is less than the standard solution, the sample will pass. Reason: -  Stannous chloride is used for complete evolution of arsine.  Zinc, KI & Stannous chloride is used as reducing agent.  HCl 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.