History of pharmacopoeia limit tests

1. 1
Presented by:
Budhe Urmila Ashok
Asst.Prof
SAJVPM’s College of Pharmaceutical Science and
Research Center
Pharmaceutical
Inorganic Chemistry

2. Syllabus
Unit I
 Impurities in pharmaceutical substances
• History of pharmacopoeia
• Sources and types iof impurities
• Principle involved in the limit test for
- Chloride
- Sulphate
- Iron
- Arsenic
- Lead and Heavy metals
- Modified limit test for Chloride and sulphate
 General methods of preparation
• Assay for the compounds superscripted with asterisk(*),
• Properties and medicinal uses of inorganic compounds 2

3. Inorganic Chemistry
 It is the study of all the elements and their compounds except carbon
and its compounds (which studied under organic chemistry).
 It describes the characteristics of substances obtained from non-living
things/matter and minerals which are found in the earth except the class
of organic compounds.
• Application : Chemical industry- Synthesis of drugs, pigments,
sulrfactants and agricultural products
• Examples: Sodium chloride(Nacl) used as table salt, Silicon
dioxide(SiO2) used in computer chips, Sulphuric acid (H2SO4) used in
production of fertilizers.
• In short, Inorganic chemistry is the branch of chemistry that deals with
inorganic compounds. 3
Branches
Co-ordination
Chemistry
Bioinorganic
chemistry
Organometallic
compounds
Synthetic
inorganic
chemistry

4. Inorganic compounds
 Organic compounds are those that are found in biological systems. In general
organic chemists that any molecule containing carbon is an organic
compounds and hence this means that inorganic chemistry that deals with the
compounds or molecules with lack carbon atom.
 Inorganic compounds are found in nature as minerals. These compounds are
synthesized for use as drug such as cisplatin, magnesium hydroxide,
catalysts such as vanadium oxide or titanium chloride.
 Study of pharmaceutical applications of inorganic compounds led to the
establishment of a new avenue called Pharmaceutical Inorganic Chemistry.
 It deals with the study of both essential and non-essential elements, their
preparation, standards of purity, test for identification, limit tests to be
performed for determining the quality, extent of purity, different
formulations, their storage conditions and therapeutic uses.
 The term pharmaceutical is used for any chemical substance useful in
preventive or therapeutic or which finds use in the preparation of
medicament.
 Quality of these pharmaceuticals must be carefully controlled. For this
reason, specifications of quality are mentioned for each pharmaceutical and
are reported in pharmacopoeia.
4

5. Importance of Inorganic Pharmaceuticals
Inorganic Pharmaceuticals are useful in any of the following ways:
1. For therapeutic purpose: e.g. Astringents, antimicrobials
2. As pharmaceutical aids: Bentonite, Talc
3. To change the reaction of body fluid either by acidifier or alkaliser. e.g.
Antacids, alkalies
4. Replacing the normal content of body fluids. e.g. Sodium, potassium,
calcium
5. As reagents to carry out the reactions. e.g. catalysts(platinum, nickel),
oxidizing and reducing agents
6. In pharmaceutical analysis: Titrants such as potassium permanganate,
EDTA
5

6. Introduction to Pharmacopoeia
 The word derives from the Greeek “ Pharmacopoiia” from pharmako-
“drug” followed by the verb stem poi- “make” & finally the abstract noun
ending –ia.
 These three elements together can be rendered as “drug-making” or “to
make a drug”.
 The pharmacopoeia which means the book containing the standards of drug
& other related substances are known as pharmacopoeia.
 The pharmacopoeias or formularies contain a list of drugs and other related
substances regarding their sources, descriptions, standard tests, formulae for
preparing the same, action and uses, doses, storage conditions etc.
 These books are prepared under the authority of the Government of the
respective countries. These books are revised from time to time to introduce
the latest information available as early as possible after they become
established.
 The books containing the standards for drugs and other related substances
are known as pharmacopoeia and formularies- collectively these books are
known as the drug compendia.
6

7. Classification of compendia
 Drug compendia are classified as:
1. Official compendia
These are the complications of drug and other related substances which are
recognized as legal standards for purity, quality and strength by a government
agency of respective countries of their origin.
Examples:
• British pharmacopoeia(BP)
• British pharmaceutical codex(BPC)
• Indian Pharmacopoeia(IP)
• United state Pharmacopoeia(USP)
• National Formulary(NF)
2. Non-official compendia
The books other than official drug compendia which are used as secondary
reference sources for drugs and other related substances are known as non-official
drug compendia.
7

8. Examples:
• Merck Index
• Extra Pharmacopoeia(Martindale)
• United State Dispensatory
Monograph
In simple way monographs are descriptions of pharmaceutical
preparations. The way it is a reference work for pharmaceutical drug
specifications.
It is a complete description of a specific pharmaceutical, which includes
chemical formulae, atomic and molecular weight, definition, statement of
content, category, dose, usual strength, description, solubility, identification
tests, assay, limits of impurities, quantities, and conditions for storage.
The appendices include standards for apparatus, reagents and solutions,
indicators, reference substances, test animals, calculation of results, other
chemical techniques, processes of the concerned pharmaceuticals.
Based on the content of information, Pharmacopoeia is divided into three
different major parts:
Part I : Generally the drugs that have similar use or actions are complied
together by part I of pharmacopoeia.
8

9. Part II: Monographs of new drugs, drugs under investigation, drugs
which are not easily classified and obsolescent drugs still of interest are
presented in part II of pharmacopoeia. It also provides details regarding
effects of required drug therapy.
Part III: Composition of the proprietary medicines that are advertised to
the public in different countries is documented with omission of herbal
medicine in part III of pharmacopoeia.
History of Pharmacopoeia
The term pharmacopoeia first appears as a distinct title in a work
published in Basel, Switzerland in 1561 by Dr.A. Foes, but does not
appear to have come into general use until the beginning of the 17th
century.
On 15th December 1820, the first united state pharmacopoeia (U.S.P) was
released.
In 1864, the first british pharmacopoeia (B.P) was published with
inclusion of monographs on benzoic acid , gallic acid, tartaric acid,
camphor and seven alkaloids along with their salts.
Todays pharmacopoeias mainly focus on assurance of quality of products
by various tools of analytical sciences. 9

10. Indian Pharmacopoeia (IP)
IP is an official document which meant for overall quality control and
Assurance of pharmaceutical products marketed in India, by way of
contributing on their safety, efficacy and affordability.
It contains a collection of authoritative procedures of analysis & specifications
for drugs.
IP prescribes standards for identity, purity & strength of drugs essentially
required from health care perspective of human beings & animals.
IP standards are authoritative in nature.
During quality assurance and the time of dispute in the court of law the IP
standards are legally acceptable.
• The historical developments of pharmacopoeia in India traces back to 1563.
• The idea of indigenous Indian pharmacopoeia was convinced in 1837.
• The Bengali & Hindi version of London pharmacopoeia was made available
in India from 1901 onwards.
• The indian pharmacopial list, published in 1946 formed the seeding for the
true official Indian pharmacopoeia published in 1955.
• The first edition of Indian pharmacopoeia was published in 1955, but
actually the process was started as early as 1944.
10

11.  The Indian Pharmacopoeial List, 1946:
The list of drugs both included and not included in the British
Pharmacopoeia along with standards to secure their usefulness, tests for
identity and purity was prepared by the committee and was published by
the Government of India under the name ‘ the Indian Pharmacopoeial
List 1946’.
The committee constituted under the chairmanship of sir R.N. Chopra
along with nine members, prepared the list of drugs with the following
details.
a) Drug of plant origin
b) Drugs of animal origin
c) Biological products
d) Insecticides
e) Coloring agents
f) Synthetics
g) Drugs for veterinary use
11

12.  British pharmacopoeia:
The British Pharmacopoeia has provided official standards for the
quality of substances and articles used in medicine since its first
publication.
Cartwright explores how these standards have been achieved through a
comprehensive review of the history and development of
pharmacopoeias in the UK.
The book which, places the British Pharmacopoeia in its global context
as an instrument of the British empire, will be of value to historians of
medicine and pharmacy and practitioners of medicine, pharmacy, and
pharmaceutical analytical chemistry.
In 1864 first British pharmacopoeia was published.
In 1867 second British pharmacopoeia was published.
In year 1874 the addendum to BP 1867 was published.
The third BP was published in 1885.
In 1890 the addendum to BP 1885 was published.
Forth BP was published in 1898. separate parts such as preparations of
compounds were included in BP and the contents had been arranged
alphabetically.
12

13. Highlights of BP
• Legally effective from 1 January 2014
• 40 new BP monographs are included
• 272 amended monographs
• Three new supplementary chapters are included
• Four new BP monographs are included
• Free in-year updates in April and July to harmonize with the European
pharmacopoeia.
European pharmacopoeia
An official standards adopted by Germany, France, Italy, Netherland,
Switzerland and Belgium is European pharmacopoeia.
In July 1964, the council of Europe issued an order, to frame out European
pharmacopoeia.
1969 onwards in the respective member countries it was appeared as
official standard book for medicinal substances and other drugs.
Later on it was revised continuously to keep the information up to date.
13

14. The United State Pharmacopoeia(USP)
The USP was originally published in 1820 under the authority of united
state pharmacopoeial convention.
The national Formulary(NF) was published in 1888 under the guidance of
American pharmaceutical Association.
In 1974 the NF was purchased by the United state Pharmacopoeial
Convention and from 1980 onward only one official book of drug standards
was published under the heading The United State Pharmacopoeia and The
National Formulary(NF).
In January 1990, the 22nd edition of USP combined with 17th edition of NF
was published.
The latest edition of USP published on November 1,2012 in English and
became official from May 1,2013.
Highlights and features
• It contains more than 4600 monographs with specifications for identity,
strength, quality, purity, packaging and labelling for substances and dosage
forms.
• It also compromise more than 260 general chapters providing clear, step-
by-step guidance for assays, tests and procedures.
14

15. • More over it also focuses specific charts and combined index which help us
to find the information.
Extra Pharmacopoeia(Martindale)
History- The extra pharmacopoeia was first produced in 1883 by William
Martindale and is known as ‘Martindale’.
Produced by-
The royal pharmaceutical society of Great Britain.
Meant for-
Medical practitioners and pharmacists all over the world.
Sources of information-
Journals and periodicals, licensed product literature, WHO publications
government reports and legislations and other official and standard
publications.
Contain information of-
Drug and medicines, selected investigations and veterinary drugs, herbal
medicines, pharmaceutical excipients, vitamins and nutritional agents,
vaccines, radiopharmaceuticals, contrast media and diagnostic agents,
medicinal gas, drugs of abuse and recreation drugs, toxic substances,
disinfectant and pesticides.
15

16. Impurity
What is impurities……?
 A foreign unwanted matter present in a compound which are differ from the
actual molecular formula.
 According to ICH, an impurity in a drug substance is defined as “any component
of new drug substance that is not the chemical entity defined as the new drug
substance”.
 Chemically a compound is impure if it contains undesirable foreign matter i.e.
Impurities. Thus chemical purity is freedom from foreign matter.
Classification of Impurities
A] According to ICH guidelines, impurities associated with API’s are classified into the
following categories:
• Organic impurities
• Inorganic impurities
• Residual solvents
B] United state Pharmacopoeia
The USP classifies impurities in various sections:
• Impurities in official articles
• Ordinary imourities
• Organic volatile impurities
16

17. Effects of impurities:
Most pure substance is difficult to prepare & that some amount of impurity
always remain in the material. The impurities present in the substances may
extent the following effects:
i. Impurities which are toxic can be injurious when present above certain
limits.
ii. Impurities even present in traces may exert a cumulative toxic effect
after some time.
iii. Impurities can cause incompatibility with other substances.
iv. Impurities may cause a change in physical and chemical properties of
substances thereby making it medicinally useless.
v. Impurities are sometimes harmless. However if present in such large
proportions that the active strength of the substance get lowered, its
therapeutic effect gel decreased.
vi. Impurities though harmless in nature may cause changes in odour,
thereby making the use of substance unhygienic.
vii. Impurities may decrease the shelf life of the product.
viii. Impurities may bring out the technical difficulties in the formulation,
and use of the substance. 17

18. Tests for purity
 Pharmacopoeia prescribes the “test for purity” for pharmaceutical
substances to check their freedom from undesirable impurities.
 Pharmacopoeia will decide and fix the limit of tolerance for these
impurities.
 For certain common impurities for which pharmacopoeia prescribes
the test of purity are:
 Colour, odour, taste
 Physicochemical constants(Iodine value, saponification value,
melting point, refractive index)
 Acidity, alkalinity, Ph
 Humidity (estimation of moisture)
 Cations and anions
 Ash
 Arsenic or lead
 Loss on drying
 Loss on ignition
18

19. Sources of impurities
 Impurities are the substances, present in a confined amount of either in
liquid, gas or solid preparation which differ from the original chemical
compositionof material or compound. These either naturally occurring or
added during synthesis of a chemical product.
 Impurities found in medicinal preparations can bring about the following
changes:
1. Depress the activity of API.
2. Impart colouring or flavouring substances, e.g.sodium salicyalate.
3. Depressed shelf life of API.
4. Physical and chemical property changes in API.
5. Impurities cause incompatibility among ingredients.
A list of impurities which are likely to be present in a given pharmaceutical
substance can be easily complied from the knowledge of the raw materials
employed, the manufacturing process and stability of the final product.
The various sources of impurities in pharmaceutical substances are as
follows:
19

20. Source of
impurities
Raw materials
Method of
manufacturing
- Reagents used
- Intermediate
products
- Reagents used to
eliminate impurity
- Solvents used
- Atmosheric
contamination
Manufacturing
hazards
- contamination from
matter
- cross
contamination
- Contamination by
microbes
- Errors in
manufacturing
Instability of
propducts
- Chemical
instabilities
- Physical
instabilities
- Reaction with
container
- temperature
20

21.  Control of impurities
Pharmacopoeial method: official monographs for pharmaceutical substances
provide description & information in addition to prescribing standards for the
product and its storage conditions. An official monograph for a pharmaceutical
substance generally includes the following:
1. Title
2. Chemical formula
3. Chemical names
4. Category
5. Dose
6. Description
7. Solubility
8. Storage
9. Standards as determined by the assay
10. Identification tests
11. Test for purity including limit tests
12. Assay 21

22. Limit Tests
Why Limit tests???
inorganic impurities are toxic at low levels, and these impurities should be
monitored to ensure safety
Sources of inorganic impurities include those that are deliberately added to
the process (e.g., catalysts),
undetected contaminants from starting materials or reagents
leaching from pipes and other equipment
from naturally derived plant or mineral sources
the level of each inorganic impurity should not exceed the limit
defined in or otherwise specified in the individual monograph.
22

23.  What is Limit Test.......?
Limit = A value or amount that is likely to be present in a substance.
Test = to examine or to investigate
Impurities = a foreign matter present in a compound.
Limit test is defined as quantitative or semi-quantitative test designed to be
identify and control small quantities of impurity which is likely to be present in
the substance.
It is generally carried out to determine the inorganic impurities present in the
compound. These is nothing but to identify the impurities present in the
substance and but also to compare it with the standards.
Specificity of the test
Any test used as a limit test must, give some form of selective reaction with the
trace impurity. Many tests used for the detection of inorganic impurities in
official inorganic chemicals are based upon the separations involved in
inorganic quantitative analysis.
23

24. Sensitivity of the test
The degree of sensitivity required in a limit test varies enormously according to
the standard of purity demanded by the monograph.
The sensitivity of most tests is dependent upon a number of variable factors like
concentration of the solute, precipitating reagent, duration of the reaction,
temperature and the nature and concentration of other substance present in
solution.
Method use for limit tests
Comparison method
Test of this type require a standard containing a definite amount of impurity, to
be set up at the same time and under the same conditions as the test experiment.
In this way the extent of the reaction is readily determined by direct comparison of
the test solution with a standard of known concentration.
E.g. limit test of chlorides, sulphates , arsenic etc.
24

25.  Limit test for Chlorides
Principle: Based on the simple reaction between simple nitrate and soluble
chloride to obtain silver chloride which is insoluble in dilute nitric acid.
Cl- + AgNO3 Dil HNO3 AgCl + NO3
The silver chloride produced the presence of dil.HNO3 makes the test solution
turbid, the extent of turbidity depending upon the amount of chloride present in
the substance.
The standard solution prepared and compare with test solution.
Reasons: Nitric acid is added in the limit test fo chloride to make solution acidic
and helps silver chloride precipitate to make solution turbid at the end of process.
Apparatus required:
1) Nessler cylinder
2) Glass rod
3) Stand
Chemicals required:
1) Dilute nitric acid (10%)
2) Silver nitrate (5%)
3) Sodium chloride
25

26. 26

27. Test sample Standard sample
1. Specific weight of compound is
dissolved in water or solution is
prepared as directed in the
Pharmacopoeia and transferred in
Nesseler cylinder
1. Take 1ml of 0.05845% w/v
solution of sodium chloride in
Nessler cylinder.
2. Add 1ml of dilute nitric acid 2. Add 1ml of dilute nitric acid
3. Dilute to 50ml in Nesseler
cylinder
3. Dilute to 50ml in Nessler cylinder
4. Add 1ml of AgNO3 solution 4. Add 1ml of AgNO3 solution
5. Stir with glass rod and Keep aside
for 5 min
5. Stir with glass rod and Keep aside
for 5 min
6. Observe the opalescence/turbidity 6. Observe the opalescence/turbidity
27
Procedure:
The opalescence in the test and standard solution are compare.

28. Observation :
The opalescence produced 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.
Results and conclusion :
If the opalescence in the sample is less than the standard, it passes the test. If
it is more than the standard, it fails the test.
For comparison opf turbidity for different substances to be used is varied, and
not the standard turbidity. Pharmacopoeia do not give a numerical value to
the limits, as is not practicable as its content will be influenced to great
extent, by large quantities of other substances present.
28

29.  Modified chloride limit test
Glassware's required:
1) Nessler cylinder
2) Measuring cylinder
3) Glass rod
Chemicals required:
1) Dilute nitric acid
2) 0.1M Silver chloride
3) Conc. HCl
4) Distilled water
Principle:
limit test of chloride is based on the precipitation reaction. The precipitates of
chlorides develop on reaction of soluble chloride withsilver nitrate in the
presence of dilute nitric acid to form silver chloride, which appears as solid
particles (opalescence) in the solution. The intensity of turbidity depends on the
amount of chlorides present in test substance.
29

30. Procedure:
With reference to International Pharmacopoeia 6th edition 2016, the limit
test of chloride has been modified in the context of standard solution
preparation. Earlier the standard solution of chloride was prepared by
dissolving sodium chloride(NaCl, known Cl- impurity) but now it has been
modified by using hydrochloric acid (HCl) instead of sodium chloride(NaCl).
HCl + AgNO3 AgCl + HNO3
Hydrochloric Silver Silver Nitric acid
acid nitrate Chloride
Conclusion :
If opalescence produced in sample solution is less than the standard solution,
the sample will pass the limit test of chloride.
30

31. 31
 Limit test for Sulphate
Principle:
The principle involved in the limit test of sulphates is precipitation method and
comparison of test solution with standard solution containing a known amount of
sulphates. The sulphates are precipitated as barium sulphate by reacting them with
barium chloride in the presence of hydrochloric acid. The HCl used prevents the
reaction of other acid radicals with barium chloride. In presence of hydrochloric
acid, only sulphates are precipitated.
SO2-
4 + BaCl2 BaSO4 + 2Cl-
Sulphate Barium Barium Chloride
ion chloride sulphate ion
Due to formation of precipitates, the solution appears turbid and the extent of
turbidity depends on the amounts of sulphates present. If the turbildity produced by
the test is less than that of standard, it means that the sample contains sulphates
within the prescribed limits.
Reagent preparations:
• Barium sulphate Reagent: Dissolve 12 g of BaCl2 .2H2O in 1000 ml of water
to make 0.05 M barium chloride solution. To 15 ml of the prepared solution,
add 55 ml water, 20 ml alcohol, 5 ml of 0.0181% w/v K2SO4 solution and
makeup the volume upto 100 ml.

32. • Standard potassium sulphate solution: Accurately weigh 0.1089 g of
K2SO4 and the volume was makeup up to 100 ml with water.
Procedure:
32
Test sample Standard sample
1. Specific weight of compound is
dissolved in water or solution is
prepared as directed in the
Pharmacopoeia and transferred in
Nesseler cylinder
1. Take 1ml of 0.1089% w/v
solution of potassium sulphate in
Nessler cylinder.
2. Add 2 ml of dilute hydrochloric
acid
2. Add 2 ml of dilute hydrochloric
acid
3. Dilute to 45 ml in Nesseler
cylinder
3. Dilute to 45 ml in Nessler
cylinder
4. Add 5 ml of barium sulphate
reagent
4. Add 5 ml of barium sulphate
reagent
5. Stir with glass rod and Keep
aside for 5 min
5. Stir with glass rod and Keep
aside for 5 min
6. Observe the
opalescence/turbidity
6. Observe the
opalescence/turbidity

33. 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 supersaturation.
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 solutions, the sample will pass the limit test of sulphate.
Conclusion:
If opalescence produced in the standard is more than that of test, the
sample complies with the limit test of sulphate as per IP. 1996.
33

34.  Modified limit test for sulphates
Glassware required:
1) Nessler cylinder
2) Measuring cylinder
3) Glass rod
Chemicals required:
1) Potassium sulphate
2) Test substance
3) Hydrochloric acid
4) Barium sulphate reagent
5) Distilled water
Principle:
The principle involved in the limit test for sulphate is precipitation method.
The sulphates are precipitated as barium sulphate by reacting with barium
chloride in the presence of hydrochloric acid. The HCl used prevents the reaction
of other acid radicals with barium chloride as in the presence of hydrochloric
acid, only sulphates are precipitated.
34

35. SO2-
4 + BaCl2 BaSO4 + 2Cl-
Sulphate Barium Barium Chloride
ion chloride sulphate ion
Due to the formation of precipitates, the solution appears turbid and the extent
of turbidity depends on the amount of sulphates present. If the turbidity produced
by the test is less than thst of standard, it means that the sample contains sulphates
within prescribed limits.
Procedure:
From IP.1996 onwards limit test for sulphate has been modified to great extent.
It has done away the requirement of barium sulphate reagent. However, it still uses
alcohol along with barium chloride to produce comparable turbidity.
Reagent preparation:
• Barium sulphate reagent: Dissolve 12 g of BaCl2 .2H2O in 1000 ml of water to
make 0.05 M barium chloride solution. To 15 ml of the prepared solution, add
55 ml water, 20 ml alcohol, 5 ml of 0.0181% w/v K2SO4 solution and makeup
the volume upto 100 ml.
• Standard potassium sulphate solution: Accurately weigh 0.1089 g of K2SO4
and the volume was makeup up to 100 ml with water.
35

36. • Test sample: Sodium chloride: Dissolve 2 g of sodium chloride in 20 ml of
water.
• Sodium bicarbonate: Dissolve 2 g of sodium bicarbonate in little quantity
of water.
Conclusion:
If opalescence produced in the standard is more than that of test solution,
the sample complies the limit test of sulphate as per IP. 1996.
36

37.  Limit test for Iron
Principle:
The test based upon the reaction of iron in an ammoniacal solution with
thioglycollic acid in a solution buffered with ammonium citrate ether which
forms to deep reddish purple color complex of iron specified amount of substance
from the test is compared by viewing vertically with a standard.
2HSCH2CO2H + Fe+ —————–> Fe [HSCH2COO]2 + 2H+
Thioglycolic acid Ferrous thioglycollate
Iron present as an impurity in the sample reacts with thioglycollic sacid to
form ferrous thioglycollate which is responsible for the appearance of purple
colour in the solution. This reaction will only occur if the given sample contains
iron as an impurity even in trace amount.
Note:
1. Citric acid reacts with ammonia solution, giving ammonium citrate, which act
as a buffer. The citrate ion also prevents the precipitation of iron by ammonia
by forming a complex with it.
2. Thioglycollic acid not only react with ferrous ions, but it also reduces ferric
ions to ferrous ions. 37

38. Test sample Standard sample
1. Sample is dissolved in specific
amount of water & then volume is
made up to 40 ml
1. 2 ml of standard solution of iron
diluted with water up to 40 ml
2. Add 2 ml of 20% w/v of citric acid
(iron free).
2. Add 2 ml of 20% w/v of citric acid
(iron free).
3. Add 2 drops of thioglycollic acid. 3. Add 2 drops of thioglycollic acid.
4. Add ammonia to make the
solution alkaline and adjust the
volume to 50 ml
4. Add ammonia to make the
solution alkaline and adjust the
volume to 50 ml
5. Stir with glass rod and Keep aside
for 5 min
5. Stir with glass rod and Keep aside
for 5 min
6. Color developed is viewed
vertically and compared with
standard solution.
6. Color developed is viewed
vertically and compared with
standard solution.
38
Procedure:
Earlier amoniumthiocynate reagent was used for the limit test of iron. Since
thioglycollic acid is more sensitive reagent, it has replaced ammonium
thiocyanatein the test.

39. Observation:
The purple colour produce in sample solution should not be greater than
standard solution. If purple colour produces in sample solution is less than the
standard solution, the sample will pass the limit test of iron.
Reasons:
• Citric acid helps precipitation of iron by ammonia by forming a complex
with it.
• Thioglycollic acid helps to oxidize iron(II) to iron(III).
• Ammonia to make solution alkaline.
Conclusion:
If the intensity of purple colour in standard is more than that in the test, the
sample complies limit test of iron as per IP. 1996
39

40.  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 thiocarbazone
(dithizone) in alkaline solution to form lead dithizone complex which is red in colour.
Dithizone is green in colour in chloroform and lead-dithizone complex is violet in
colour, so the resulting colour at the end of process is red.
Lead ion Dithizone Lead dithizonate
Lead present as an impurities in the sample reacts with dithizone to form lead
thizonate which is responsible for the appearance of violet colour in the solution. This
reaction will only occur if the given sample contains lead as an impurity even in trace
amount. In this test, nesseler cylinder are not used, instesd it is performed by
extraction with the help of separating funnel.
40

41. The original colour of dithizone in chloroform is green while the lead dithizonate
complex is violet in colour. The intensity of the violet colour of the complex
depending upon quantity of lead present in the solution is compared with that of the
standard colour produced by trating standard solution containing definite amount of
lead in the similar manner.
Intensity of this violet colour is compared with the both tests and standard
solution in a chloroform solvent medium.
Reagent Preparation:
• Preaparation of standard lead solution (1 PPM Pb): Dissolve 0.400 g of lead
nitrate in water containing 2 ml of dilute nitric acid and add sufficient water to
produce 250 ml. this gives standard lead solution (1% Pb). Satndard lead solution
(1 PPM Pb) is prepared by dilute in 1 volume of standard lead solution (1% Pb) to
1000 volumes with water.
• Preparation of dithizone extraction solution: Dissolve 30 mg dithizone in 1000
ml of chloroform and add 5 ml of ethanol (95%). The solution is stored in
refrigerator. Before use, the solution is shaked with about half of its volume of
1% v/v nitric acid solution and the acid is discarded.
• Preparation of dithizone standard solution: Dissolve 10 mg of dithizone in
1000 ml of chloroform.
41

42. Procedure:
42
Test sample Standard compound
1. A known quantity of sample solution
is transferred in a separating funnel
1. A standard lead solution is prepared equivalent to
the amount of lead permitted in the sample under
examination
2. Add 6ml of ammonium citrate 2. Add 6ml of ammonium citrate
3. Add 2 ml of potassium cyanide and 2
ml of hydroxylamine hydrochloride
3. Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 drops of phenol red Add 2 drops of phenol red
4. Make solution alkaline by adding
ammonia solution.
4. Make solution alkaline by adding ammonia
solution.
5. Extract with 5 ml of dithizone until it
becomes green
5. Extract with 5 ml of dithizone until it becomes
green
6. Combine dithizone extracts are
shaken for 30 mins with 30 ml of nitric
acid and the chloroform layer is
discarded
6. Combine dithizone extracts are shaken for 30
mins with 30 ml of nitric acid and the chloroform
layer is discarded
7. To the acid solution add 5 ml of
standard dithizone solution
7. To the acid solution add 5 ml of standard
dithizone solution
8. Add 4 ml of ammonium cyanide 8. Add 4 ml of ammonium cyanide
9. Shake for 30 mins 9. Shake for 30 mins
10. Observe the color 10. Observe the color

43. Observation:
The intensity of the colour complex, is depends on the amount of lead in the
solution. The colour produce in sample solution should not be grater than standard
solution. If colour produces in sample solution is less than the standard solution, the
sample will pass the limit test of lead.
Reasons:
Ammonium citrate, potassium cynide, 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 colour 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.
Conclusion:
If colour produced in the sample solution is less than the standard solution, the
sample will pass the limit test of lead.
43

44.  Limit test for Heavy Metals
Principle:
Methods I, II and III are based upon the reaction of heavy metal ion with
hydrogen sulphide (in methods I and II) or szodium sulphide (in method III)
leading to theformation of heavy metal sulphides. The metal sulphides remain
distributed in a colloidal state and give rise to brownish colour. The colour
produced in the test solution is compared with that of the standard solution
containing a definite amount of heavy metal.
Reagent preparation is based upon the precipitation of relatively insoluble and
characteristically coloured sulphides of heavy metals when aqeuous solutions are
treated with alkali metal sulphides.
Heavy metal Hydrogen sulphide heavy metal sulphide
Procedure:
The Indian Pharmacopoeia has adopted three methods for the limit test of
Heavy metals.
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45. 45

46. Method I: Use for the substance which gives clear colorless solution under the
specific condition.
Observation:
The color produce 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 heavy metals 46
Test sample Standard sample
1. Dissolve 4 g of sample (dextrose) in
25 ml of water and transferred in
Nessler’s cylinder
1. Take 2 ml of standard lead solution
and dilute to 25 ml with water and
transferred to Nesseler’s cylinder.
2. Adjust the pH between 3 to 4 by
adding dilute acetic acid ‘Sp’ or dilute
ammonia solution ‘Sp’(Sp= specificity
for laboratopries)
2. Adjust the pH between 3 to 4 by
adding dilute acetic acid ‘Sp’ or dilute
ammonia solution ‘Sp’
3. Dilute with water to 35 ml 3. Dilute with water to 35 ml
4. Add freshly prepared 10 ml of
hydrogen sulphide solution
4. Add freshly prepared 10 ml of
hydrogen sulphide solution
5. Dilute with water to 50 ml 5. Dilute with water to 50 ml
6. Allow to stand for five minutes 6. Allow to stand for five minutes
7. View downwards over a white surface7. View downwards over a white surface

47. Method II: Use for the substance which do not give clear colorless solution under the
specific condition.
47
Test sample Standard compound
1. Weigh 2 g of sample(Paracetamol), moisten
with sulphuric acid and ignite on a low flame till
completely charred.
a) Add few drops of nitric acid and heat to 500 °C.
Allow to cool and add 4 ml of hydrochloric acid
and evaporate to dryness
b) Moisten the residue with 10 ml of hydrochloric
acid and digest for two minutes
c) Neutralize with ammonia solution and make
just acid with acetic acid
1. Take 2 ml of standard lead solution and dilute
to 25 ml with water
2. Adjust the pH between 3 to 4 and filter if
necessary
2. Adjust the pH between 3 to 4 by adding dilute
acetic acid ‘Sp’ or dilute ammonia solution ‘Sp’
3. Dilute with water to 35 ml 3. Dilute with water to 35 ml
4. Add freshly prepared 10 ml of hydrogen
sulphide solution
4. Add freshly prepared 10 ml of hydrogen
sulphide solution
5. Dilute with water to 50 ml 5. Dilute with water to 50 ml
6. Allow to stand for five minutes 6. Allow to stand for five minutes
7. View downwards over a white surface 7. View downwards over a white surface

48. Observation:
The color produce 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 heavy metals and vice versa.
Method III: Use for the substance which gives clear colorless solution in sodium
hydroxide solution.
48
Test sample Standard sample
1. Solution is prepared as per the
monograph and 25 ml is transferred in
Nessler’s cylinder or weigh specific
amount of substance and dissolve in 20
ml of water and add 5 ml of dilute sodium
hydroxide solution
1. Take 2 ml of standard lead solution
2. Make up the volume to 50 ml with water 2. Add 5 ml of dilute sodium hydroxide
solution and make up the volume to 50 ml
with water
3. Add 5 drops of sodium sulphide solution 3. Add 5 drops of sodium sulphide solution
4. Mix and set aside for 5 min 4. Mix and set aside for 5 min
5. View downwards over a white surface 5. View downwards over a white surface

49. Observation:
The color produce 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 heavy metals.
Reagent preparation:
• Standard solution: Pipette out 10 ml of either standard lead solution (1 PPM Pb) or
standard lead solution (2 ppm pb) into a small nessler cylinder labelled as “standard”.
Add 2 ml of test solution and mix.
• Test solution: Prepare as directed in the individual monograph and pipette out 12 ml
into a nessler cylinder labelled as “test”.
• Preparation of standard lead solution: Dilute 1 volume of standard lead solution
(20 ppm pb) to 10 volumes with water.
• Preparation of thioacetamide reagent: To 0.2 ml of 4% w/v thioacetamide
solution, add 1 ml of mixture of 15 m,l of 1M sodium hydroxide, 5 ml of water and
20 ml of glycerine (85%). Heat on a water bath for 20 seconds, cool and use
immediately.
• Preparation of acetate buffer of Ph 3.5: Dissolve 25 g of ammonium acetate in 25
ml of water and add 38 ml of 7M hydrochloric acid. Adjust the ph to 3.5 by using 3M
hydrochloric acid or 6M ammonia and dilute upto 100 ml with water.
49

50. 50