NIET (PHARMACY INSTITUTE) Pharmaceutical Inorganic Chemistry (IPC) BP-104T

Unit 1
Impurities in pharmaceutical Substances: History of Pharmacopoeia, Sources and types of
impurities, principle involved in the limit test for Chloride, Sulphate, Iron, Arsenic, Lead and
Heavy metals, modified limit test for Chloride and Sulphate.

History of Pharmacopoeia
Pharmacopoeia
The word “pharmacopoeia” is derived from the two Greek words ‘Pharmakon’ meaning ‘a drug
or medicine or charm’ and ‘poiein or poeia’ means ‘to make’ or ‘how to make’. Literally it
means that it is a list of medicinal substances, crude drugs and formulae for making preparations
from them.
Father of Pharmacopoeia: Charles Rice (also he was Father of National Formulary).
Father of First Pharmacopoeia: Su Jing who works with a team of 23 Pharmaceutical
Scientist in 659 AD of Tang Dynasty (618-907 AD).
In 1833 a committee of East Indian Company’s Dispensary recommended the publication of
Pharmacopoeia and Bengal Pharmacopoeia and general conspectus of medicinal plants was
published in 1844 which contains only indigenous remedies (native; naturally from a particular
place).

United States of Pharmacopoeia

The United States Pharmacopoeia (USP) is a pharmacopoeia (compendium of drug information)
for the United States published annually by the United States Pharmacopoeial Convention, a
nonprofit organization that owns the trademark and copyright.
A monograph published in any USP compendium (in a book, CD-ROM, or online) provides:
 Name of a substance and its definition,
 Package, storage, and labeling requirements
 Information on test needed to ensure the substances is of the appropriate strength, quality
and purity.
The First edition of USP was published on 15th December 1820 in both Latin & English
languages.

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 It was published at Ten years intervals from 1820 to 1942.

 It was published at Five years intervals from 1942 to 2000.
 It was published annually from 2002.
 Current version of USP-NF was published in 1st May 2022.
In 1888 the first National formulary was published by American Pharmaceutical Association. A
single official standard publication for prescription drugs has been published each year since
1980 by United States Pharmacopoeial Convention, under the name United States
Pharmacopoeia and the National Formulary (USP-NF).
Year Edition Volumes Supplements/ Description
Addendum
January USP21- - 08 Last supplement in 1988
1985 NF16 supplements
1990 USP22- 1 - Third revision that consolidates USP
NF17 & NF into a single volume
1992 USP-NF - - Electronic version on floppy disks
1994 USP23- 1 10 Published in Mumbai as an Asian
NF18 supplements Edition .
First supplement in January 1995
and last in May 1999
2000 USP24- 1 - -
NF19
2006 USP29-NF 1 - In Spanish language
May 2007 USP30- 3 2  It contains Scientific standards for
NF25 drugs, dietary substances,
biological products and excipients
used in dosage forms.
 It contains 4,100 monographs and
200 general chapters.
 Volume I contains general chapters
& Volume II & III contains

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monographs. Each volume have

index and table of contents.
 First supplement in August
November & Second in 2007
(official from 2008)
 Convenient slipcase for easy
access and storage (English edition
only).
2008 USP32- 3 -  More than 4,200 monographs.
NF27  Includes over 200 general chapters
covering general test and assays.
 Creates in-house standards for
operating procedures and
specifications.
 Enables validation of test results
against proven benchmarks.
2009 USP33- 3 -  More than 4,400 monographs
NF28  Helpful guides and charts that
make it easy to find focus specific
information.
 Facilitates new product
development and approval.
2010 USP34- 3 -  More than 4,500 monographs.
NF29  More than 230 general chapters
with current guidelines for the full
range of laboratory tests and
established processes for validating
methods.
2017 USP40- 4 2  More than 4,900 monographs with
NF35 specifications for identity, quality,

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purity, packaging, and labeling for

substances and dosage forms.
 More than 300 general chapters.
 Include general chapters of 800
hardazous drugs handling in
healthcare settings

Extra Pharmacopoeia
Martindale: The Complete Drug Reference is the alternate reference book for drugs and
medicines.
It originally produced by William Martindale in 1883 and now published by the
Pharmaceutical Society of Great Britian, contains information on the drugs presently used in
Great Britian.
Martindale contains information on drugs in clinical use as well as:
a) Selected investigational and veterinary drugs
b) Herbal and complementary medicines
c) Pharmaceutical excipients
d) Vitamins and nutritional agents
e) Vaccines
f) Radiopharmaceuticals
g) Contrast media and diagnostic agents
h) Medicinal agents
i) Drugs of abuse and recreational drugs
j) Toxic substances
k) Disinfectants and pesticides
Aim of Martindale:
 To update information for pharmacist, physician in all subjects.
 To provide information of official, unofficial, proprietary preparations currently in use.
General section of EP contains about 1,400 pages of drug descriptions listed in alphabetical order
under English titles.

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Monographs of EP include
1. Chemical Abstracts Service (CAS)
2. Anatomical Therapeutic Chemical Classification System (ATC) numbers
3. FDA Unique Ingredient Identifiers (UNII)
38th Edition of Martindale: The Complete Drug Reference
This was published in June 2017. It is arranged into two main parts followed by three extensive
indexes:

1. Monographs
 On drugs and ancillary substances, listing over 6,000 monographs arranged in 49
chapters based on clinical use with the corresponding disease treatment reviews.
 Monographs summarized the nomenclature, properties, and actions of each
substance.
 A chapter on supplement drugs and other substances covers some 1190
monographs on new drugs, those not easily classified, herbals and drugs no longer
clinically but still of interest.
 Monographs of some toxic substance are also included
2. Preparations: including over 1,80,000 items from 43 countries and regions including
china.
3. Directory of Manufactures: listing some 20,000 entries.
4. Pharmaceutical terms in various languages: list nearly 5,600 pharmaceutical terms and
routes of administration in 13major European languages as an aid to the no-native
speaker in interpreting packaging, product information, or prescriptions written.
5. General Index: prepared from 1,75,000 entries it includes approved name, synonyms
and chemical names, a separate Cyrillic section lists nonproprietary and proprietary
names in Russian and Ukrainian.
6. Digital Versions: include an additional 1,000 drug monographs, 60,000 preparation
names, and 5,000 manufactures.
Uses:
 It provides a useful source of information for patients arriving from abroad to identify
their existing medications

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 This may reveal that a currently taken proprietary preparation is available under another
brand name.
 Alternatively if the drug is not available, the class of agent can be determined allowing a
pharmacist or doctor to determine which alternative equivalent drugs can be substituted.

Impurities in Pharmaceutical Substances

Impurities
A compound is said to be impure if it is having foreign matter. Pure chemical compound refer to
that compound which is having no foreign matter i.e., impurities.
Impurity is any material that affects the purity of the material of interest. Presence of impurities
in the pharmaceutical substances may produce toxic effects on the body and may also lower
down the active strength of the pharmaceutical substances.
Types of Impurities
According to ICH guidelines, impurities related to drug substances can be classified into three
main categories: organic impurities, inorganic impurities, and residual solvents.
1. Organic Impurities: Organic impurities can arise in APIs or drug product formulations
during the manufacturing process or during the storage of drug substances. They may be
known, unknown, volatile, or nonvolatile compounds with sources including starting
materials, intermediates, unintended byproducts, and degradation products. They may
also arise from racemization— the contamination of one enantiomeric form with another.
In all cases, they can result in undesired biological activity or toxicity.
2. Inorganic Impurities: Elemental impurities are unwanted elements that occur in drug
formulations. They can arise from active pharmaceutical ingredients, raw materials,
synthetic additives, excipients, catalysts, and production processes and equipment used
during manufacturing.
Ex- Iron, Sulphate, Chloride, Arsenic, Cadmium, Lead, etc
3. Residual Solvents: Residual solvents are the volatile organic chemicals used during the
manufacturing process, or generated during drug production. Several organic solvents
used in the synthesis of pharmaceutical products are known to have toxic or
environmentally hazardous properties, and their complete removal can be very difficult.
Ex- Benzene, carbon tetrachloride, toluene, pyridine, etc.

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Sources of Impurities in Pharmaceuticals substances

There are following sources of impurities in pharmaceuticals are listed below:
1. Raw materials employed in the manufacturing of the Pharmaceutical substances
2. Reagents employed in the manufacturing process
3. Method/process used in manufacture or method of manufacturing
4. Chemical processes used in the manufacture
5. Atmospheric contamination during the manufacturing process
6. Intermediate products in the manufacturing process
7. Defects in the manufacturing process
8. Inadequate Storage conditions
9. Decomposition of the product during storage
10. Manufacturing hazards
11. Accidental substitution or deliberate adulteration with spurious or useless materials.

Raw material employed in the manufacturing of the Pharmaceutical substances

Impurities known to be associated with these chemicals may be carried through the
manufacturing process and contaminate the final product.
Example
a. Rock salt used for the preparation of sodium chloride is contaminated with small amounts
of calcium sulphate and magnesium chlorides, so that sodium chloride prepared from
rock salt will definitely contain traces of calcium and magnesium compounds impurities.
Rock Salt CaSO4 (Calcium Sulphate )+ MgCl2 (Magnesium Sulphate) = NaCl

b. Impurities such as arsenic, lead and heavy metals are present in raw materials and hence
are found in substances. Copper turnings are known to have Iron and Arsenic as
impurities.
So, it is necessary to use pure chemicals and substances as raw materials for the manufacturing
process.
Reagents employed in the manufacturing process
If reagents employed in the manufacturing process are not completely removed by washing,
these may find entry into the final products.

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Example

a. Calcium carbonate contains ‘soluble alkali’ as impurity which arises from the sodium
carbonate (Na2CO3) employed in the process. Calcium carbonate is prepared by the
interaction of a soluble calcium salt with a soluble carbonate. Therefore, the final product
(CaCO3) is liable to contain small amount of ‘soluble alkali’ as impurities which were
not removed by the washing process.

CaCl2 + Na2CO3 = CaCO3 + 2 NaCl

(Soluble) (Soluble) (Precipitate) (Soluble)

b. Ammoniated mercury may be prepared by adding a solution of Mercuric chloride to

dilute ammonia solution.
HgCl2 + 2 NH4OH = NH2HgCl + NH4Cl + 2H2O
Soluble Soluble Precipitate Soluble
Method/process used in manufacture or method of manufacturing
The process or method of manufacture may introduce new impurities into the final product
arising due to contamination by reagents, catalysts and solvents employed at various stages of
the manufacturing process. The new impurities may also arise from the reaction vessels and
reaction intermediates.
Such as
a. Reagents employed in the manufacturing process
Ex- Soluble alkali in calcium carbonate arises from sodium carbonate used in the process.
b. Reagents added to remove other impurities
Ex- Potassium bromide contains traces of Barium, which is added in the manufacturing
process to remove excess of sulphate.
c. Solvents: Ex- Water
d. Action of solvents and reagents on reaction vessel.
Chemical processes used in the manufacture
For the synthesis of drugs, many chemical reactions such as nitration, halogenations, oxidation,
reduction, and hydrolysis are involved in which different chemicals are used.

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Example: Tap water is generally used in the various processes and it is often having Cl-, Mg+2,
Ca+2 ions which are generally found in the substances which is being manufactured.
Atmospheric contamination during the manufacturing process
Atmosphere may contain dust aluminum oxide, sulphur, silica, soot etc.) and some gases like
carbon dioxide, sulphur dioxide, arsine and hydrogen sulphide. These may contaminate the final
product during the manufacturing process. Some substances which are susceptible to action by
atmospheric carbon dioxide and water may get contaminated with them during their preparation
Example
a. Sodium hydroxide readily absorbs atmospheric carbon dioxide when exposed to
atmosphere.
2NaOH + CO2 → Na2CO3 + H2O
b. Calcium hydroxide solutions can absorb carbon dioxide from the atmosphere to form
calcium carbonate.
Ca(OH)2 + CO2 → CaCO3 + H2O

Intermediate products in the manufacturing process

Sometimes, an intermediate substance produced during the manufacturing process may
contaminate the final product.
Example
a. Sodium bromide is prepared by reaction of sodium hydroxide and bromine in slight
excess.
6NaOH + 3Br2 → NaBrO3 + 5NaBr + 3H2O
The sodium bromate an intermediate product is reduced to sodium bromide by heating
the residue (obtained by evaporating the solution to dryness) with charcoal.
NaBrO3 + 3C → NaBr + 3CO
(Sodium bromate) (Sodium bromide)
If sodium bromate is not completely converted to the sodium bromide then it is likely to
be present as an impurity.
Manufacturing Hazards
If the manufacturer is able to control and check impurities from the all above mentioned sources
there exists certain manufacturing hazards which can lead to product contamination. The various
manufacturing hazards can lead to:

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(i) Contamination from the particulate matter: The unwanted particulate matter can arise by a
number of ways, such as accidental inclusion of dirt or glass, porcelain, plastic or metallic
fragments from sieves, granulating, tabletting and filling machines and the product container.
The particulate contamination mainly arises from the wear and tear of the equipments. It may
also arise from the bulk materials used in the formulation or from dirty or improperly maintained
equipments .
e.g., metal particles found in eye ointments packed in metal tubes made up of tin and aluminium.
(ii) Cross-contamination of the product: This manufacturing hazard has to be considered in the
preparation of solid dosage forms. Cross-contamination of product can occur by air-born dust
arising out of handling of powders, granules and tablets in bulk. Cross-contamination is
dangerous particularly in case of steroidal and other synthetic hormones and therefore, it should
be carefully controlled. Precautions, such as use of face mask and special extraction equipment
can minimize these undesirable contaminations.
(iii) Contamination by microbes: Many products, like liquid preparations and creams intended
for topical applications are liable to contamination by microbes from the atmosphere during
manufacturing. For all products intended for parenteral administration and ophthalmic
preparations, sterility testing is done and it provides an adequate control for microbial
contaminations in such preparations. Microbial contamination can be controlled by adding
suitable antimicrobial and antifungal agents.
(iv) Errors in the manufacturing process: Sometimes in a liquid preparation, there is
incomplete solution of the solute. This ought to be detected by the normal analytical methods as
it can lead to major error. A proper check on the efficiency of mixing, filling, tabletting,
sterilization etc. should be exercised in order to obtain a product of maximum purity and desired
quality. Special precautions are required to be observed to avoid mixing and filling errors in the
preparation of low dosage forms such as tablets and capsules containing highly potent
medicaments.
(v) Errors in the packaging: Similar looking products, such as tablets of the same size, shape
and colour, packed in similar containers can result in mislabeling of either or both of the
products. Adequate care should be taken to avoid the handling of such products in the close
proximity
Decomposition of the product during storage

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A. Chemical instability: Impurities can also arise during storage because of chemical
instability of the pharmaceutical substance. Many pharmaceutically important substances
undergo chemical decomposition when storage conditions are inadequate. This chemical
decomposition is often catalyzed by light, traces of acid or alkali, traces of metallic
impurities, air oxidation, carbon dioxide and water vapours.
B. Changes in physical properties: Pharmaceuticals may undergo changes in physical
properties during storage. There can be changes in crystal size and shape, sedimentation,
agglomeration and caking of the suspended particles. These physical changes are not
always avoidable and may result in significant changes in the physical appearance,
pharmaceutical and therapeutic effects of the product.
C. Reaction with container material: The possibility of reaction between the container
material and the contents cannot be ruled out as it constituents a safety hazard.
Preparations susceptible to reaction with metal surfaces e.g., salicylic acid ointment must
not be packed in metal tubes.
D. Temperature: The rate of chemical decomposition and physical changes of stored
products depends upon the temperature. The susceptible substances may have
temperature storage requirements assigned to them in order to protect them against
undesirable decomposition.

Limit Test

 Limit test is defined as quantitative or semi quantitative tests designed to identify and
limit/control small quantities of impurity which is likely to be present in the substance.
Limit test is generally carried out to determine the inorganic impurities present in
compound.
 All the limit test that are prescribed in the pharmacopoeias are based on the comparison
of standard turbidity or colour with that of the sample under test.
 Limits are prescribed in parts per million (ppm).
 For the preparation of standard turbidity or colour the pharmacopoeias prescribe the limit
of particulate impurities for substances and it varies for different compounds.

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 The amount of test samples to be taken in mentioned in the individual monograph of the
pharmacopoeias.
Types of Limit Test
There three types of limit test as:
1. Test in which there is no visible reaction: Testing as prescribed, there is no colour,
opalescence or precipitate.
2. Comparison Methods: Compare the amount of impurity in the substance with a standard
of known concentration and determine whether impurity is within or the excess of the
limit prescribed.
3. Quantitative Determination: Amount of impurity present in actually determined and
compared with the numerical limit given in Pharmacopoeia.

Limit Test of Chlorides

Principle: The limit test of chloride involves the reaction of silver nitrate with soluble
chlorides to form the precipitate of silver chloride which is insoluble in dilute nitric acid. The
extent of precipitation (turbidity/opalescence) depends upon the amount of silver chloride
formed. i.e. on the amount of chloride ions present in the substance. The opalescence
produced is compared with a reference opalescence obtained by a standard silver nitrate
solution, under the same experimental conditions.
NaCl + AgNO3 = AgCl + NaNO3
Soluble Precipitate

Preparation of reagents
1. 0.1M Silver nitrate solution: Dissolve 17.0 g of silver nitrate in sufficient water to 1000
mL. (Store in light resistant containers)
2. Dilute Nitric acid: contains approximately 10 % w/w of HNO3. Dilute 106 mL of nitric
acid to 1000 mL with water.

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Procedure
Standard Solution Test Solution
1 mL of a 0.05845 % w/v solution of sodium Dissolve the specified quantity of the
chloride in Nessler cylinder substance in distilled water and transfer to
Nessler cylinder
Add 10 mL dilute nitric acid Add 10 mL dilute nitric acid
Diluted to 50 mL with water and add 1 mL of Diluted to 50 mL with water and add 1 mL of
silver nitrate solution silver nitrate solution
Stirr immediately with a glassrod and stand for Stirr immediately with a glassrod and stand for
5 minutes 5 minutes

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: Nitric acid is added in the limit test of chloride to make solution and helps silver
chloride precipitate to make solution turbid at the end of process. It prevents the precipitation
caused by silver carbonate or silver hydroxide which may results due to alkaline impurities in the
solution.

Limit Test of Sulphates

Principle: This test involves the reaction between barium chloride and soluble sulphate in the
presence of dilute hydrochloric acid. The turbidity formed by a given amount of sample is
compared with a reference turbidity obtained from an authentic amount of the sulphate under the
same experimental conditions.

BaCl2 + Na2SO4 = BaSO4 + 2 NaCl

Soluble Precipitate

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Preparation of Reagents

1. Barium sulphate reagents: 15 mL of 0.5 M barium chloride, 55 mL of water, and 20

mL of sulphate-free alcohol are mixed, 5 mL of a 0.0181 %w/v solution of potassium
sulphate are added, diluted to 100 mL with water and mixed. It must be freshly prepared.
2. 0.5 M Barium chloride: Barium chloride dissolved in water to contain in 1000 mL 122.1
gm of BaCl2.2H2O.

Procedure

Standard Solution Test Solution

1mL of a 0.1089 % w/v solution of potassium The specific quantity of the substance is
sulphate in a Nessler cylinder dissolved in water and transfer to a Nessler
cylinder.
Add 2 mL of dilute hydrochloric acid Add 2 mL of dilute hydrochloric acid
Diluted to 45 mL with water Diluted to 45 mL with water
Add 5 mL of Barium sulphate reagent Add 5 mL of Barium sulphate reagent
Stirr immediately using a glass rod and Stirr immediately using a glass rod and
allowed to stand for 5 minutes. allowed to stand for 5 minutes.
Observed turbidity in the solution Observed turbidity in the solution

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

1. Hydrochloric acid helps to make solution acidic.

2. Potassium sulphate is used to increase the sensitivity of the test by giving ionic.
3. Concentration in the reagent and alcohol helps to prevent super saturation.

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Limit Test of Iron

The limit test for iron depends on the reaction of iron in ammonical solution in the presence of
citric acid, with thioglycolic acid to obtain a pale pink to deep reddish purple colour. Citric acid
forms a complex with iron. Citric acid forms a complex with iron which is not precipitated by
ammonia. The colour is obtained due to formation of a ferrous salt Fe (S.CH2COO-)2, which
disappears in air due to oxidation.

Principle: Limit test of iron is based on the reaction of iron in ammonical solution with
thioglycolic acid in presence of citric acid to form iron thioglycolate which is pale pink to deep
reddish purple in colour.

2 HSCH2COOH + Fe3+ = Fe (HSCH2COO)2 + 2 H+

Thioglycollic acid (HS.CH2COOH) is a sulphur derivative of glycolic acid. It is a colourless

liquid with unpleasant odour. The coloured produced from a known amount of the substance is
compared with the standard colour obtained from a known amount of iron under the same
experimental conditions.

Preparation of Reagents

Standard Iron solution: 0.1726 g of ferric ammonium sulphate is weighed and dissolved in 10
mL of 0.1 N sulphuric acid and sufficient water to produce 1000 mL. Each mL of this solution
contains 0.02 mg of Fe.

Procedure

Standard Solution Test Solution

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2 mL of standard solution of iron diluted with The specific amount of substance is dissolved
water upto 40 mL in a Nessler cylinder in water upto 40 mL in Nessler cylinder
Add 2 mL of 20 % w/v of citric acid (iron free) Add 2 mL of 20 % w/v of citric acid (iron free)
Add 0.1 mL (2 drops) of thioglycollic acid Add 0.1 mL (2 drops) of thioglycollic acid
Add ammonia to make the solution alkaline Add ammonia to make the solution alkaline
and adjust the volume to 50 mL with water and adjust the volume to 50 mL with water
Allowed to stand for 5 minutes Allowed to stand for 5 minutes
Colour developed is observed Colour developed is observed compared with
standard solution

Observation

The purple (violet) 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

1. Citric acid helps precipitation of iron by ammonia by forming a complex with it.
2. Thioglycollic acid act as reducing agent which do reduction and converts iron (III) to iron
(II) [Ferric to Ferrous].
3. Ammonia use to make solution alkaline.

Limit Test for Heavy Metals

The test for heavy metals is designed to determine the content of metallic impurities that are
coloured by sulphide ion, under specified conditions. The limit for heavy metals is indicated in
the individual monographs in terms of the parts of lead per million parts of the substance (by
weight), as determined by visual comparison of the colour produced by the substance with that of
a control prepared from a standard lead solution.
The amount of heavy metals is determined by one of the following methods:
A. Method A: Used for substance that yields clear, colourless solutions under the specified
test condition.

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B. Method B: Used for the substance that do not yield clear, colourless solutions under the
test conditions specified method A, or for substance which by virtue of their complex
nature, interfere with the precipitation of metals by sulphide ion.
C. Method C: Used for substances that yield clear, colourless solution with sodium
hydroxide solution.
Preparation of Reagents
1. Dilute acetic acid: contains approximately 6 % w/w of acetic acid. Dilute 57 mL of
glacial acetic acid to 1000 mL with water.
2. Dilute ammonia Solution: contains approximately 10 % w/w of ammonia. Dilute 425
mL of strong ammonia solution to 1000 mL. Store in well-closed containers in a cool
place.
Procedure
Method A
Standard Solution Test Solution
2 mL standard solution of heavy metals is The sample solution is prepared as per the
pipette out and transfers into a Nessler monograph and 25 mL of solution is
cylinder. transferred into a Nessler’s cylinder.
Add dilute acetic acid or ammonia solution to Add dilute acetic acid or ammonia solution to
adjust the pH between 3.0 to 4.0. adjust the pH between 3.0 to 4.0.
Dilute to 35 mL with distilled water and mix Dilute to 35 mL with distilled water and mix
well. well.
Add 10 mL of freshly prepared hydrogen Add 10 mL of freshly prepared hydrogen
sulphide solution and mix. sulphide solution and mix.
Dilute to 50 mL with distilled water. Dilute to 50 mL with distilled water.
Allow to stand for 5 min and view downwards Allow to stand for 5 min and view downwards
over a white background. over a white background.

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Method B
Standard Solution Test Solution
2 mL standard solution of heavy metals is Transfer the prescribed quantity of sample into
pipette out and transfers into a Nessler a crucible.
cylinder.
Add dilute acetic acid or ammonia solution to Moisten the sample with sulphuric acid and
adjust the pH between 3.0 to 4.0. ignite carefully at low temperature until
complete charring of the sample.
Dilute to 35 mL with distilled water and mix Add 2 mL of nitric acid and 5 drops of
well. sulphuric acid heated cautiously until white
fumes are no longer.
Add 10 mL of freshly prepared hydrogen Ignited on a muffle furnance at 500 to 600 °C
sulphide solution and mix. until carbon completely burnt and cool it.
Dilute to 50 mL with distilled water. Add 4 mL of dilute hydrochloric acid and
digest for 2 minutes on water bath (evaporate
to dryness).
Allow to stand for 5 min and view downwards Add dilute acetic acid or ammonia solution to
over a white background. adjust the pH between 3.0 to 4.0.
Dilute to 35 mL with distilled water and mix
well.
Add 10 mL of freshly prepared hydrogen
sulphide solution and mix.
Dilute to 50 mL with distilled water.
Allow to stand for 5 min and view downwards
over a white background.

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Method C
Standard Solution Test Solution
2 mL of standard solution is pipette out and The required quantity of sample is dissolved in
transfer into the Nessler cylinder. Add 5 mL the mixture of 20 mL water and 5 mL sodium
sodium hydroxide solution. hydroxide solution as per monograph.
Make up to 50 mL with distilled water. Make up to 50 mL with distilled water.
Add 5 drops of sodium sulphide solution, mix Add 5 drops of sodium sulphide solution, mix
well and kept aside for 5 minutes. well and kept aside for 5 minutes.
View downwards over a white background. View downwards over a white background.

Observation
If the colour produced in the test solution is not more intense than that of standard solution, the
sample complies with the standards of IP or vice versa.

Reasons
1. Acetic acid and Ammonia is used for adjusting the pH of solution.
2. Dil. Hydrochloric acid used for digestions in the limit test of heavy metals.

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