Impurities in pharmaceutical substances

IMPURITIES IN PHARMACEUTICAL SUBSTANCES
By
Mr. Dukre Tushar Pradip
M. Pharmacy
Assistant Professor
Shri Swami Samarth Institute of
Pharmacy (B. Pharm),
Malwadi, Bota.

POINTS ON UNIT – I:
 History of Pharmacopoeia,
 Sources and types of impurities,
 Principle involved in the limit test
 Chloride,
 Sulphate,
 Iron,
 Arsenic,
 Lead and Heavy metals, &
 Modified limit test for Chloride and Sulphate

History of Pharmacopoeia
 The word Pharmacopoeia is derived from Greek words ‘pharmakon’
= a drug and ‘poein’ = to make or create.
 Pharmacopoeia is book containing directions for identification of
samples and the preparation of compound medicines, and published
by the authority of a management or a medical or pharmaceutical
society.
 History:
 Each country has some rules on pharmaceutical preparations which
sets standards and required quantity indices for medicament, raw
materials and preparations employed in the manufacture of drugs.
These regulations are presented in separate articles.
 General and specific matters relating to individual drugs are
published in the form of a book called a Pharmacopoeia.

• 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
monograph on benzoic acid, gallic acid, tartaric acid, tannic acid,
camphor, lactose, sucrose, and seven alkaloids along with their salts.

• Indian Pharmacopoeia (I.P.):
• Govt. of India constituted a permanent Indian Pharmacopoeia
Committee in 1948 under the chairmanship of Col. R. N. Chopra
for the preparation of the Indian Pharmacopoeia and established
the central Indian Pharmacopoeia Laboratory at Ghaziabad, Uttar
Pradesh to keep up to date.
• Under the Drugs and Cosmetic Act, 1940, fulfill the requirements.
• The first edition of I.P. was published in the year 1955 under the
chairmanship of Dr. B. N. Ghosh.

• Indian Pharmacopoeia (I.P.):
• After that near about 7 edition was published under the
chairmanship of different person.
• The latest edition was published in 2018 under the chairmanship
of D. P. L. Saha.
• British Pharmacopoeia (B.P.):
• European Pharmacopoeia (E.P.):
• International Pharmacopoeia:
• U.S.P.:

• Official Monograph:
• The monograph in a pharmacopoeia are the treaties on drug and
formulations, which give description, assay, assay limits and other
details necessary for maintaining requisite standards.
• A monograph in I.P. includes the following.
1. Title: The main title for a drug substance is the International Non-
proprietary Name (INN) approved by WHO
2. Chemical formulae: Chemical structure, molecular formulae and
International Union of Pure and Applied Chemists (IPUAC) name.

3. Atomic and molecular weight: It is shown at top right hand corner of the
monograph.
4. Definition: The opening statement of a monograph is definition of the
substance.
5. Category: It express the pharmacological, therapeutic, pharmaceutical
application of the compound.
6. Dose: The average range of quantity which is suitable for adult.
7. Usual strength: Indicates the strength (s), information of pharmacists.
8. Description: Substance nature, colour, odour and taste etc.

9. Solubility: The approximate solubility mentioned at temp between 15 to
300C.
10. Test methods:
11. Identification: IR absorption spectroscopy, UV, MP or BP, chemical test.
12. Test and assay:
13. Limits: Designed for identify and control small quantities of impurities,
which are likely present in the substance.
14. Assay: Detail the analytical method for substance in order to determine
percentage content of a particular chemical in given test sample.
15. Storage: Necessary for preserving the activity of the chemical.

16. Storage containers:
• Well closed container: protection from dust, dirt, insects, etc.
• Tightly closed container: prevention form atmospheric condition, moisture,
carbon dioxide.
• Light resistant container: protection from light (amber or dark coloured)
• Single dose container:
17. Labeling: It is govern by the Drugs and Cosmetics Acts 1945.
18. Appendix: it contains general information.

What is Impurities..........?
 Impurities can have unwanted pharmacological or toxicological effects that
seriously impact product quality and patient safety. Potential sources and
mechanisms of impurity formation are discussed for both drugs. The International
Conference on Harmonization (ICH) has formulated a workable guideline regarding
the control of impurities.
 Impurities in pharmaceuticals are the unwanted chemicals that remain with the
active pharmaceutical ingredients (APIs), or develop during formulation, or upon
aging of both API and formulated APIs to medicines. The presence of these
unwanted chemicals even in small amounts may influence the efficacy and safety of
the pharmaceutical products.

•According to ICH, an impurity in a drug substance is defined as-
“any component of the new drug substance that is not the
chemical entity defined as the new drug substance”.
• There is an ever increasing interest in impurities present in APIs
recently, not only purity profile but also impurity profile has
become essential as per various regulatory requirements.

• The presence of the unwanted chemicals, even in small amount,
may influence the
 efficacy and safety of the pharmaceutical products.
• “In the pharmaceutical world, an impurity is considered as any
other organic material, besides the drug substance, or ingredients,
arise out of synthesis or unwanted chemicals that remains with
API’s”

Common Name of Impurities’..…..
 By product;
 Degradation product;
 Interaction product;
 Intermediates;
 Related product;
 Transformation product;
 Penultimate product;

Source and Types of Impurities’……
 The purpose of drug substances and drug formulations (pharmaceuticals) is
primarily for the well-being of humans.
 They cure patients of diseases, disorders or deficiencies.
 They cure due to potency and therapeutic efficacy.
 However, these properties are also related to another important
characteristics; and that is purity.
 A compound is said to be impure if it has foreign matter/impurities. These
impurities affects its potency.

 Effects of Impurities on Pharmaceuticals:
1. Some impurities if present beyond certain tolerance limits can cause
untoward side effects that can lead to unpleasant reactions.
2. Some impurities may be able to catalyse the degradation, thereby
shortening the self life of the drug substance.
3. Some impurities by their chemical nature can interact with the drug
substance to affect its purity and potency.
4. Some impurities by virtue of their unstable nature link hygroscopic nature,
oxidisable nature etc., can bring about change in the physical properties
like appearance, taste, odour, stability, etc., of drug causing technical
difficulties in its use as well as formulation.

 Impurities may enter or are formed in a drug substance during any of
the following three stages:
1. During manufacturing.
2. During purification and processing.
3. During storage.

 During manufacturing:
(a) Raw material employed: Impurities present in raw materials may be
carried through the manufacturing process to contaminate the final
products.
 Ex.: Heavy metals, chlorides associated with Na compound, H2SO4 with
CuSO4 and HCl with FeCl3.
(b) Reagents used in manufacturing process: The quality and purity of
reagents used for manufacturing the drug substances are very important. If
it contain some impurities they may find entry into the final products.
 Ex.: Sulphuric acid is used in many chemical processes.

(c) Solvents used in the manufacturing process: The manufacturing
processes may involve a single step or multiple steps (unit operations).
Naturally, solvents play an important role next to the main reagents as most
of the chemical reactions involved in these processes are solvent based. If
proper quality/purity of solvents is not assured, they may add to the
impurities.
Ex.: Alcoholic solvents also may be contaminated with water and ethyl acetate
can contain acetic acid in small amounts. Thus, quality of solvents needs to
be assured and controlled.

(d) Reaction vessels: The reaction vessels employed in the manufacturing
process may be metallic (cast iron, mild steel, stainless steel) or mild steel
with glass lining. Nowadays, wooden or other metallic vessels are not used
in pharma industry. Some solvent and reagents reacts with the metal of the
reaction vessels, causing to corrosion and passing traces of metal impurities
into the solution, contaminating the final products.

(e) Intermediate products in manufacturing process: Some intermediates
which are produced during the manufacturing may be carried out through
the final product as impurities. Intermediates are products of (i) incomplete
conversion of reactants to final products or (ii) side or competing reactions
or (iii) decomposition of products formed due to poor process control.
Ex.: In the manufacturing process of KI, the intermediate iodate is the main
impurity.

(f) Defects in manufacturing process: Defects like imperfect mixing, non-
adherence to optimum reaction conditions (proper temp, pressure and pH)
may lead to impurities.
Ex.: Improper heating (failing to achieve bright red temperatures) in process of
manufacturing of zinc oxide can lead to un oxidised metallic Zn as an
impurities.
2 Zn + O2 2 ZnO + Zn (impurity)

(g) Manufacturing hazards: In the industrial area the atmosphere is
contaminated with dust particles (Silica glass, carbon, gases like H2S, CO2,
SO2, etc.). During the manufacturing of the final products these impurities
may enter into the final products.

 During Purification and Processing: Often if not properly controlled,
impurities also get added during the purification processes, mainly through
the purifying reagents, solvents or vessels used.
(a) Reagents used to remove other impurities: Sometimes some chemicals
are added to remove or to precipitate another substance. This may be also
give rise to source of impurities.
Ex.: Barium chloride (BaCl2) is added to remove excess of sulphate in
Aluminium chloride (AlCl3), hence AlCl3 is likely to contain Barium (Ba)
as an impurity.

 During Purification and Processing:
(b) Solvents used in the process of purification: Often the solvents used for
purification can be sources of impurities. These solvents range from
organic solvents to acids (organic as well as mineral) and of course water.
Ex.: Water is the cheapest solvent and most widely used. Therefore it is known
as universal solvent.

(b) Solvents used in the process of purification:
 Types of water used as:
(i) Tap water: It contains impurities of sodium, calcium, magnesium, etc., which when used
appear as impurities in the final products.
(ii) Softened water: It contains sodium and calcium ions as impurities which when used may
appear as impurities in the final products.
(iii) Demineralised water: It is free from all above inorganic ion- impurities it still contains
organic impurities like salts of carboxylic acid, etc,.
(iv) Distilled water: Considered to be the best. It is pure water and is free from all inorganic
and organic impurities but cost of it’s production is very high.

(c) Contamination due to vessels and equipment used for purification:
During the purification processes, if the vessels are defective or not
perfectly cleaned and dried they may add impurities like metallic ions, rust,
glass particles, moisture etc.
The other equipment mainly the filters, centrifuges, dryer etc., also needed
to be clean and dry.

 During Storage and Packaging:
(a) Errors in the packaging or use of substandard packaging material:
During the process of packaging or filling and sealing, whether applicable
for solid dosage forms or liquid dosage forms or API, proper material
which can ensure complete foolproof packaging without access to
atmosphere and light will ensure the stability of the products. Thus, the
quality and strength of packaging material is very important.
Ex.: Aluminium foil for tablet strip or cap for a liquid formulation bottle is of
substandard quality it can add to impurities.

(b) Faculty packaging processes: Most of the pharmaceutical packaging
processes are assembly lined automated process, generally involving
pressing and sealing with heat.
Ex.: Nowadays most of the parenteral products are in polymer containers using
FFS (Form-Fill-Sealing) processes which involve proper heating, filling,
sealing and congealing cycles. Any changes in process parameters can be
hazardous.

(c) Microbial contamination: Microbial contaminations, mainly in the form of
fungal and bacterial growth may be due to the result of improper storage
conditions as well as faculty packaging. The products for parenteral
administration and ophthalmic preparation have to undergo sterility tests.

Official method for Test for purity.......................
1. Color, Odor & Taste
2. Physicochemical constant
3. Acidity, Alkalinity & pH
4. Anions & Cations
5. Limits of insoluble matter
6. Limit of soluble matter
7. Limit of non-volatile matter
8. Ash value
9. Limit of residue on ignition

What is Limit Test.......?
1. It is a type of test in which compared opalescence turbidity or color
with the standard as a prescribed in pharmacopoeias.
2. The extent of opalescence, turbidity and color is affected by the
presence of other impurities present in the substance, variation in
time and method of performance of the test. Therefore no
numerical values for the limit in these tests are prescribed in the
pharmacopoeia.
3. Limit test are performed in an aqueous solution or extract or in
media as specified in monograph of pharmacopoeia.

Definition .......
 Limits tests are quantitative or semi quantitative tests designed to identify and
control small quantities of impurity, which are likely to be present in the
substances.
 Limits: 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 compound.

Importance of Limit Test…..
To find out the harmful amount of impurities.
To find out avoidable/ Unavoidable amount of impurities.
Types:
1. Tests in which there is no visible reaction
2. Comparison methods
3. Quantitative determinations

General principle:
 If the sample is lighter (in colour/turbidity/opalescence) than the standard
solution then it is within the pharmacopoeial limit (accepted).
 If the sample is darker/ heavier than the standard solution then it is above
the pharmacopoeial limit (rejected).
 Specificity of Limit test: A given limit test for a trace impurity should
involve some selective reaction of the reagent with the trace impurity under
consideration/ detection specifically characteristic only to it.

General principle:
 Sensitivity of Limit test: As most of the limit tests involve dilute solutions
and results are based on concentration of the trace impurity, the results may
take longer duration to become observable or appreciable. Thus,
consideration of duration of test need to be prime consideration in designing
the limit test.

 It is a clear glass cylinder with normal capacity of 50 ml, the
overall height is about 15 cm, the external height to be 50
ml marked 11.0 to 12.4 cm, the thickness of wall is around
1.0 to 1.5 mm and the thickness of the base is about 1.0 to
3.0 mm.
 The external height to the 50 mark of cylinders used for the
test must not differ by more than 1 mm.
Nessler Cylinder (IP appendix VII A127)

1. The liquid used must be clean and filtered if necessary.
2. The nessler cylinder must be made of colourless glass and
of the same inner diameter.
3. Detecting opalescence or colour development must be
performed in daylight.
4. Comparison of turbidity, it should be done against a black
background.
5. Comparison of colour, it should be done against a white
background.
General Precautions…..

 Apparatus Required:
 Nessler cylinders.
 Glass rod.
 Stand.
 Reaction:
Limit Test for Chlorides
 Chemicals Required:
 Sodium chloride.
 Silver nitrate (5%).
 Dilute nitric acid (10%).

 Principle: It is based upon the chemical reaction between silver nitrate and
soluble chloride in the presence of dilute nitric acid to give opalescence of
silver chloride. The opalescence produced is compared with standard solution.
If the opalescence in the sample is less than standard, it passes the test. If it is
more than the standard, it fails the test.
Procedure: Take two 50 ml Nessler cylinders. Label one as “Test” and other as
“Standard”.

Sr. No. Standard Test
1. Place 1 ml of 0.05845% w/v solution of
NaCl in a Nessler cylinder
Dissolve the specified quantity of the
substance in distilled water and transfer to
Nessler cylinder.
2. Add 10 ml of dilute nitric acid. Add 10 ml of dilute nitric acid.
3. Dilute to 50 ml with water and add 1 ml
of silver nitrate solution.
Dilute to 50 ml with water and add 1 ml of
silver nitrate solution.
4. Stir immediately with a glass rod and
allow to stand for 5 minutes.
Stir immediately with a glass rod and allow
to stand for 5 minutes.
5. Observe the opalescence developed and
compare with that of the sample.
Observe the opalescence developed and
compare with that of the standard.
Video of Limit Test for Chloride:

 Observation:
 The opalescence produce in sample solution should not be greater than
standard solution. If opalescence produces in sample solution is less than the
standard solution, the sample will pass the limit test of chloride and visa
versa.
 Reasons:
 Nitric acid is added in the limit test of chloride to make solution acidic and
helps silver chloride precipitate to make solution turbid at the end of
process. Nitric acid dissolved other impurities & AgCl precipitate is
insoluble in Nitric acid.
 B.P. Make use of 1 mL of 0.01 N HCl as standard solution & 1 mL 5% Silver
nitrate solution.

 Glass rod.
 Stand.
 Chemicals Required:
 Dilute hydrochloric acid.
 0.5 M Barium chloride: 122.1 gm of BaCl2. 2H2O dissolved in distilled water.
 Barium sulphate reagent containing 0.5 M barium chloride in 1000 ml of water. (This
prepared as follows: Mix 15 ml of 0.5 M BaCl2, 55 ml of water and 20 ml of sulphate free
alcohol. Add 5 ml of 0.0181% w/v potassium sulphate. Dilute to 10 ml with water and
mix.)
Limit Test for Sulphates
 Reaction:

 Principle: The limit test of sulphate depends on the precipitation of the
sulphate with barium sulphate with barium chloride as barium sulphate in
presence of hydrochloric acid & traces of barium sulphate. Or
Test based on the reaction of soluble sulphate with barium chloride in presence
of dil. HCl to form barium sulphate which appears as solid particles
(turbidity) in the solution.
Procedure: Take two 50 ml Nessler cylinders. Label one as “Test” and other as
“Standard”.
Limit Test for Sulphates

1. Place 1 ml of 0.1089% w/v solution of
K2SO4 in a Nessler cylinder
Nessler cylinder.
2. Add 2 ml of dilute HCl. Add 2 ml of dilute HCl.
3. Dilute to 45 ml with water and add 5 ml
of barium sulphate reagent.
Dilute to 45 ml with water and add 5 ml of
barium sulphate reagent.
4. Stir immediately with a glass rod and
allow to stand for 5 minutes.
Stir immediately with a glass rod and allow
to stand for 5 minutes.
5. Observe the opalescence developed and
compare with that of the sample.
Observe the opalescence developed and
Limit Test for Sulphate
Video of Limit Test for Sulphate:

Limit Test for Sulphate
 Barium sulphate reagent contains barium chloride, sulphate free alcohol & small amount of potassium
sulphate.
 Observation:
 The turbidity produce in sample solution should not be greater than standard solution. If turbidity
produces in sample solution is less than the standard solution, the sample will pass the limit test of
sulphate and vice versa.
 Reasons:
 Hydrochloric acid helps to make solutionacidic.
 Potassium sulphate is used to increase the sensitivity of the test by giving ionic concentration in the
reagent.
 Alcohol helps to prevent super saturation.

 Glass rod.
 Stand.
 STD Iron solution: Ferric ammonium sulphate (1.726 gm) dissolved in 10 ml of 0.1 N H2SO4 and
sufficient water to produce 1000 ml.
 Sulphuric acid (0.1 N): 10.0 ml.
 Iron-free citric acid solution (20% w/v): 2.0 ml.
 Thioglycollic acid: 0.1 ml.
 Iron-free ammonia solution: 20 ml.
Limit Test for Iron
 Reaction:

 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 a pink to deep reddish purple color complex of iron-
thioglycollate . The color produce from specified amount of substance from
the test is compared by viewing vertically with a standard.
 Procedure: Take two 50 ml Nessler cylinders. Label one as “Test” and other
as “Standard”.
Limit Test for Iron

1. Dilute 2 ml of standard iron solution
with 20 ml of water in a Nessler
cylinder.
Nessler cylinder.
2. Add 2 ml of 20% w/v solution of iron-
free citric acid and 0.1 ml of
thioglycollic acid and mix.
Add 2 ml of 20% w/v solution of iron-free
citric acid and 0.1 ml of thioglycollic acid
and mix.
3. Make alkaline with iron-free ammonia
solution.
Make alkaline with iron-free ammonia
solution.
4. Dilute to 50 ml with water. Dilute to 50 ml with water.
5. Observe the intensity of the purple
colour developed by viewing vertically
and compare with that of the sample
Observe the intensity of the purple colour
developed by viewing vertically and
Limit Test for Iron
Video of Limit Test for Iron:

Limit Test for Iron
 Earlier ammonium thiocyanate reagent was used for the limit test of iron. Since thioglycollic
acid is more sensitive reagent, it has replaced ammonium thiocyanate in the test.
 Observation:
 The purple color produce in sample solution should not be greater than standard solution. If
purple color produces in sample solution is less than the standard solution, the sample will
pass the limit test of iron and vice versa.
 Reasons:
 Citric acid helps precipitation of iron by a ammonia by forming a complex with it.
 Thioglycollic acid helps to oxidize iron (II) to iron (III).
 Ammonia to make solution alkaline

 Arsenic Limit test apparatus.
 HgCl2-Paper: smooth white filter paper. (having thickness NLT 25 mm).
 Soaked in a saturated solution of HgCl2, pressed to get rid of excess of
solution and dried at about 600C in the dark.
 Lead acetate solution: 10.0 w/v solution, KI: 10 gm, Zn: 10 gm, Dilute
arsenic solution; Standard stains, Test solutions are prepared according to
the Indian Pharmacopoeia 1996.
Limit Test for Arsenic

 Principle: Limit test of Arsenic is based on the reaction of arsenic gas with
hydrogen ion to form yellow stain on mercuric chloride paper in presence of
reducing agents like potassium iodide. It is also called as Gutzeit test and.
requires special apparatus Arsenic, present as arsenic acid in the sample is
reduced to arsenious acid by reducing agents like potassium iodide, stannous
acid, zinc, hydrochloric acid, etc.
Arsenious acid is further reduced to arsine (gas) by hydrogen and reacts with
mercuric chloride paper to give a yellow stain.

 Reactions: Various chemical reaction involved may be expressed by the following equations.
H3AsO4 + H2SnO2 → H3AsO3 + H2SnO3
Arsenic acid Arsenious acid
H3AsO3 + 3H2 AsH3 + 3H2O
Arsenious acid Arsine
The depth of yellow stain on mercuric chloride paper will depend upon the quality of arsenic
present in the sample.
Procedure: Take two 50 ml Arsenic LT apparatus. Label one as “Test” and other as “Standard”.

1. A known amount of dilute arsenic
solution is kept in the wide mounted
bottle of apparatus.
Test solution: Dissolving specific amount
of sample in water and stannate HCL (as
free) and kept in the wide mouthed bottle
of the apparatus.
2. Add 1 gm of KI, 5 ml of stannous
chloride and 10 gm of zinc (all these
reagents should be arsenic free).
Add 1 gm of KI, 5 ml of stannous chloride
and 10 gm of zinc (all these reagents
should be arsenic free).
3. Keep the solution aside for 40 minutes. Keep the solution aside for 40 minutes.
4. Compare the stain obtained on the
mercuric chloride paper with that in the
apparatus containing test solution.
Compare the stain obtained on the mercuric
chloride paper with that in the apparatus
containing standard solution.
Video of Limit Test for Arsenic:

 Apparatus:
 A wide-mouthed glass bottle capable of holding about 120 ml is fitted with a
rubber bung through which passes a glass tube.
 The latter, made from ordinary glass tubing, has a total length of 200 mm and an
internal diameter of exactly 6.5 mm (external diameter about 8 mm).
 It is drawn out at one end to a diameter of about 1 mm and a hole not less than 2
mm in diameter is blown in the side of the tube, near the constricted part.

 When the bung is inserted in the bottle containing 70 ml of liquid, the constricted
end of the tube is kept above the surface of the liquid, and the hole in the side is
below the bottom of the bung.
 The upper end of the tube is cut off square, and is either slightly rounded or
ground smooth.
 The rubber bungs (about 25 mm × 25 mm), each with a hole bored centrally and
through exactly 6.5 mm in diameter, are fitted with a rubber band or spring clip
for holding them tightly in place.

Arsenic Limit Test Apparatus
Reasons:
Stannous chloride is used for complete evolution of
arsine
Zinc, potassium iodide and stannous chloride is used
as a reducing agent.
Hydrochloride acid is used to make the solution acidic
Lead acetate pledge or papers are used to trap any
hydrogen sulphide which may be evolved along with
arsine.

 Lead is one of the most undesirable impurities and enters through storage
containers like bottle caps, as well as some apparatus.
 It has two different variants of limit tests. The first one is more specific for
lead.
 Test – 1: This test is official in IP and USP.
 Test – 2: It is described under Limit Test for Heavy Metals after this limit test.
Limit Test for Lead

Principle: Limit test of lead is based on the reaction of lead and diphenyl
thiocabazone (dithizone) in alkaline solution to form lead dithizone complex
which is red in color.
Dithizone is green in color in chloroform and lead-dithizone complex is
violet in color, so the resulting color at the end of process is red..
Limit Test for Lead

1. A standard lead solution (1 ppm Pb) is
prepared equivalent to the amount of
lead permitted in the sample under
examination.
A known quantity of the sample solution is
transferred in a separating funnel.
2. Add 6 ml of ammonium citrate. Add 6 ml of ammonium citrate.
3. Add 2 ml of potassium cyanide and 2
ml of hydroxylamine hydrochloride.
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride.
4. Make solution alkaline by adding
ammonia solution.
Make solution alkaline by adding ammonia
solution.
5. Extract with 5 ml of dithizone in
chloroform solution, until it becomes
green.
Extract with 5 ml of dithizone in
chloroform solution, until it becomes
green.
Limit Test for Lead

6. Dithizone extracts are shaken for 30
minutes with 30 ml of nitric acid and
the chloroform layer is discarded.
Dithizone extracts are shaken for 30
minutes with 30 ml of nitric acid and the
chloroform layer is discarded.
7. To the acid solution add 5 ml of
standard dithizone solution.
To the acid solution add 5 ml of standard
dithizone solution.
8. Add 4 ml of ammonium cyanide. Add 4 ml of ammonium cyanide.
9. Shake for 30 minutes. Shake for 30 minutes.
10. Observe the colour. Observe the colour.
Limit Test for Lead

Limit Test for Lead
 Observation:
 The intensity of the color of complex, is depends on the amount of lead in the solution. 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 lead and vice versa.
 Reasons:
 Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is used to make pH
optimum so interference and influence of other impurities have been eliminated.
 Lead present as an impurities in the substance, gets separated bye extracting an alkaline
solution with a dithizone extraction solution.

Limit Test for Heavy Metals
 It is a limit test of the quantity of heavy metals contained as impurities in
drugs.
 The heavy metals are the metallic inclusions that are darkened with sodium
sulphate (TS) in acidic solution or hydrogen sulphide saturated solution, as
their quantity is expressed in terms of the quantity of lead (Pb).
 Apparatus: Nessler cylinders, Glass rod, Stand.
 Chemicals: Dilute CH3COOH (10% v/v), Dilute ammonia (10% v/v),
Hydrogen sulphide solution, Standard lead solution, Lead nitrate stock
solution.

Principle: Limit test of heavy metals is based on the reaction of metallic
impurities with hydrogen sulphide in acidic medium to form brownish
colour solution. Metals that response to this test are lead, mercury, bismuth,
arsenic, antimony , tin, cadmium, silver, copper, and molybdenum.
 The metallic impurities in substances are expressed as parts of lead per
million parts ofthe substance. Theusual limit as per Indian Pharmacopoeia is
20 ppm.
 Procedure: Take two 50 ml Nessler cylinders. Label one as “Test” and other
as “Standard”.

1. 2 ml of standard lead solution is taken
in a Nessler cylinder and diluted to 25
ml with water.
substance in distilled water diluted to 25 ml
with water and transfer to Nessler cylinder.
2. Adjust the pH to 3-4 by dilute acetic
acid or dilute ammonia solution.
Adjust the pH to 3-4 by dilute acetic acid
or dilute ammonia solution.
3. Dilute further to 35 ml with water. Dilute further to 35 ml with water.
4. Add 10 ml of freshly prepared H2S
solution.
Add 10 ml of freshly prepared H2S
solution.
5. Dilute to 50 ml with water. Dilute to 50 ml with water.
6. Mix and allow to stand for five minutes Mix and allow to stand for five minutes
7. Observe the quantity of the black ppt of
lead sulphide formed and compare with
that of the test.
Observe the quantity of the black ppt of
lead sulphide formed and compare with
that of the standard.
Video of Limit Test for Heavy Metals:

Qualitative Tests for Alkali and Alkaline Earth
Metals
1. Barium: Presence of barium is confirmed by addition of dilute sulphuric acid to the test
solution, which should yield precipitate of barium sulphate.
2. Calcium: Presence of calcium is confirmed by addition of dilute ammonium oxalate to the
ethanolic test solution, which should yield precipitate of calcium oxalate.
3. Magnesium: Confirmed by formation of magnesium 8-hydroxyquinoline (oxine) complex.
Alternatively, if calcium is present it is to be removed as calcium oxalate, and then
magnesium is precipitated as sulphate.
4. Aluminium: Confirmed by formation of aluminium 8-hydroxyquinoline (oxine) complex
and measurement of its fluorescence.

Modified Limit Tests for Chlorides and
Sulphates
 Normally a sample substance is weighed in specified amount and dissolved
in distilled water and the volume is made up to the 50 ml mark in a Nessler
cylinder for the limit tests for chlorides and sulphates. However, the nature
of sample varies depending upon its physicochemical characteristics. Thus,
few modifications are suggested in the limit tests of such samples.
 Alkaline nature of sample: To be dissolved in dilute acid (HNO3 or HCl,
respectively) instead of distilled water.

Sulphates
 Water insoluble sample: (e.g. light kaolin, magnesium trisilicate etc) are
generally boil with corresponding acid and distilled water (to get extracted)
and filtered (as insoluble matter modified the opalescence). The filtrate is
used for the test.
 Samples which react with nitric acid (for limit test of chloride): Sample
like MgO, MgCO3, Ca(OH)2, etc., are to be dissolved in acetic acid for
making sample solution.
 In case of metallic salts of organic acid (e.g. sodium benzoate, sodium
salicylate etc.): The free acid gets liberated on shaking with the mineral
acid prescribed in the limit test. The filtrate is employed for the limit test.

Sulphates
 Samples which are reducing agents (e.g. hypo-phosphorous acid): Such
sample can react with silver nitrate reagent in the test for chlorides and
therefore should be pre-oxidized by boiling with nitric acid before carrying
the limit test.
 Coloured samples: Samples like KMnO4 are pre-reduced by boiling with
alcohol prior to the test; while other coloured substances like crystal violet,
malachite green are carbonized to ash prior to the test and the ash is
extracted with distilled water, filtered and the filtrate is used as the sample.

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