Pharmaceutical Inorganic Chemistry Sources of impurities Limit tests

1. UNIT I
IMPURITIES IN PHARMACEUTICAL
SUBSTANCES
MR.DHIRAJ NIKAM
ASST.PROF
BVCOP

2. INTRODUCTION
• Inorganic chemistry(Def.):
It is study of all elements and their compounds except carbon
and its compounds obtained from non living things and
minerals found in earth.
• Application:
Chemical industry: Synthesis of drugs, pigments, surfactants
and agricultural products
Branches
Coordination
chemistry
Bioinorganic
chemistry
Organometallic
compounds
Synthetic
inorganic
chemistry

3. Introduction
• Pharmaceutical inorganic chemistry (Def.):
It deals with study of essential and non essential elements their preparation,
standards of purity, test for identification limit test to be performed for
determining quality, extent of purity, different formulations, their storage
conditions and therapeutic uses.
Importance
Therape
utic
• Astringents, Antimicrobials
Pharmace
uticalAid
• Bentonite, Talc
Acidifier
And
Alkaliser
• Antacids, Alkalies, Mineral acids
Repleni
sher
• Body fluids e.g. Na,K,Ca,Cl,PO4
Reagent
• Platinum, Nickel, Oxidizing and reducing agents-Li, Al

4. Pharmacopoeia
Pharmcon-Drug or medicine
Poeia- To make
Prepared by; Government authority of respective countries
The book containing
the standards of drug
and other related
substances are
known as
pharmacopoeia and
formularies
Collective name-
Drug compendia
Book revised from
time to time
Contains:
1.List of drugs
2.Related substances
3.Sources,descriptio
n,standard Test,
formulae for
preparing same,
mode of action, uses,
doses,
storage condition
etc.
Preparation of book:
Experts in the field
like
Medical
practitioners,
Teachers,
scientist,
pharmaceutical
manufacturers

5. Classification of compendia
Compendia
Official
1.British Pharmacopoeia (BP)
2.British Pharmaceutical codex(BPC)
3.Indian pharmacopoeia (IP)
4.United states pharmacopoeia (USP)
5.National Formulary (NF)
6.Pharmacopoeia of other countries
Non official
1.Merc Index
2.Extra pharmacopoeia (Martindale)
3.United state Dispensatory

6. History of Pharmacopoeia
The term Pharmacopoeia first distinct title in
work published in Basel, Switzerland in 1561 by
Dr. A.Foes
On 15th December 1820,First United state
pharmacopoeia was released (U.S.P)
In 1864 the first British Pharmacopoeia(B.P)
was published inclusion on few monographs
Today’s pharmacopoeias focus mainly on
assurance of quality of products by various
tools of analytical sciences.

7. Indian Pharmacopoeia
Contribute:
Quality,
Safety,
efficacy,
affordability
Indian
Pharmacopoeia:
Official
document meant
for overall quality
control and
assurance
Contains:
Collection of
authorative
procedures of
analysis and
specifications
of drugs
Legal Aspect:
Drugs and
cosmetic act
1940 and rules
under 1945
Enforcement:
Regulatory
authorities of
Indian
Government
Prescribes:
Standards for
identity, purity
and strength of
drugs

8. Indian Pharmacopoeia
• Historical development:
1953
Garciada
Orta
Portugues
Physcian-
cum-Teacherr
1837
Idea
conceived in
and bore
fruit in
1841
Bengal
Pharmacopo
eia and
Conspectus
of drugs
1901
Bengali and
Hindi version
of London
Pharmacopo
eia was made
available in
India
.1944
Indian
Government
Asked
Technical
Advisory
Board to
prepare list of
drugs with
sufficient
medicinal
value
1946
Indian
Pharmacopo
eial List
Published.
1955
Official
Indian
Pharmacopo
eia published

9. Indian Pharmacopoeia
Indian Pharmacopoeial List 1946:
• List of drugs included and not included in British Pharmacopoeia
along with standards published by Government of India under
name of “ The Indian Pharmacopoeial list”
• Committee constituted under chairmanship of Col. Sir. R. N.
Chopra along with Nine members
• List Consist of Following details
a) Drugs of plant and animal origin
b) Biological products
c) Insecticide
d) Colouring agents
e) Synthesis
f) Miscellaneous
g) Drugs of veterinary use

10. Indian Pharmacopoeia
• Development and amendments:
Year Establishment, Amendments and
Feature edition
Highlights
1946 Indian Pharmacopoeial list Committee Constituted by Sir.R.N.Chopra
48 monographs of crude drugs
90 drugs of plant origin ,10 animal
origin,05 biological,07 insecticide,03
colouring, 05 synthesis,15
miscellaneous,02 veterinary
1948 Indian Government constituted
permanent Indian Pharmacopoeial
committee .
Committee constituted after
independence on 23rd November 1948
1955 FIRST EDITION of Indian
Pharmacopoeia (IP) published
Published in English and 986
monographs covered in Latin
1960 Supplement of IP 1955.Revision
and compilation of new edition
done under chairmanship of Dr. B.
N.Ghosh. After 1958 Dr. B.Mukherji
Director of CDRI appointed as
chairman
Context of supplement to first edition IP
was published and includes many new
drugs introduced in market

11. Indian Pharmacopoeia
Year Establishment, Amendments and Feature
edition
Highlights
1966 SECOND EDITION of IP was published 890 Monographs and 41
appendices with titles of
monographs written in English
1975 A supplement of IP 1966 was published Contains 126 new monographs and
also 250 new monographs of
second edition was amended.
1978 New reconstitution of committee by Govt.
under chairmanship of Dr. Nityanand
Director of CDRI
Reconstitution of IP Committee by
Government of India and Ministry
of Health and Welfare
1985 THIRD EDITION of IP published in 2 Volumes
by Controller of Publication
Volume I- Legal Notices, Preface,
Acknowledgement, Introduction, Notices
and Monographs Volume
Volume II- Monographs from P to Z,
Appendices and index
Two Volumes along with
appendices and 261 new
monographs
1989 Addendum(I) to IP 1985 was published 46 monographs amended 126
monographs

12. Indian Pharmacopoeia
Year Establishment, Amendments and
Feature edition
Highlights
1991 Addendum(II) to IP 1985 includes new
drugs manufactures and marketed
Effective from 1st Jaunary 1992 covers
62 monographs
1996 FOURTH EDITION of IP published Contains 1149 monographs and 123
appendices. Includes 294 new
monographs. 110 monographs were
deleted
2000 Addendum(I) to IP 1996 was Published 42 new monographs were added to IP
1996.cabrbazine monograph
undergone major changes
2002 Addendum(II) to IP 1996 was published 19 new monographs added. Emphasis
given on veterinary supplement 2000.
2005 Addendum(III) to IP 1996 was published.
IP committee decided to delete obsolete
or less used products monographs and
added monographs on therapeutic merit.
Includes large number of antiretroviral
drugs and raw plants used in making
medicinal products.

13. Indian Pharmacopoeia
Year Establishment, Amendments and Feature edition Highlights
2007 FIFTH EDITION of IP 2007 was published Contains 271 new
monographs focus on drugs
covered in national
healthcare programme and
National essential medicine
2008 Addendum(I) to IP 2007 was Published
Focus on drugs covered in national healthcare
programme and National essential medicine
Contains 72 new
monographs. IPC has become
fully financed autonomous
body from January 2009
located at NCR
2010 SIXTH EDITION of IP 2010 published.
Volume I-Comprise Notice, preface about IPC,
acknowledgements, Introduction, General
chapters and reference data
Volume II- General Notice, dosage forms, drug
substances, dosage forms and aids from(A to M)
Volume III- Dosage forms and (N to Z),vaccines,
immune sera for human use and herbal products,
blood products, biotechnological products
Contains total 2000
monographs including 287
new monographs and more
than 600 updated
monographs

14. Year Establishment, Amendments
and Feature edition
Highlights
2012 Addendum(I) to IP 2010 was
published
52 new monographs were added
2014 SEVENTH EDITION of IP 2014
was published Includes 4
volumes.
Includes biotechnological herbal
products, veterinary vaccines,
antiretroviral dugs and
formulations, fixed dose
combinations.
Contains- 2548 monographs,
577 new monographs contains
API, excipients, dosage forms,
antibiotic, insulin products
19 new monographs of
radiopharmaceuticals
Total 2548 monographs out of which
577 new monographs,
134 API monographs,163 formulation
monographs,
18 excipient,43 NDS, 10 antibiotic,19
anticancer,11 antiviral,31 herbal,05 vaccine,07
biotech products.
19 General chapters,200 new IR spectra
19 new radiopharmaceuticals
Separate volume for veterinary contains 143
monographs
Standards reaching 3000 which is at par of
other pharmacopoeia

15. Pharmacopoeial description/presentation
A) Introduction including general notices
B) Monographs of official drugs
C) Appendices and index
A) Introduction including general
notices
• Summarize different changes
including addition/deletion
• Comparison bet current and last
edition
• Avoids misinterpretation and
misunderstanding the text
attention should be paid to
general notices.
b) Monographs:
• Written study of subject implied by
word “monograph”
• General monographs of dosage form
and API grouped together at beginning
of Vol. II of IP 2010 in alphabetical
order.
• Monographs related to herbal
products, blood and blood related
products, biotechnological products,
veterinary products given in separate
sections Volume III of IP 2010.

16. Pharmacopoeial description/presentation
• Monographs Contains:
1.Main Title: Main Name of substances
2.Synonym: Common name of substance
3.Chemical Formula and Mol.Wt: As per IUPAC
4.Category: Uses of drug e.g Antibacterial,
5.Doses: Average range of quantity for adults
6.Description: General physical properties

17. Pharmacopoeial description/presentation
• Monographs Contains:
7.Solubility: Approximate part of solvent for 1 part of solute
8.Standards: Standards for purity and strength e.g NaHCO3 NLT
99 and NMT 100.5 %
9.Identification: Specific and Non specific test for identity
10.Test for Purity: m.p, b.p, wt per ml, limit test, sulpahted ash
11.Assay Method: Quantitative determination of principle
ingredient
12.Storage: Conditions for storage to prevent deterioration

18. Pharmacopoeial description/presentation
C) Appendices and Index: Includes general notices and
monographs apparatus needed for the tests and assay it
includes
Appendices
and
Index
Infra-red spectra
Apparatus for test and assay
Biological tests and determinations
Chemical test and assay
Chromatography and electrophoresis
Clarity and color solutions
Dissolution and disintegration
Microbiological assay
Limit Test of particulate matter

19. Impurity
Impurity
These are
substances which
are present in
confined amount
either in liquid,
gas or solid
preparation
which differ from
the original
chemical
composition of
material or
compound.
Impurity (Def.)
As per ICH guide
line:
Any component
of new drug
substance that is
not chemical
entity defined as
new drug
substance.
or
Substance in the
API are not API
itself.
Effect:
1.Toxic above certain
limit
2.Incompatibility
3.Change physical or
chemical properties
4.Decrease shelf life
5.Causes technical
difficulty in
formulation

20. Classification of Impurities
A) According to ICH guideline, impurities associated with API.
• Organic impurities (Process and drug related)
• Inorganic impurities
• Residual solvents
B) United state Pharmacopoeia
• Impurities in official articles
• Ordinary impurities
• Organic volatile impurities

21. Types of impurities
1.Organic impurities:
a) By product impurities:
b) Degradation Product

22. Types of impurities
c) Intermediate product:
6KOH + 3 I2 5KI + KIO3 + 3H2O
The resulting solution evaporate to dryness
KIO3 + 3C KI +3CO
KIO3 potassium iodate remaining impurity up to final product.
d) Reagents, ligands and catalyst:
Rare and needs proper care during manufacturing to avoid.
e) Enantiomeric impurities:
Stereoisomers are related drug products similar to drug
substance having potential toxicological effect. e.g.
Levofloxacin (S-ofloxacin), Lavelbutol (R-albuterol)

23. Types of impurities
2. Inorganic impurities:
Reagents,
ligands and
catalyst
• Rare but in some processes it creates problem
• e.g. CaCl2 + Na2CO3 CaCO3 + 2NaCl
• The ppt of CaCO3 washed to remove excess of Na2CO3 if it is not
properly washed it remains as impurity.
Heavy
metals:
• Water is main source and reactors like stainless steel causes
acidification or acid hydrolysis to avoid it by using demineralized
water ,reverse osmosis water
Other
Materials
• Filter aids, Charcoal commonly found during manufacturing
• Filters or filtering aids such as centrifuge bags, activated carbon,
fibers and black particles in bulk drugs are essential to avoid.

24. Types of impurities
3. Residual Solvents
Class I
• Benzene(1-2 ppm) and CCl4(1-4ppm)
• Avoid due to carcinogenic and toxicity
Class II
• Methylene chloride (600 ppm),methanol (3000 ppm)
• Pyridine(200 ppm) Tolune (890 ppm) Acetonitrile (410 ppm)
Class III
• Acetic acid, acetone, isopropyl alcohol, butanol, ethanol
• Ethyl acetate permitted daily exposure of 50 mg or less per
day

25. Types of impurities
• Other Impurities
Polymorp
h
• Phase Transformation during manufacturing and storage
• e.g. Ritonavir with unwanted polymorph
Genotox
ic
• Highly reactive molecules used in various steps have high potential of Side
reactions e.g. Dibromoethane, mesylate
Excipien
t
• Peroxides, aldehydes, heavy metals alkaline residues added in final product
• e.g. Lactose phosphate in lactose benzaldehyde in benzyl alcohol
Element
al
• Excipient impurities or during manufacturing as catalysts for oxidation and
hydrolysis. e.g. Al, As, Sn,Cd, Cr, Cu
Packaging
material
• Leachable and extractable substance from primary packaging from reaction
products e.g. Alkaline oxides from glass( Na2O,SiO2,MgO)

26. Sources of Impurities
1.Raw
Material
In
Preparation
of ZnSO4
ZnO +H2SO4
ZnSO4 +H2O
Zinc Oxide
contains Al, Cu,
Mg, Mn, Ni,As
and Fe as
impurities
2.Method of
Manufacturing
a) Reagent employed in manufacturing
CaCl2 + Na2CO3 CaCO3 + 2NaCl
Calcium carbonate contains alkali as impurity arise from sodium
carbonate
b) Reagent used to eliminate other impurities
Barium used in preparation of KBr to remove sulphate impurity but later
KBr contaminated by traces of Barium
b) Solvents
i) Tap water-contain Ca,Mg,Na,Cl,CO3 and SO4 impurity in traces
ii) Softened water- Free from Ca and Mg but contain Na and Cl as
impurity
iii) Demineralized water- free from ions but may contain pyrogens,
bacteria.
Iv Distilled water- Free from all impurities best solvent

27. Sources of Impurities
3.Reaction vessel
Vessel made up
of Cu, Galvanized
Fe, Glass and
silica reacts with
some reagents
leading to traces
of impurities.
e.g. Strong acid
leaches alkali
from glass,
copper and zinc
4.Intermediates
In the preparation
of NaBr
6 NaOH +3Br2
NaBrO3 + NaBr +3CO
NaBrO3 as
intermediate
reduced to NaBr
by heating but if
NaBrO3 is not
completely
converted to NaBr
then it remains as
impurity
5.Atomstpheric contamination
during manufacturing
Dust, gases like
CO2,SO2,As ,H2S
may contaminate
the final product
as impurity
e.g.
NaOH absorbs
atmospheric CO2

28. Sources of Impurities
• 6.Manufacturing hazards:
Particulate
matter
• Arise from wear and tear or Dirty improperly maintained
equipment or bulk material
• Contamination of eye ointment packed in metal container
Cross
contaminati
on
• Air borne dust from bulk powders, granules and tablets
• e.g. In case of steroidal and synthetic hormones
Microbes
contaminati
on
• Creams for topical, parenteral application are liable to be
contaminated by microbes.
Errors in
manufacturi
ng
• 2Zn + O2 2ZnO if Zn metal not completely converted to
ZnO then it remains as impurity.

29. Sources of Impurities
7. Instability of products
Temperature
• Chemical decomposition and physical changes occurred due to
temperature condition.
• This can be avoided by storing products as specific temperature condition
Chemical
Instability
• Chemical decomposition catalyzed by light, traces of acid and alkali,
impurities and air oxidation,CO2 and water vapours
Changes in
physical state
• Change in crystal size, shape, sedimentation, caking and agglomeration of
suspended particles leads to physical changes and affect therapeutic
effect.

30. Limit Test
Limit Test (Def.):
It is defined as
quantitative or
semiquntitative test
designed to identify
small amount of
impurity which is likely
to be present in the
substance
Test:
To examine or to
investigate the
compound
Limit:
A value or amount that
is likely to be present
in substance Impurities:
Any foreign matter
present in material

31. Limit Test
.General principle
• If the sample is
lighter than the
standard solution
then it is within
the pharmacopeia
limit (accepted)
• If the sample is
darker/heavier
than the standard
solution then it is
above the
pharmacopoeia
limit (rejected)

32. Limit Test
General precaution
The liquid used must be clean and filtered if necessary
The Nessler cylinder must be made of colorless glass and of the
same inner diameter
Detecting opalescence or color development must be performed
in daylight
When comparing turbidity it should be done against black
background
When comparing color it should be done against white
background

33. Chloride limit Test
Principle:
• Limit test for chloride based upon precipitation reaction.
Interaction of soluble chloride impurity with silver nitrate in
the presence of dilute nitric acid leads to precipitation of
silver chloride. The precipitation of silver chlorides appears as
opalescence. It is compared under uniform conditions of
illumination with standard opalescence.
Reaction:
• NaCl + AgNO3 dilute HNO3 AgCl + NaNO3
Standard:
Dissolve 0.05845 g of NaCl to 100 ml of distilled water.(25 PPM)

34. Chloride limit Test
Sr TEST STANDARD REASONS
1 Place 1 ml of Test
solution in Nessler’s
cylinder.
Place 1ml of 0.0585% w/v
solution of sodium chloride
in Nessler’s cylinder.
The aqueous solution will leach out all
the chloride ions present in the sample
and make them ready to react with silver
nitrate.
2 Add 10 ml of dilute
nitric acid.
Add 10 ml of dilute nitric
acid.
Dilute nitric acid makes solution acidic
and helps silver chloride to precipitate to
make solution turbid at the end.
It also prevents precipitation of other
radicals. This produces common ion
effect with silver nitrate and
dissociation of silver ion is suppressed.
3 Dilute to 50ml with
chloride free water.
Dilute to 50ml with chloride
free water.
Equal volumes can be compared easily.
Add 1ml of 0.1M
AgNO3, stir and allow
to stand in dark.
Add 1ml of 0.1M AgNO3,
stir and allow to stand in
dark.
The Ag+ ion reacts with Cl- ion to form
opalescence of silver chloride.
Ag+ + Cl- AgCl

35. Limit Test of Sulphate
Principle:
• The principle involved precipitation method and comparison test solution
with standard solution with known amount of sulphate. The sulphate ion
precipitated as barium sulphate by reaction with barium chloride in presence
of hydrochloric acid. Hydrochloric acid prevents reaction of other radicals with
barium chloride. The turbidity produced due to formation of barium sulphate
is compared with standard.
Reaction:
• BaCl2 + SO4
-2 BaSO4
-2 + 2Cl-
Reagents:
1.Stock solution of Potassium sulphate : 0.181 %w/v solution of potassium sulphate.
2.Standard Potassium Sulphate Solution (10 ppm):
Dilute 1 volume of 0.181 % w/v g of K2SO4 up to 100 ml of distilled water
3,Ethanolic Potassium Sulphate Solution (10 ppm): Dilute 1 volume of 0.181 % w/v g of
K2SO4 in ethanol (30 %) to 100 ml with distilled water.
4.Barium sulphate reagent(25 % w/v): Dissolve 25 g of barium chloride (BaCl2.2H2O) in 50
ml of distilled water to make up the volume up to 100 ml with distilled water

36. Limit Test of Sulphate
Sr.
No
TEST STANDARD REASONS
1. Take 1ml of 25%w/v
solution of BaCl2 in
Nessler’s cylinder.
Take 1ml of 25%w/v solution
of BaCl2 in Nessler’s cylinder.
It is a principle precipitating agent. The
barium ions react with sulphate to
form opalescence of barium sulphate.
2. Add 1.5ml of ethanolic
potassium sulphate
solution (10PPM of SO4
-2).
Add 1.5ml of ethanolic
Potassium sulphate solution
(10PPM of SO4
-2).
This ethanolic solution of K2SO4
increases sensitivity of the test and
alcohols prevents the supersaturation
of barium sulphate precipitate.
3. Add 15ml of Test solution
prepared as directed in
individual monograph.
Add 15ml of standard
potassium sulphate solution
(10PPM sulphate).
It gives sulphate ion present in the
sample of sulphate ion in standard.
4. Add 0.15ml of 5M acetic
acid.
Add 0.15ml of 5M acetic acid. The acetic acid prevents the
precipitation at various anions such as
borate, oxalate, phosphate etc.
5. Make up the volume with
sulphate free water upto
50ml.
Make up the volume with
sulphate free water up to
50ml.
Equal volume opalescence are easily
compared by viewing in black
background.

37. Limit Test Iron
Principle:
• Limit test for iron is based upon the reaction of iron in ammonical solution in
presence of iron free citric acid (20%) with thioglycolic acid which results in
formation of ferrous thioglycolate complex. A purple color is produced only in
alkaline medium hence ammonia solution forms ppt. with iron to prevent this citric
acid is added to form ammonium citrate buffer which stabilizes ferrous
thioglycolate complex.
Standard iron solution (20 ppm):
• Dissolve 0.173 g of ferric ammonium sulphate in 10 ml 0.1 N sulphuric acid. Add
sufficient water to produce 1000 ml of solution. Each ml of this solution contains
0.02 mg of iron.

39. Limit test of lead
Principle:
• The limit test for lead is based on the reaction between lead and
diphenylthiocarbazone (dithizone) in an alkaline medium to form
lead–dithizonate complex.
• The lead present as an impurity in the substance is separated by
extracting an alkaline solution with dithizone extraction solution.
• The interference by other metal ions is eliminated by adjusting the
optimum pH for the extraction by using reagents like ammonium
citrate, potassium cyanide and hydroxylamine hydrochloride
• 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
the quantity of lead present in the solution is compared with
standard colour produced by standard solution.

40. Limit test of lead
Reagents
• Dithizone extraction solution: Dissolve 30 mg of dithizone
in 1000 ml of chloroform and add 5 ml of ethanol (95%). Store the
solution in a refrigerator. Before use, shake a suitable volume of the
solution with about half its volume of a 1%v/v solution of nitric acid
and discard the acid.
• Dithizone standard solution: Dissolve 10 mg of dithizone in
1000 ml of chloroform. Store the solution in a glass-stoppered,
lead- free, light-resistant bottle in a refrigerator
• Lead standard solution (0.1% Pb): Dissolve 0.400 g of lead nitrate in
water containing 2 ml of nitric acid and add sufficient water to
produce 250.0 ml.
• Lead standard solution (1 ppm Pb): Dilute 1 volume of lead standard
solution (10 ppm Pb) to 10 volumes with water

41. Limit test of leadLimit test of lead
Sr Test Standard Reasons
1 The required quantity of
sample is dissolved in water and
transferred into a separating
funnel.
Transfer required amount of lead
standard solution (1 ppm lead)
equivalent to the amount of lead
permitted in the substance being
examined into a separating funnel
Suspected sample
have impurity
2 Add 6 ml of ammonium citrate
solution
(Sp).
Add 6 ml of ammonium citrate
solution (Sp).
Maintains optimum
pH and prevent
formation of
undesired
precipitates
3 Add 2ml potassium cynide and 2
ml of hydroxylamine
hydrochloride solution
Add 2ml potassium cynide and 2 ml
of hydroxylamine hydrochloride
solution
Cynide forms
complex with all
interfering metals
4 Add two drops of phenol red
solution.
Add two drops of phenol red
solution
Used as indicator to
develop colour at
the end of process
5 Make the solution just alkaline
by the addition of strong
ammonia solutions cool it if
necessary.
Make the solution just alkaline
by the addition of strong ammonia
solution cool it if necessary.
Ammonia provides
basic condition to
develop red colour

42. Limit test of lead
Sr Test Standard Reasons
6
Extract immediately with 5 ml of
dithizone extraction solution until
it becomes green.
Extract immediately 5 ml of
dithizone extraction solution until it
becomes green.
Perform 2-3 extractions
in separating funnel and
mix all extracts in end
7 Combine the dithizone extracts
and shaken for 30 seconds with 30
ml of 1 % v/v solution of nitric acid
and discard the chloroform layer.
(Dithizone remains in chloroform
layer, lead nitrate in aqueous layer).
Combine the dithizone extracts and
shaken for 30 seconds with 30
ml of 1 % v/v solution of nitric
acid and discard the chloroform
layer (dithizone remains in
chloroform layer, lead nitrate in
aqueous layer).
Acidic layer (contains
free Lead) separated
from chloroform. Nitric
acid makes lead free
from chloroform layer
8 To this acid solution add 5 ml
of standard dithizone solution.
To this acid solution add 5 ml of
standard dithizone solution.
Violet colour appears
due to lead-dithiazone
complex
9 Add 2 ml of potassium cyanide
solution
Add 2 ml of potassium cyanide
solution.
Maintains optimum pH
10 Shake well for 30 min and
observe the color of chloroform
layer after separation
Shake well for 30 min and
observe the color of chloroform
layer after separation.
Comparison of colour

43. Limit Test of Heavy Metals
Principle:
• Method A, B and C are based upon the reaction of the heavy metal ion with
hydrogen sulphide (in method A and B) or sodium sulphide (in method C) leading
to the formation of heavy metal sulphides.
• The metal sulphide remain distributed in a colloidal state and give rise to a
brownish colouration.
• The colour produced in the test solution is compared with that of standard
solution containing a definite amount of the lead.
• Method D is based upon the precipitation of relatively insoluble and
characteristically coloured sulphides of heavy metals when aqueous solutions
are treated with alkali metal sulphides (NaSH).
• The colour is compared by keeping the two Nessler cylinders side by side and
viewing vertically downwards against a white background. The usual limit for
heavy metals as per I.P. is 20 ppm.
• Method A: It is used for the substance which gives a clear, colorless solution
under specified conditions.
• Method B: It is used for the substance which does not give a clear, colorless
solution.
• Method C: It is used for the substance which gives a clear, colorless
solution in sodium hydroxide medium.

44. Limit Test of Heavy Metals
Reaction:
Heavy metal(M) + H2S/Na2S Heavy metal sulphide(MS) + 2H+
Reagents:
1. Lead Standard solution (0.1% Pb): Dissolve 0.400 g of lead nitrate
in water containing 2 ml of nitric acid and add sufficient water to
produce 250.0 ml.
2. Lead Standard solution (100 ppm Pb): Dilute 1 volume of lead
standard solution (0.1% Pb) to 10 volumes with water.
3. Lead Standard solution (20 ppm Pb): Dilute 1 volume of lead
standard solution (100 ppm Pb) to 5 volumes with water.

45. Limit Limit Test of Heavy Metals Test of Heavy
Metals
Method A
Test solution Standard solution
The sample solution is prepared as per
the monograph and 25 ml of solution is
transferred into a Nessler’s cylinder.
Transfer 1.0 ml of standard lead solution
and dilute to 25 ml with water.
Add dilute ammonia or acetic acid
solution to adjust the pH between 3 to 4
Add dilute ammonia or acetic acid
solution to adjust the pH between 3 to 4
Dilute to 35 ml with distilled water and
mix well.
Dilute to 35 ml with distilled water and
mix well.
Add 10 ml of freshly prepared hydrogen
sulphide solution, mix, dilute to 50 ml
with water.
Add 10 ml of freshly prepared hydrogen
sulphide solution, mix, dilute to 50 ml
with water.
Allow to stand for 5 min and view
downwards over a white background.
Allow to stand for 5 min and view
downwards over a white background.

46. Limit Test of Heavy Metals
Method B
Test solution Standard solution
1.Weigh 2g sample (Paracetamol)
moisten with sulphuric acid on low flame
till completely charred.
1.Take 2 ml of standard lead solution and
dilute to 25 ml with water
a) Add few drops of nitric acid and heat
to 500 C .Allow to cool and add 4 ml
of HCl and evaporate to dryness.
b) Moisten the residue with 10 ml of
HCl and digest 2 for min.
c) Neutralize with ammonia solution
Adjust the pH 3 to 4, filter if
necessary. Dilute to 35 ml with water.
Adjust the pH 3 to 4, filter if
necessary. Dilute to 35 ml with water.
Add 10 ml of freshly prepared
hydrogen sulphide solution. Dilute
to 50 ml with water.
Add 10 ml of freshly prepared
hydrogen sulphide solution. Dilute to
50 ml with water.
Allow to stand for 5 min and
view downwards over a white
background.
Allow to stand for 5 min and view
downwards over a white background.

47. Limit Test of HLimit Test of Heavy Metalseavy Metals
Method C
Test solution Standard solution
The required quantity of sample
is dissolved in 20 ml of water; add 5
ml of sodium hydroxide solution or
the sample solution is prepared as per
monograph.
The standard solution is prepared
by using 1.0 ml of standard lead
solution; add 5 ml of sodium
hydroxide solution.
Make up to 50 ml with water. Make up to 50 ml with water.
Add 5 drops of sodium sulphide
solution
mix well and kept aside for 5 min.
Add 5 drops of sodium sulphide
solution
mix well and kept aside for 5 min.
View downwards over a white
background.
View downwards over a white
background.

48. Limit Test of Arsenic
Principle:
• The limit test for arsenic is based on the reduction of the
arsenic in the arsenious state to the arsine gas (AsH3) with zinc
and hydrochloric acid. The arsine gas stains the mercuric
chloride paper yellow.
• The sample is dissolved in acid whereby the arsenic present as
impurity in the sample gets converted to arsenic acid.
• The arsenic acid is reduced to arsenious acid by reducing
agents like stannous acid, potassium iodine etc. The nascent
hydrogen formed during the reaction further reduced
arsenious acid to the arsine gas. The arsine gas reacts with
mercuric chloride paper to produce a yellow stain.
• The depth of the yellow stain depending upon the amount of
arsenic present in the sample, is compared with that of
standard stain produced from a known amount of arsenic.

49. Limit Te Limit Test of Arsenic st of Arsenic
Reaction

50. Limit Test of Limit Test of Arsenic senic
Sr Test Standard Reasons
1 The test solution which is
prepared and directed in
monographs placed in
bottle
Take 1 ml of arsenic
standard solution(10 ppm
As) is diluted to 50 ml with
water
1.Suspected sample is taken
which might have impurity.
2 Add 5 ml of 1 M
potassium iodide, 5 ml of
stannated hydrochloride
solution AsT and 10 g of
zinc AsT
Add 5 ml of 1 M potassium
iodide, 5 ml of stannated
hydrochloride solution AsT
and 10 g of zinc AsT
1.Stannous chloride used
for complete evolution of
arsine gas
2.Zinc , KI and stannous
chloride acts as reducing
agent
3.HCl makes solution acidic
3 Immediately apparatus is
assembled and the flask is
immersed in water bath at
temperature of 400 C.
Immediately apparatus is
assembled and the flask is
immersed in water bath at
temperature of 400 C.
Uniform evolution of arsine
gas
4 After 40 min any stain
produced on mercuric
chloride paper is observed
After 40 min any stain
produced on mercuric
chloride paper is observed
Arsine gas reacts with
mercuric chloride paper
and make yellow stain

51. Modified limit test for chloride
Principle:
• Limit test for chloride based upon precipitation reaction.
Interaction of soluble chloride impurity with silver nitrate in the
presence of dilute nitric acid leads to precipitation of silver
chloride. The precipitation of silver chlorides appears as
opalescence. It is compared under uniform conditions of
illumination with standard opalescence.
Conditions:
• If limit test for colored compound which can not be done from
normal method. e.g. KmNO4
• As KmNO4 forms purple color in aqueous solutions that interfere in
the comparison of opalesence or turbidity, so aqueous solution must
be decolorized.
• KmNo4 is oxidizing agent and ethanol is reducing agent which allows
redox reaction in presence of heat and KmNo4 reduced to MnO2
forming brown precipitate which is filtered .
• The filtrate obtained is clear colorless and subjected to limit test

52. Modified limit test for chloride
Reaction:
• 2KmNO4 + 3 C2H5OH Heat 2MNO2+ 2KOH +2CH3CHO +2H2O
Reagents:
1. Chloride standard solution (250 ppm): Dilute 50 ml of 0.0824%w/v
solution of sodium chloride to 100 ml with distilled water.
2. Dilute Nitric acid: Dilute 1.06 ml conc. nitric acid to 100 ml with
distilled water.
3. 0.1 M AgNO3: Dissolve 1.7 g of silver nitrate in 100 ml of distilled
water.
4. Test Solution: Dissolve 1.5 g KmNO4 in 50 ml of distilled water, heat
on water bath and add gradually 6 ml of ethanol(95%),cool dilute to
60 ml with distilled water and filter

53. Modified limit test for chloride
Sr.
No
Test Standard Reason
1 Take 40 ml of test
solution
Take 10 ml of standard
solution and add 5 ml
distilled water
Suspected sample is taken
which might have impurity
2 Add 1 ml of Dilute
nitric acid
Add 1 ml of Dilute
nitric acid
Dilute nitric acid makes
solution acidic and helps silver
chloride to precipitate to make
solution turbid at the end.
3 dilute to 50 ml with
distilled water
and dilute to 50 ml
with distilled water
Equal volumes can be
compared easily.
4 Add 1 ml of 0.1 M
AgNO3 solution
Add 1 ml of 0.1 M
AgNO3 solution
The Ag+ ion reacts with
Cl- ion to form
opalescence of silver
chloride.
5 Stir immediately with glass rod and alow stand for 5 min protected from light