Impurities and their limit test

Impurities, sources of impurities and limit test

1. Impurities in Pharmaceuticals and Limit test
Esha Shah 1
EshaBhavinShah
Assistant Professor
Department of Pharmaceutical Chemistry
and Quality Assurance
BabariaInstitute of Pharmacy
Bits Edu Campus
Email: [email protected]

2. Outline of the chapter
Definition of impurity
Sources of Impurity
Definition of Assay
Impurities commonly found in
medicinal preparations
Definition of limit test
Importance of Limit test in pharmaceuticals
Limit test for Iron, Arsenic, Chloride, Lead,
Sulphate, Heavy metals
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3. Impure Chemical Compound:
A compound is said to be impure if it is having foreign matter i.eImpurities.
Pure Chemical Compound:
A pure chemical compound refers to that compound which is having no foreign matter i.e
impurities.
Chemical purity means freedom from foreign matter.
Analytically 100 % pure substances are not available
and traces of impurities must be present.
Normally undesirable foreign materials are present in
the pharmaceutical substances.
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4. Impurity means
undesired particles
What is impurity?
Any material that affects the purity of the material of interest.
PresenceofImpuritiesinthepharmaceuticalsubstancesmay
producetoxiceffectsonthebodyandmayalsolowerdown
theactivestrengthofthepharmaceuticalsubstance.
Impuritiescommonlyinchemicalsubstancesincludesmall
quantitiesoflead,Arsenic,Iron,Chlorideandsulphate.
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5. Impurities commonly found in medicinal preparations:
Impurities which have toxic effects on body and bring about unpleasant reactions when
present beyond certain limits. e.gLead and Arsenic salts.
The impurities which are able to make substance incompatible with other substances.
The impurities which if present beyond the limit, affect the storage property of the
pharmaceuticals.
The impurities whichare harmless, but if present beyond the limit, it will lower the
active strength of the medicinal compound. E.gSodium salt in potassium salt.
The impurities which may bring about technical difficulties in the use of the substance.
Impurities such as taste, odour, colour or appearance which can be easily detected by the
senses and make the substance unhygienic and unaesthetic. E.g. Sodium chloride becomes
damp because of the presence of traces of magnesium salts. Also phenolic impurities
present in sodium salicylate alters its odour.
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6. Sources of Impurities in Pharmaceuticals
Thetypeandamountofimpuritypresentinthechemicalsorpharmaceutical
substances,dependsuponseveralfactorslikethoselistedbelow:
1)Rawmaterialusedinmanufacture
2)Reagentsusedinmanufacturingprocess
3)Method/processusedinmanufactureormethodofmanufacturing
4)Chemicalprocessesusedinthemanufacture
5)Atmosphericcontaminationduringthemanufacturingprocess
6)Intermediateproductsinthemanufacturingprocess
7)Defectsinthemanufacturingprocess
8)Manufacturinghazards
9)InadequateStorageconditions
10)Decompositionoftheproductduringstorage
11)Accidentalsubstitutionordeliberateadulterationwithspuriousor
uselessmaterials
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[GTU important]

7. 1)Rawmaterialsemployedinmanufacture
•Impuritiesknowntobeassociatedwiththesechemicalsmaybecarriedthroughthe
manufacturingprocessandcontaminatethefinalproduct.
•Example
Rocksalt--------CalciumSulphate(CaSO
4)+MagnesiumChloride(MgCl
2)=NaCl
prepared
RocksaltcontainssmallamountsofCalciumsulphateandMagnesiumchloride.
ThusSodiumchloridepreparedfromthissourcewillcontaintracesofCalcium
andMagnesiumcompounds.
Impurities such as Arsenic, Lead and Heavy metals are present in raw materials
and hence are found in substances. So, it is necessary to use pure chemicals and
substances as raw materials for the manufacturing process.
Calcium sulphate
and Magnesium
chloride
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8. •Example:
Coppersulphatemaybepreparedbytheactionofsulphuricacidon
copperturnings:
Cu+2H
2SO
4------------------------CuSO
4+2H
2O+SO
2
CopperturningsareknowntohaveIronandArsenicasimpurities.
IfLargequantitiesofimpuritiesarepresentintherawmaterial
(e.gCopperturnings),theymayenterthefinalproduct.(CuSO
4.5H
2O)
DuetothisI.P.prescribeslimitoftoleranceforArsenicasimpuritytobe
notmorethan8partspermillionincoppersulphate.Similarlyit
prescribesalimitofIronasimpurity.
Copper turnings
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9. 2)Reagents used in the manufacturing process:
If reagents used in the manufacturing process are not completely removed by washing,
these may find entry into the final products.
Example:
Ammoniated mercury may be prepared by adding a solution of Mercuric chloride to dilute
ammonia solution.
HgCl
2+ 2NH
4OH-------------NH
2HgCl + NH
4Cl + 2 H
2O
Soluble soluble Ammoniated mercury (ppt) (soluble)
The precipitate of Ammoniated mercury (Final Product)contains ammonium hydroxide.
Thus, this precipitate is washed with cold water to remove ammonium hydroxide.
If it is not removed completely by washing with water, the final product may contain in it
Ammonium hydroxide as impurity.
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10. 3) Method or the process used in the
manufacture:
Many drugs and chemicals (usually organic) are manufactured from different raw
materials, by using different methods or processes.
Some impurities are incorporated into the materials during the manufacturing process.
The type and amount of impurity present in the drug/ chemical varies.
In certain drugs , a multiple-step-synthesis procedure is used , which produces
intermediate compounds.
The purification of intermediates is also important, otherwise the impurities present in
the intermediate will get incorporated in the final product.
Usually side reactions occur during the synthesis.
Impurities of the product side reactions also occur in the substances. This may
introduce new impurities due to contamination by reagents and solvents at various
stages of the process as described below:
a)Reagents employed in the process
b)Reagents added to remove other impurities
c)Solvents
d)Action of solvents and reagents on reaction vessels.
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11. C) Solvents:
Water is the cheapest solvent available and has been used wherever possible.
Tap WaterIt has Ca
+2
, Mg
+2
, Na
+
, Cl
-
, SO
4
-2
and CO
3
-2
as
impurities in small amounts
Softened water It is obtained by allowing the tap waterto pass
through the sodium form of Zeolite which
removes divalent cationslike Ca
+2
and Mg
+2
from tap water in exchange of sodium.
So, softened water contains Na+,Cl
-
ions as
impurity.
De-mineralised
water
It is obtained by passing tap water through
columns packed with ion exchange resin.The
water obtained from this process is free from
Ca
+2
, Mg
+2
, Na
+
, Cl
-
, SO
4
-2
and CO
3
-2
Thus the final product is free fromthese
impurities.
The water obtained from this source may still
contain organic impurities and so final product
contains organic impurities.
Distilled waterIt is considered the best but it is very costly.
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12. a)Reagents employed in the manufacturing process:
•Soluble alkali in Calcium carbonate arises from sodium carbonate used in the
process.
•Calcium carbonate is obtained by interaction of a soluble calcium salt and a
soluble carbonate and therefore the product will contain traces of soluble alkali,
which the washing process has failed to remove.
b) Reagents added to remove other impurities:
•Potassium bromide contains traces of Barium, which is added in the
manufacturing process to remove excess of sulphate.
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13. Esha Shah 13

14. d) Action of solvents and reagents on reaction vessels:
During manufacturing process, some of the solvents and reagent may undergo reaction with
metals of reaction vessel and may dissolve these metals, which appear as impurities in the
final product.
Example:
Iron is known to contain Arsenic impurity.
The inorganic compounds manufactured in Iron vessel will contain Arsenic and Iron as
impurities.
Thus IP has prescribed limit test for Arsenic and Iron for most inorganic compounds.
Iron vessel
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15. 4) Chemical process used in the manufacture:
For the synthesis of drugs, many chemical reactions such as Nitration, Halogenation,
Oxidation, reduction, hydrolysis are involved.
In these chemical processes, different chemicals are used.
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 substance which is being manufactured.
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16. 5) Atmospheric contamination during the manufacturing
process
In the industrial areas, the atmosphere is contaminated with dust particles and
some gases like Hydrogen sulphide, Sulphur dioxide, and black smoke.
During the manufacture or purification of the pharmaceutical products, these
impurities enter the final products.
There are many pharmaceutical products which when manufactured are
contaminated with atmospheric CO
2 and water vapour. E.gNaOHabsorbs
atmospheric CO
2.
2NaOH + CO
2 --------------------------------Na
2CO
3 + H
2O
Due to this reaction, NaOHshould not be kept open for a longer time during its
manufacture.
Therefore, IP has prescribed that Sodium hydroxide should not contain more than
3% of sodium carbonate.
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17. 6) Defects in the manufacturing process:
In many manufacturing processes, there are defects like imperfect mixing, incompleteness,
non-adherence to proper temperature, pressure, pH or reaction conditions, which may
give chemical compounds with impurities in them.
Example:
•Zinc oxide may be prepared by heating metallic zinc to bright redness in a current of air.
The vapours of Zinc burn to form Zinc oxide which is collected as a fine white powder.
•But if there is less heat or air or both, zinc metal is not completely converted to zinc
oxide.
•Thus the final product, Zinc oxide may still contain metallic zinc as impurity.
•So, IP has prescribed a test for Zinc metal in zinc oxide.
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18. 7) Intermediate products in the manufacturing process:
Therearesomeintermediateswhichareproducedduringthemanufacturingprocess.
Sometimestheseintermediatesmaybecarriedthroughtothefinalproductasimpurity.
Example:
PotassiumiodideispreparedbyreactingIodinewithPotassiumhydroxide.
6KOH+3I
2--------------------5KI+KIO
3+3H
2O
Theresultingsolutionisfirstevaporatedandthenheatedwithcharcoal.
KIO
3+3C----------KI+3CO
Inthisprocessiftheintermediateproduct(KIO
3)isnotcompletelyconvertedintoKI,
thenitmaybecarriedthroughtothefinalproductasanimpurity.
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19. 8) Manufacturing hazards:
Particulate
contamination
Process errors
Cross
contamination
Microbial
contamination
Packing errors
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20. Particulate contamination:
The presence of unwanted particulate matter can arise due to dirt, dust, glass,
porcelain or plastic fragments from sieves, granulating or tableting machines or
from product containers.
Ware and tare of equipment or improperly cleaned equipment may also cause
particulate contamination.
Clarity of solutions for injection is particularly important.
E.gMetal particles which have been found in eye ointments packed in metal
tubes.
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21. Process errors:
Gross errors arising from incomplete solution of a solute in a liquid preparation must be
detected readily by the normal analytical control procedures.
Minor errors arise if the manufacturing tolerance for the quantity of active ingredient in
the product has been wide.
Cross contamination:
The handling of powders, granules, and tablets in large bulk creates air-borne dust,
which leads to cross contamination of the product.
So, face masks and special extraction equipment are used to protect operators from
harmful effects of drugs.
E.gpenicillin preparation requires special handling during its manufacture.
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22. Microbial contamination:
•Parenteral preparations and ophthalmic preparations require special care against
microbial contamination.
•Many liquid preparations and creams are liable to bacterial and fungal contamination. So
care should be taken.
•Eg. Acacia, senna, tragacanth---They should be controlled for Salmonellae.
Packing errors:
•Products of similar appearance such as tablets of same size, shape, colour packed in
similar containers can constitute a potential source of danger.
•Improper labelling or destruction of stock of unused labels also constitutes a major
packaging hazard.
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23. 9) Storage conditions:
The chemical substances when prepared have to be stored in different types of containers
depending upon:
Nature of the material
Batch size
Quantity
Many types of materials are used for storage purpose like plastic, polythene, iron vessels,
stainless steel and aluminium.
Leachingouteffect:Alkaliesstoredinordinaryglasscontainersextractleadfromit,
whichinfoundasimpurityinthefinalproduct.
StrongchemicalsreactwithironcontainersandextractIronanimpurityinfinalproduct.
Reaction of these
substances
With materials of
storage vessels
The products
formed are
found as
impurities in the
stored materials
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24. Inadequate storage and their effects are as follows:
a)Filth:Stored products may become contaminated with dust, bodies of insects, animal and
insect excreta.
b)Chemical instability: decomposition because of light, traces of acid or alkali, air
oxidation, water vapour, CO2 and traces of metallic ions.
e.glight sensitive materials should be stored in amber coloredbottles.
c)Reactions with container materials:e.gsalicylic acid ointment must not be stored in
metal tubes.
d)Physical changes: The occuranceof changes in the physical form of drug like change in
crystal size can lead to change in efficiency of product.
e)Temperature effect:Chemical and physical changes occur if materials are not stored at
proper temperature.
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25. 10)Decompositionoftheproductduringstorage:
Chemicaldecomposition,analysisorbreakdownistheseparationofachemical
compoundintoelementsorsimplercompounds.Itissometimesdefinedastheexact
oppositeofachemicalsynthesis.Chemicaldecompositionisoftenanundesiredchemical
reaction.
Somesubstancesdecomposeonstoringduetopresenceofair,lightandoxygen.So,
thefinalproductiscontaminated.
Deliquescentsubstances,absorbwaterfromtheatmosphereandgetliquefied.
Decompositionproductsappearasimpuritiesinthesubstances.
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26. 11) Accidental substitution or deliberate adulteration with spurious or
useless materials:
It is possible to avoid accidental substitution by storing the toxic substances together
separately or in a locked cupboard.
Many pharmaceutical chemicals are adulterated with cheaper substances.
E.gThe expensive potassium may be adulterated with sodium bromide.
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27. Effect of Impurities:
Theimpuritiespresentinthesubstancesmaygivefollowingeffects:
Impuritieshavingtoxiceffectsmaybeinjurioustohealth,ifpresentabove
certainlimits.
Tracesofimpurities,mayexertacumulativetoxiceffectafteracertaintime.
Impuritiesmaylowertheactivestrengthofthesubstance.
Impuritymaydecreaseshelflifeofsubstance.
Impuritymaycauseincompatibilitywithothersubstances.
Impuritiesmaycauseaphysicalorchemicalchangeinthepropertiesofthe
substance,somakingthesubstancemedicinallyuseless.
Maycausechangeincolor,odourandtaste.
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28. Test for purity:
Pharmacopoeia prescribes the “Test for purity” for pharmaceutical substances to
check their freedom from undesirable impurities.
Pharmacopoeia will decide and fix the limit of tolerance for these impurities.
For certain common impurities for which pharmacopoeia prescribes the test of
purity are:
Colour, odour, taste
Physicochemical constants (Iodine value, saponification value, melting point,
refractive index etc.)
Acidity, alkalinity, pH
Humidity (Estimation of moisture)
Cations and anions
Ash
Arsenic or lead
Loss on drying
Loss on ignition
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29. Impurity means
undesired particles
What is impurity?
Any material that affects the purity of the material of interest.
PresenceofImpuritiesinthepharmaceuticalsubstancesmay
producetoxiceffectsonthebodyandmayalsolowerdown
theactivestrengthofthepharmaceuticalsubstance.
Impuritiescommonlyinchemicalsubstancesincludesmall
quantitiesoflead,Arsenic,Iron,Chlorideandsulphate.
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30. Limit tests:
Tests being used to identify the impurity.
Tests being used to control the impurity.
Definition:Limittestsarequantitativeorsemiquantitativetest
designedtoidentifyandcontrolsmallquantitiesofimpuritieswhich
arelikelytobepresentinthesubstances.
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31. Factors affecting limit tests:
Specificity of the tests
Sensitivity
Control of personal errors (Analyst errors)
•Test in which there is no visible reaction
•Comparison methods
•Quantitative determination
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32. Types:
Tests in which there is no visible reaction
Comparison methods
Quantitative determinations
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33. Turbidity means cloudiness or haziness.
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34. Limit test for IRON:
LimittestofIronisbasedonthereactionofironinammonicalsolutionwith
thioglycollicacidinpresenceofcitricacidtoformironthioglycolate(Ferrous
thioglycolatecomplex)whichproducespalepinktodeepreddishpurplecolorinalkaline
media.
Thioglycolicacidisusedasreducingagent.
ThecoloroftheFerrousthioglycolatecomplexfadesinthepresenceofairdueto
oxidation.
Also,thecolorisdestroyedinpresenceofoxidizingagentsandstrongalkalis.
Thepurplecolorisdevelopedonlyinalkalinemedia.Soammoniasolutionisused.
Butammoniareactswithiron,formsprecipitateofferroushydroxide.
ThuscitricacidisusedwhichpreventstheprecipitateofironwithAmmoniabyforming
acomplexwithironasironcitrate.
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35. Test sample Standard compound
Sampleisdissolvedinspecificamount
ofwaterandthenvolumeismadeupto
40ml
2mlofstandardsolutionofirondiluted
withwaterupto40ml
Add2mlof20%w/vofcitricacid
(ironfree)
Add2mlof20%w/vofcitricacid
(ironfree)
Add2dropsofthioglycollicacid Add2dropsofthioglycollicacid
Addammoniatomakethesolution
alkalineandadjustthevolumeto50ml
Addammoniatomakethesolution
alkalineandadjustthevolumeto50ml
Keepasidefor5min Keepasidefor5min
Colordevelopedisviewedvertically
andcomparedwithstandardsolution
Colordevelopedisviewedverticallyand
comparedwithstandardsolution
Procedure:
Note: All the reagents used in the limit test for Iron should themselves be iron free.
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36. Observation:
Thepurplecolorproduceinsamplesolutionshouldnotbegreaterthanstandardsolution.If
purplecolorproducesinsamplesolutionislessthanthestandardsolution,thesamplewill
passthelimittestofironandviceversa.
Reasons:
Citricacidformscomplexwithmetalcationandhelpsprecipitationofironby
ammoniabyformingacomplexwithit.
Thioglycolicacidhelpstooxidizeiron(II)toiron(III).
Ammoniaisaddedtomakesolutionalkaline.Thepalepinkcolorisvisibleonly
inthealkalinemedia.Thecolorisnotvisibleinacidicmediaasferrous
thioglycolatecomplexdecomposesinhighacidicmedia.
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37. Limit test for Chloride:
The test is used to limit the amount of Chloride as an impurity
in inorganic substances.
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38. Limit test for CHLORIDE:
Principle:
Limit test of chloride is based on the reaction of soluble chloride with silver nitrate in
presence of dilute nitric acid to form silver chloride, which appears as solid particles
(Opalescence) in the solution.
Cl
-
+ AgNO
3--------------------------AgCl + NO
3
-
Soluble
chloride
present as
impurity
ThesilverchlorideproducedinthepresenceofdiluteNitricacidmakesthetestsolution
turbid,theextentofturbiditydependingupontheamountofChloridepresentinthe
substanceiscomparedwiththestandardopalescenceproducedbytheadditionofSilver
nitratetoastandardsolutionhavingaknownamountofchlorideandthesamevolumeof
dilutenitricacidasusedinthetestsolution.
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silver chloride

39. Esha Shah 39

40. Test sample Standard compound
Specific weight of compound
is dissolved in water
or solution is prepared as
directed in the pharmacopoeia
and transferred in Nessler
cylinder
Take 1 ml of 0.05845 % W/V
solution of sodium chloride in
Nesslercylinder
Add1mlofnitricacid Add1mlofnitricacid
Diluteto50mlinNesslercylinderDiluteto50mlinNesslercylinder
Add1mlofAgNO
3solutionAdd1mlofAgNO
3solution
Keepasidefor5min Keepasidefor5min
Observe the
Opalescence/Turbidity
ObservetheOpalescence/Turbidity
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41. Observation:
Theopalescenceproduceinsamplesolutionshouldnotbegreaterthanstandard
solution.Ifopalescenceproducesinsamplesolutionislessthanthestandard
solution,thesamplewillpassthelimittestofchlorideandvisaversa.
Reasons:
Nitricacidisaddedinthelimittestofchloridetomake
solutionacidicandhelpssilverchlorideprecipitateto
makesolutionturbidattheendofprocessasDiluteHNO
3
isinsolubleinAgCl.
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42. Pharmacopoeiadoesnotprescribeanynumericalvalueoflimittestforchlorides,
sulphateandironbecauselimittestisbasedonthesimplecomparisonof
opalescenceorcolourbetweenthetestandstandardsolutionprescribedaccording
topharmacopoeia.
Inthistypeoflimittest,theextentofturbidityoropalescenceorcolourproduced
ininfluencedbythepresenceofotherimpuritiespresentinthesubstanceandalso
byvariationintimeandmethodofperformanceoftest.
Thusthepharmacopoeiadoesnotprescribeanynumericalvalueofthelimittest.
Pharmacopoeianotprescribeanynumericalvalueforlimittestforchlorides,
sulphateandironbecauselimittestisbasedonsimplecomparisonofopalescenceor
colorbetweentestandstandardsolutionprescribedaccordingtopharmacopoeia.
Thevariationinthepermissiblelimitsforvarioussubstancesisobtainedbytaking
varyingquantitiesofthesubstancesundertest.Inthistypeoflimittest,theextentof
opalescenceorturbidityorcolorproducedisinfluencedbythepresenceofother
impuritiespresentinthesubstanceandalsobyvariationintimeandmethodof
performanceoftestsandhencethepharmacopoeiadonotprescribeanynumerical
valuesforthelimittestinthesetest.
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43. The limit test involve simple comparisons of opalescence, turbidity, or colour with
standard.
These are semi-qualitative reactions in which extent of impurities present can be estimated
by comparing visible reaction response of the test and standard.
By this way, extent of reaction is readily determined by direct comparison of test solution
with standard. So pharmacopoeia prefers comparison methods.
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44. Limit test for sulphate:
TheSulfateLimitTestisdesignedtodeterminetheallowablelimitofsulfatecontainedina
sample.
Principle:
Limit test of sulphate is based on the reaction of soluble sulphate with barium chloride in
presence of dilute hydrochloric acid to form barium sulphate which appears as solid
particles (turbidity) in the solution.
Thencomparisonofturbidityisdonewithastandardturbidityobtainedfromaknown
amountofSulphateandsamevolumeofdiluteHydrochloricacidhavebeenaddedtoboth
solutions.
ThebariumchloridetestsolutionintheIPhasbeenreplacedbyBariumsulphatereagent
whichishavingbariumchloride,sulphatefreealcoholandasolutionofpotassium
sulphate.Potassiumsulphatehasbeenaddedtoincreasethesensitivityofthetest.
SO
4
-2
+ BaCl
2---------------------------BaSO
4+ 2Cl
-
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45. Test sample Standard compound
Specific weight of compound is
dissolved in water or solution is
prepared as directed in the
pharmacopoeia and transferred in
Nesslercylinder
Take 1 ml of 0.1089 % W/V solution of potassium
sulphate in Nesslercylinder
Add 2 ml of dilute hydrochloric acidAdd 2 ml of dilute hydrochloric acid
Dilute to 45 ml in Nesslercylinder Dilute to 45 ml in Nesslercylinder
Add 5 ml of barium sulphate reagentAdd 5 ml of barium sulphate reagent
Keep aside for 5 min Keep aside for 5 min
Observe the Turbidity Observe the Turbidity
Procedure:
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46. Barium sulphate reagent contains barium chloride, sulphate free alcohol and 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 solution acidic.
Potassium sulphate is used to increase the sensitivity of the test by giving ionic
concentration in the reagent.
Alcohol helps to prevent super saturation and so produces a more uniform opalescence.
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47. Limit test for Arsenic:
Arsenicisawellknownundesirableandharmfulimpuritywhichispresentinmedicinal
substances.
Allpharmacopoeiasprescribealimittestforit.
PharmacopoeialmethodisbasedontheGutzeittest.
AllthespecialreagentsusedinthelimittestforArsenicaremarkedanddistinguishedby
letter‘AsT’,whichmeansthattheyallshouldbeArsenicfreeandshouldthemselves
conformtothetestforArsenic.
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48. Principle:
LimittestofArsenicisbasedonthereactionofarsenicgaswithhydrogeniontoform
yellowstainonmercuricchloridepaperinpresenceofreducingagentslike
potassiumiodide.ItisalsocalledasGutzeittestandrequiresspecialapparatus.
Arsenic,presentasarsenicacid(H
3AsO
4)inthesampleisreducedtoarsenious
acid(H
3AsO
3)byreducingagentslikepotassiumiodide,stannousacid,zinc,
hydrochloricacid,etc.Arseniousacidisfurtherreducedtoarsine(gas)(AsH
3)
byhydrogenandreactswithmercuricchloridepapertogiveayellowstain.
Substance + dilHCl-------------------------H
3AsO
4
(contains Arsenic impurity) Arsenic acid
H
3AsO
4+ H
2SnO
2-------------------→ H
3AsO
3+ H
2SnO
3
Arsenic acid Arsenious acid
H
3AsO
3+ 6[H] ---------------------→AsH
3+ 3H
2O
Arsenious acidnascent hydrogen Arsine gas
Thedepthofyellowstainonmercuricchloridepaperwilldependuponthe
quantityofarsenicpresentinthesample.
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49. Whenthesampleisdissolvedinacid,theArsenicpresentinthesample
getsconvertedtoArsenicacid.
ByactionofreducingagentslikePotassiumiodide,stannousacidetc.,
Arsenicacidgetsreducedtoarseniousacid.
Thenascenthydrogenformedduringthereaction,furtherreduces
ArseniousacidtoArsinegas,whichreactswithmercuricchloridepaper,
givingayellowstain.
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50. Esha Shah 50

51. Apparatus:
Itishavingawidemouthedglassbottleof120mLcapacityhavingmouthofabout
2.5cmindiameter.Thisbottleisfittedwitharubberbungthroughwhichpassesaglass
tube,20cmlong.
Externaldiameter=0.8cm
Internaldiameter=0.65cm
Thetubeisconstrictedatitslowerendextremitytoabout1mmdiameterandthereis
blownahole,notlessthan2mmindiameter,inthesideofthetubeneartheconstricted
part.
Theupperendoftheglasstubeisfittedwithtworubberbungs(25mmx25mm),each
havingaholeboredcentrallyandexactly6.5mmindiameter.
Oneofthebungshasbeenfittedtotheupperendofthetube,whilethesecondbunghas
tobefitteduponthefirstbunginsuchawaythatthemercuricchloridepapergets
exactlysandwichedbetweenthecentralperforationofthetwo.
Thebungsarekeptinclosecontactbyusingrubberbandorspringclipinsuchamanner
thatthegasevolvedfromthebottlemusthavetopassthroughthe0.65mminternal
circleofmercuricchloridepaper.
Duringthetest,theevolvedgaseshavebeenpassingthroughthesidehole,thelower
holeservingasanexitforwaterwhichcondensesintheconstrictedpartofthetube.
AnimportantfeaturehasbeenthestandardizationoftheareaofMercuricchloridepaper
whichisexposedtothereactionofarsinegas.
Esha Shah 51

52. Test sample Standard compound
Thetestsolutionispreparedby
dissolvingspecificamountinwater
andstannatedHCl(arsenicfree)and
keptinawidemouthedbottle.
Aknownquantityofdilutearsenic
solutioninwaterandstannatedHCl
(arsenicfree)iskeptinwidemouthed
bottle.
1 g of KI 1 g of KI
5 ml of stannous chloride acid solution5 ml of stannous chloride acid solution
10 g of granulated zinc is added (all
this reagents must be arsenic free).
Keep the solution aside for 40 min
10 g of zinc is added (all this reagents
must be arsenic free).
Keep the solution aside for 40 min
Stainobtainedonmercuricchloridepaperiscomparedwithstandardsolution.
Standardstainmustbefreshlypreparedasitfadesonkeeping.
Inference:Ifthestainproducedbythetestisnotdeeperthanthestandard
stain,thensamplecomplieswiththelimittestforArsenic.
Esha Shah 52

53. 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 paper are used to trap any hydrogen sulphide which may be evolved
along with arsine.
Esha Shah 53

54. Use of stannatedHydrochloric acid:
If pure zinc and HClare used, the steady evolution of gas does not occur. This produces
improper stain (e.gslow evolution produces short but intense stain while rapid evolution of
gas produces long but diffused stain.)
So, to get steady evolution of gas, stannatedhydrochloric acid is used.
Use of Lead Acetate solution:
H
2S gas may be formed during the experiment as zinc contains sulphides as impurities. It
gives black stain to HgCl
2 paper and so will interfere the test.
Hence
,gases evolved are passed through cotton wool plug moistened with lead acetate,
where H
2S gas is trapped as PbS.
Use of Potassium iodide:
KI is converted to HI which brings about reduction of unreacted pentavalent arsenic to
trivalent Arsenic. Thus, reproducible results can be obtained. If it is not used then some
pentavalent Arsenic may remain unreacted.
Esha Shah 54

55. Limittestforheavymetals
Thelimittestforheavymetalsisdesignedtodeterminethecontentofmetallicimpurities
thatarecolouredbyhydrogensulphideorsodiumsulphideundertheconditionofthetest
shouldnotexceedtheheavymetallimitsgivenundertheindividualmonograph.
Theheavymetals(metallicimpurities)maybeiron,copper,lead,nickel,cobalt,bismuth,
antimonyetc..Thelimitforheavymetalsisindicatedintheindividualmonographin
termofppmofleadi.e.thepartsofleadpermillionpartsofthesubstancebeing
examined
Insubstancestheproportionofanysuchimpurity(Heavymetals)hasbeenexpressedas
thequantityofleadrequiredtoproduceacolorofequaldepthasinastandardcomparison
solutionhavingadefinitequantityofleadnitrate.
Thequantityisstatedastheheavymetallimitandisexpressedaspartsoflead(by
weight)permillionpartsofthetestsubstance.
Thelimittestforheavymetalshasbeenbaseduponthereactionofthemetalion
withhydrogensulphide,undertheprescribedconditionsofthetestcausingthe
formationofmetalsulphides.
Theseremaindistributedincolloidalstate,andgiverisetoabrownishcoloration.
Esha Shah 55

56. Esha Shah 56
I.Plimitforheavymetalsin20ppm.
Thetestsolutioniscomparedwithastandardpreparedusingaleadsolution(astheheavy
metal).Themetallicimpuritiesinsubstanceareexpressedaspartsofleadpermillionparts
ofsubstance.
IPhasadopted3methodsforthis:
MethodI:Themethodisapplicableforthesampleswhichgiveclearcolourlesssolutions
underspecifiedconditionsoftest.
MethodII:ThemethodisapplicableforthesampleswhichDONOTgiveclear
colourlesssolutionsunderspecifiedconditionsoftest.
MethodIII:Usedforsubstanceswhichgiveclearcolourlesssolutionsinsodium
hydroxidemedium.

57. Esha Shah 57
Limittestforlead:
Leadisamostundesirableimpurityinmedicalcompounds
andcomesthroughuseofsulphuricacid,leadlined
apparatusandglassbottlesuseforstorageofchemicals.
Principle:
Limittestofleadisbasedonthereactionofleadanddiphenyl
thiocabazone(dithizone)inalkalinesolutiontoformlead
dithizonecomplexwhichisredincolor.

58. Esha Shah 58
Dithizoneinchloroform,isabletoextractleadfromalkalineaqueoussolutionsas
aleaddithizonecomplex(Redincolour)
Theoriginaldithizoneishavingagreencolourinchloroformwhilethelead-
dithizoneishavingavioletcolor.So,resultingcolorattheendoftheprocessis
read.
Theintensityofthecolorofcomplexisdependantupontheamountofleadin
thesolution.
Thecolorofthelead-dithizonecomplexinchloroformhasbeencomparedwitha
standardvolumeofleadsolution,treatedinthesamemanner.
Inthismethod,theleadpresentasanimpurityinthesubstances,getsseparatedby
extractinganalkalinesolutionwithadithizoneextractionsolution.
Theinterferenceandinfluenceoftheothermetalionshasbeeneliminatedby
adjustingtheoptimumpHfortheextractionbyemployingAmmonium
citrate/potassiumcyanide.

59. Esha Shah 59
Method:
•Sample solution is transferred to a separating funnel.
•To it 6 ml of ammonium citrate, 2 ml potassium cyanide and 2 ml of hydroxalamineHCl
are added.
•2 drops of phenol red
•Solution is made alkaline by adding ammonia solution.
•This is then extracted with 5 ml portions of dithizonesolution until it becomes green.
•The combined dithizoneextracts are shaken for 30 seconds with 30 ml of nitric acid and
chloroform layer is discarded.
•To the acid solution 5 ml of standard dithizonesolution is added and 4 ml ammonium
cyanide and solution is shaken for 30 sec.
•Similarly prepare standard.

60. Esha Shah 60
Observation:
The intensity of the colorof complex, is depends on the amount of lead
in the solution. The colorproduced in sample solution should not be
greater than standard solution. If colorproduces in sample solution is less
than the standard solution, the sample will pass the limit test of lead and
vice versa.
Reasons:
Ammonium citrate,potassiumcyanide,hydroxylamine
hydrochlorideisusedtomakepHoptimumsointerferenceand
influenceofotherimpuritieshavebeeneliminated.
Phenolredisusedasindicatortodevelopthecolorattheendofprocess
Leadpresentasanimpuritiesinthesubstance,getsseparatedby
extractinganalkalinesolutionwithadithizoneextractionsolution.

61. Esha Shah 61
A known quantity of sample solution is
transferred in a separating funnel
A standard lead solution is prepared equivalent to
the amount of lead permitted in the sample under
examination
Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 drops of phenol red Add 2 drops of phenol red
Make solution alkaline by adding ammonia solution.Make solution alkaline by adding ammonia solution.
Extract with 5 ml of dithizoneuntil it becomes
green
Extract with 5 ml of dithizoneuntil it becomes green
Combine dithizoneextracts are shaken for 30 mins
with 30 ml of nitric acid and the chloroform layer is
discarded
Combine dithizoneextracts are shaken for 30 mins
with 30 ml of nitric acid and the chloroform layer is
discarded
To the acid solution add 5 ml of standard dithizone
solution
To the acid solution add 5 ml of standard dithizone
solution
Add 4 ml of ammonium cyanide Add 4 ml of ammonium cyanide
Shake for 30 mins Shake for 30 mins
Observe the color Observe the color

62. Aq. Ammonia is added in limit test of lead:
Pb+ S-------------PbS
In limit test of lead, PbSis produced by addition of standard H
2S , to the
solution containing lead.
pH 3-4 is necessary for the precipitation of PbS. So aq. Ammonia /
acetic acid is added to maintain that pH.
Esha Shah 62

63. Modified limit test for Chlorides
Depending upon the nature of the substance,
some modifications have to be adopted for the
preparation of the solution.
Esha Shah 63

64. Modified limit test for Chlorides
(a)Alkalinesubstanceshavetobedissolvedinacidsothateffervescenceceasesand
muchofthefreeacidisleftinthesolutionasisprescribedinthetest.
(b)Insolublesubstancesaregenerallyextractedwithwaterandthenfiltered,andthe
filtrateisusedforthetest,becausethepresenceofinsolublesubstancemodifiesthe
opalescenceandcolour.
(c)Saltsoforganicacidslikesodiumbenzoate,sodiumsalicylate,etc.liberatefree
waterinsolubleorganicacidduringacidificationwhichisfilteredoffandthefiltrateis
employedforthetest.
(d)Colouredsubstanceslikecrystalviolet,malachitegreen,etc.arecarbonisedandthe
ashsoproducedisextractedinwater.
(e)Deeplycolouredsubstanceshavetobedecolourisedbeforeteste.g.,potassium
permanganateisreducedbyboilingwithalcoholandthefiltrateisused.
(f)Reducingsubstanceslikehypophosphorusacid,whichreactwithsilvernitrateinthe
limittestforchloridesshouldbeoxidizedwithnitricacidorsomeotheroxidizing
agentsbeforecarryingoutthetest.
Esha Shah 64

65. Modified limit test for Chlorides
AimToperformthelimittestforchlorideinpotassiumpermanganatesample(accordingto
IP’96)
Requirement:
Nessler’scylinder,measuringcylinder,pipette,spatula,distilledwater,dilutenitricacid,0.1M
silvernitratesolution,potassiumpermanganatesample
Principle:
Thelimittestforchloridebasedonthereactionbetweensolublechlorideimpuritiespresentinthe
substanceandsilvernitratesolutiontogivewhiteprecipitatesofsilverchloride.Thesewhite
precipitatesareinsolubleindilutenitricacidandhencegiveturbidityoropalescencetothetest
solution.Theextentoftheturbidityproduceddependsupontheamountofthechloridepresentin
thesubstancewhichiscomparedwithastandardopalescenceproducebyadditionofsilver
nitratetoastandardsolutionhavingknownamountofchlorideandthesamevolumeofthedilute
nitricacidastheuseinthetestsolution.Iftheturbiditydevelopedinthesampleislessthanthe
standardturbidity,thesamplepassesthelimittestforchlorideandvice-versa.Aspotassium
permanganategivespurplecoloraqueoussolutionthatinterferesinthecomparisonof
opalescenceorturbidity,thereforetheaqueoussolutionmustfirstbedecolorized.
Potassiumpermanganateisoxidizingagentwhileethanolisreducingagent.
Esha Shah 65

66. Whenpotassiumpermanganatesolutionistreatedwithethanolinpresenceofheat
theredoxreactionwilltakeplace,i.e.potassiumpermanganategetsreducedto
manganesedioxide(precipitates).Thefiltrateofthereactioniscolorlessthatis
subjectedtoproceedforlimittestforchloride.
Esha Shah 66

67. Esha Shah 67
Chemical Reaction:
2 KMnO
4
+ 3 C
2
H
5
OH----------2 MnO
2
+ 2 KOH + 2 CH
3
CHO + 2 H
2
O
Sr.
No.
STANDARDSOLUTION SR.
NO
TESTSOLUTION
1 Take10mlchloridestandardsolution(25ppm
chloride)andadd5mlwaterinaNessler’s
cylinder.
1 TransferthepreparedtestsolutioninNessler’s
cylinder
2 Add10mlofdilutenitricacidanddiluteto50ml
withdistilledwater
2 Add10mlofdilutenitricacidanddiluteto50
mlwithdistilledwater
3 Add1mlof0.1Msilvernitratesolutionandstir
immediatelywithglassrodandallowstanding
for5minutesprotectedfromlight.
3 Add1mlof0.1Msilvernitratesolutionand
stirimmediatelywithglassrodandallow
standingfor5minutesprotectedfromlight.
Compare the turbidity or opalescence produced in test solution with respect to
standard solution and report the result and conclusion.

68. Observation and conclusion:
Observation and conclusion will be of two types:
If the intensity of turbidity or opalescence appears to be more in test
solution than the standard solution then conclusion is impurities of
chloride in given sample is over the limit as per IP’96. Hence, sample
do not passes the limit test for chlorides.
If the intensity turbidity or opalescence appears to be less or equal in
test solution than the standard solution then conclusion is impurities
of chloride in given sample is under the limit as per IP’96. Hence,
sample passes the limit test for chloride
Esha Shah 68

69. To perform the limit test for sulphatein Potassium
permanganate sample (According to IP’96)
Principle:
It is a comparison method. It involves the comparison of opalescence or turbidity of test sample verses
standard sample which contain the definite amount of sulphateimpurities.
The limit test of sulphateis performed on the basis of reaction between the barium chloride reagent
(containing barium chloride, sulphatefree alcohol and solution of potassium sulphate(K
2SO
4) and
soluble sulphatein the sample with formation of barium sulphate(BaSO
4) white precipitates.
Sulphatefree alcoholic potassium sulphateis added to increase the sensitivity of the test. Very small
amount of barium sulphatepresent in the reagents acts as a seeding agents for precipitation of barium
sulphate, if sulphateis present in the sample under the test.
Ethanol is added to prevent the super saturation i.e. the crystallization of sulphatewith any other ion.
As potassium permanganate gives purple colored aqueous solution that interferes in the comparison of
opalescence or turbidity, therefore it requires to be decolorized. Potassium permanganate is oxidizing
agent while ethanol is reducing agent. When potassium permanganate solution is treated with ethanol
in presence of heat the redox reaction takes place, i.e. potassium permanganate gets reduced to
manganese dioxide (precipitates) and ethanol gets oxidized to form ethanal. The filtrate of the reaction
is colorless that is subjected to proceed for limit test for sulphate.
Esha Shah 69

70. Sr.
No.
STANDARDSOLUTION SR.
NO
TESTSOLUTION
1 Take1ml25%w/vbariumchlorideinNessler’s
cylinderandadd1.5mlofethanolicsulphate
standardsolution(10ppmSO
4
-2
).Mixandallowto
standfor1minutes
1 Take1ml25%w/vbariumchlorideinNessler’s
cylinderandadd1.5mlofethanolicsulphate
standardsolution(10ppmSO
4
-2
).Mixandallowto
standfor1minutes
2 Add15mlofstandardsulphatesolution(10ppm
SO
4
-2
)and0.15mlof5Maceticacid.
2 Transferpreparedtestsolutionandadd0.15mlof
5Maceticacid.
3 Addsufficientdistilledwatertoproduced50ml.
Stirreditimmediatelyandallowstandingfor5
minutes.
3 Addsufficientdistilledwatertoproduced50ml.
Stirreditimmediatelyandallowstandingfor5
minutes.
Esha Shah 70
Reaction:
2KMnO
4
+3C
2
H
5
OH--------------------2MnO
2
+2KOH+2CH
3
CHO+2H
2
O

71. •Compare the turbidity or opalescence in the
test solution by viewing transversely both
solutions against black background.
•
Esha Shah 71