This document provides an introduction to pharmaceutical chemistry and discusses sources of errors and impurities in pharmaceuticals. It describes how impurities can affect pharmacopoeial substances by making them toxic, decreasing therapeutic effects, or changing physical/chemical properties. Common impurity tests are outlined, including limit tests for chlorides, sulfates, arsenic, and iron which use chemical reactions to identify impurities and compare results to standards. Proper procedures and reasons for each test are explained in detail.

1. Introduction to
Pharmaceutical Chemistry
By: Mrs. V. S. Lokhande

2. Sources and types of errors
 Accuracy
 Precision
 Errors
 Determinate errors
 Indeterminate errors
 Significant figures

3. Impurities in Pharmaceuticals
 Sources of Impurities in Pharmaceutical Substance
1. Raw material used in Manufacturing Process
2. Method or Process used in Manufacturing
3. Reagents and Solvents used in Manufacturing Process
4. Reaction Vessels
5. Atmospheric Contaminants
6. Decomposition of the Product during storage

4. Effects of Impurities on Pharmacopoeial Substance
 Impurities which have a toxic effect can be injurious when present above certain
limits
 Impurities even when present in traces , may show cumulative toxic effect after a
certain limit
 Impurities are sometimes harmless, but are present in such large proportions, that
the active strength of the substance is lowered, thus therapeutic effect of dug is
decreased
 Impurities may bring about a change in the Physical & Chemical properties of the
substance thus making it medically useless

5. Effects of Impurities on Pharmacopoeial
Substance
 Impurities may cause technical difficulties in the formulation & use of the
substance
 Impurities may lower Shelf life of substance
 Impurities though harmless in nature may bring about changes in odour , colour ,
taste etc. thus making the use of the substance unethical as well as unhygenic

6. Test for purity:
 Pharmacopoeia prescribe the “Test for purity” for pharmaceutical substance 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, Cations and Anions
 Limit test for Chlorides, Sulphates, Arsenic, Iron, Arsenic
 Qualitative Analysis and Quantative Analysis

7. Limit Test
 Definition : Limit tests are quantitative or semi quantitative test designed to
identify and control small quantities of impurities which are likely to be present
in the substances.

8. Limit Test for Chlorides:
The test is used to limit the amount of Chloride as an impurity in
inorganic substance
 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- + AgNO3 AgCl + NO3
-
Soluble chloride Silver Chloride
present as
impurity

9. Procedure:
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 1ml of 0.05845% W/V solution of sodium
chloride in Nessler cylinder
Add 10ml of dilute Nitric Acid Solution Add 10ml of dilute Nitric Acid Solution
Dilute up to 50ml with water Dilute up to 50ml with water
Add 1ml of Silver Nitrate Solution Add 1ml of Silver Nitrate Solution
Keep aside for 5min Keep aside for 5min
Observe the Opalescence/ Turbidity Observe the Opalescence/ Turbidity

10. Observation:
The opalescence produce in sample solution should not be greater than standard
solution. If opalescence produce in sample solution is less than the standard
solution, the sample will pass the limit test of Chloride and vice versa.
Reason:
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 the process as dilute
Nitric acid is insoluble in Silver Chloride

11. Limit Test for Sulphates:
The test is used to limit the amount of sulphate as an impurity in
inorganic substance
The limit test for sulphate is based on reaction between Barium Chloride and soluble
sulphate in the presence of dilute hydrochloric acid turbidity is produced by
precipitation of Barium sulphate in a fine state of division this is compared with
turbidity produced in standard containing known quantity of sulphate and similarly
treated
HCl
SO4
2- + BaCl2 BaSO4 + Cl-
The Barium sulphate reagent is used in test which contains barium chloride, sulphate
free alcohol & small amount of potassium sulphate the addition of potassium sulphate
increases sensitivity of the test & alcohol prevents supersaturation and more uniform
turbidity is formed

12. Procedure:
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 1ml of 0.1089%W/V solution of potassium
sulphate in Nessler cylinder
Add 2ml of dilute hydrochloric acid Add 2ml of dilute hydrochloric acid
Dilute to 45ml in Nessler Cylinder Dilute to 45ml in Nessler Cylinder
Add 5ml of Barium Sulphate Reagent Add 5ml of Barium Sulphate Reagent
Keep aside for 5min Keep aside for 5min
Observe the Opalescence/ Turbidity Observe the Opalescence/ Turbidity

13. Observation:
The opalescence produce in sample solution should not be greater than standard
solution. If opalescence produce in sample solution is less than the standard
solution, the sample will pass the limit test of Chloride 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.

14. Limit test for Arsenic:
 Arsenic is well known undesirable and harmful impurity which is present in
medicinal substance
 All pharmacopeia prescribed limit test for it
 Pharmacopoeial method is based on the Gutzeit test
 All the special reagent used for limit test for Arsenic are marked and
distinguished by letter 'As T' which means that they all should be Arsenic free
and should themselves confirm for test for Arsenic

15. Principle of Limit test for Arsenic:
 Limit test of arsenic is based on the reaction of arsenic gas with hydrogen ion to form
yellow stain on Mercuric chloride paper in the presence of reducing agent like
potassium iodide , Stannous Chloride or Sulphuric acid It is also called as Gutzeit test
and requires special apparatus
 Arsenic present in arsenic acid (H3 AsO4) in the sample is converted to either
arsenious acid ( if the arsenic is trivalent) or arsenic acid ( if the arsenic is
pentavalent) then it is further treated with reducing agent and converted in to
Arsenious acid
 Arsenious acid is further reduced to arsine gas ( Arsenious hydride AsH3 )
 by nascent hydrogen which is produced by action of granulated zinc and
hydrochloric acid. When arsine comes into contact with dry paper saturated with
mercuric chloride it produces yellow stain

16. Reaction
H3 AsO4 H3 AsO3
Arsenic acid Arsenious acid
H3 AsO3 AsH3 + 3H2O
Arsine
2AsH3 + HgCl2 Hg(AsH2 )2 +2HCl
Yellow Stain
The depth of yellow stain on mercuric chloride paper will depend upon the quantity of
arsenic present in the sample

17. Gutzeit Apparatus

18. Gutzeit Apparatus
 Apparatus : it is having wide mouthed glass bottle of 120 ml capacity having
mouth of about 2.5 cm in diameter this bottle is fitted with rubber bungs
through which passes a glass tube 20 cm long
 external diameter 0.8 cm
 internal diameter 0.65 cm
 the tube is constricted at its lower end extremities to about 1 mm diameter and
there is blown a hole not less than 2 mm in in diameter in the side of the tube
near the constricted part
 the upper end of the glass tube is fitted with to rubber bumps (25mm x 25mm)
each having a hole bored centrally and exactly 6.5 mm in diameter

19.  One of the bungs has been fitted to the upper end of the tube while the second
bung has to be fitted upon the first bung in a such way that Mercury chloride
paper gets exactly sandwiched between the central perforation of the two.
 Bungs are kept in close contact by using rubber band or spring clip in a such
manner that the gas evolved from the bottle must have to pass through the 0.65
mm internal circle of Mercuric chloride paper
 During the test the evolved gases have been passing through the side hole, the
lower hole serving as and exit for water which condenses is in constructed part of
the tube
 an important feature has been standardization of the area of Mercury chloride
paper is exposed to the reaction of arsine gas

20. Procedure
Test Sample Standard Compound
The test solution is prepared by dissolving specific
amount in water and stannated HCl (arsenic
free) and kept in a wide mouthed bottle
A known amount of dilute arsenic solution is kept
in the wide mouthed bottle of the apparatus.
To this solution 1 gm of KI, 5 ml of stannous
chloride acid solution and 10 gm of zinc is added
(all this reagents must be arsenic free)
To this solution 1 gm of KI, 5 ml of stannous
chloride acid solution and 10 gm of zinc is added
(all this reagents must be arsenic free)
Keep the solution aside for 40 min Keep the solution aside for 40 min
Compare the stain obtained on mercuric chloride
paper with standard solution
Compare the stain obtained on mercuric chloride
paper with standard solution.
The stain produced by the test is not deeper than the standard solution.
Standard stain must be freshly prepared as it fades on keeping

21. Inference:
If the stain produced by the test is not deeper than the standard stain , then sample complies
with the limit test for Arsenic.
Reasons:
Stannous chloride is used for complete evolution of arsine.
Zinc, potassium iodide and stannous chloride is used as a reducing agent
Hydrochloric 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.

22. Limit test for iron
 Limit test for iron limit test of iron is based on the reaction of iron in Ammonia
solution with thioglycolic acid in the presence of citric acid to form iron
thioglycolate ferrous thioglycolate complex which produces pale pink to Deep
reddish purple colour in alkaline media
 Thioglycolic acid is used as reducing agent
 The colour of the ferrous thioglycolate complex fades in the presence of air due
to the oxidation
 Also the colour is destroyed in the presence of oxidizing agent and strong alkalis
 The purple colour is developed only in alkaline media so Ammonia solution is
used
 But Ammonia reacts with iron forms precipitate of ferrous hydroxide
 Thus citric acid used which prevents the precipitate of iron with ammonia by
forming a complex with iron as iron citrate

23. Reaction:
Fe2+ + 2CH2SH CH2SH OOC + 2H+
Fe
COOH COO HSH2C
Thioglycolic Acid Thioglycolic acid complex

24. Procedure
Test Sample Standard Compound
Sample is dissolved in specific amount of water
and then volume is made up to 40 ml
2 ml of standard solution of iron diluted with
water up to 40 ml
Add 2 ml of 20% w/v of citric acid (iron free ) Add 2 ml of 20% w/v of citric acid (iron free )
Add two drops of thioglycolic acid Add two drops of thioglycolic acid
Add ammonia to make the solution alkaline and
adjust the volume 50 ml keep aside for 5 minutes
Add ammonia to make the solution alkaline and
adjust the volume 50 ml keep aside for 5 minutes
Colour developed is viewed vertically and
compared with standard solution
Colour developed is viewed vertically and
compared with standard solution
Note: All the reagents used in the limit test for Iron should themselves be iron free

25. Observation:
The purple colour produced in the sample solution should not be creator dial
standard solution purple colour produces in sample solution is less then the standard
solution the sample will pass the limit test of iron and vice versa
Reasons:
 Citric acid prevent the precipitate of iron with ammonia by forming a
complex with it
 Thioglycolic acid helps to oxidize iron (II) iron (III)
 Ammonia is added to make a solution alkaline The Pale pink colour is visible
only in the alkaline medium the colour is not visible acidic media ferrous
thioglycolate complex decomposers in high acidic medium

26. Limit test for heavy metals
 Limit test for heavy metals is designed to determine the content of metallic
impurities that are coloured by hydrogen sulphide or sodium sulphide under the
condition of the taste should not exceed the heavy metal limits given under the
individual monograph
 The heavy metals (metallic impurities) may be iron copper lead Nickel Cobalt
Bismuth antimony etc. the limit for heavy metal is indicated in the individual
monograph in the term of ppm of lead that is the parts of lead for million parts of
the substance being examined
 In the substance the proportion of any such impurities (Heavy metals )has been
expressed as the quantity of lead required to produce the colour of equal depth as
the standard comparison solution having a definite quantity of Lead Nitrate
 The quantity is stated as the heavy metal limit and is expressed as the part of lead
(by weight) per million parts of the test substance.

27.  The limit test for heavy metals has been based upon the reaction of metal ions
with the hydrogen sulfide under the prescribed conditions of the test causing the
formation of metal sulphide
 These remain distributed in colloidal state, and give rise to brownish coloration
 I. P. limit for heavy metal is 20 ppm
 The test solution is compared with standard prepared using a lead solution (as the
heavy metal )The metallic impurities in substance are expressed as a part of lead
per million parts of substance
 IP has adapted three methods for this:
 Method I: the method is applicable for samples which gives a colourless solution
under specified condition of the test
 Method II: the method is applicable for the samples we do not give the clear
colourless solution on the specified condition of the test
 Method III: used for substances which gives clear colour solution in sodium
hydroxide medium

28. Limit test for Lead:
Lead is most undesirable impurity in medicinal compounds and comes through the
use of sulfuric acid, lead lined apparatus and glass bottles use for the storage of
chemicals:
 Principle:
 Limit test of the lead is based on the reaction of lead and diphenyl thiocarbazone
(dithizone )in alkaline solution to form lead dithizone complex which is red in
colour.
 Dithizone in chloroform is able to extract lead from the alkaline aqueous solution as
a lead dithizone complex( Red in colour )
 The original dithizone is having a green colour in chloroform while the lead
dithiozone is having a violet colour so the resulting colour at the end of the process
is red .
 The intensity of the colour of the complex is dependent upon the amount of lead in
the solution.

29. The colour of the lead-dithizone complex is chloroform has the compared with the standard
volume of lead solution, treated in the same manner.
 In this method ,the lead present as an impurity in the substance get separated by
extracting in alkaline solution with dithizone extraction solution.
 The interference and influence of the other metal ions has been eliminated by adjusting
the optimum pH for the extraction by employing Ammonium citrate /potassium cyanide
Reaction:
 Aq. ammonia is added in the limit test of lead:
 In the limit test of the lead, PbS is produced by addition of standard H2S, 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.

30. Method:
 Sample solution is transferred to the separating funnel.
 To it 6 ml of Ammonium citrate 2ml and potassium cyanide 2 ml of hydroxylamine
hydrochloride are added
 2 drops of phenol red
 Solution is made alkaline by adding Ammonia solution.
 This is then extracted with 5ml portions of dithizone solution until it becomes green
 The combined dithizone extracts are shaken for 30 seconds and with the 30 ml of
nitric acid chloroform layer is discarded.
 To the acid solution 5ml of standard dithiazone solution is added and 4 ml
ammonium cyanide and solution is shaken for 30 second.
 Similarly prepare standard

31. Observations:
The intensity of the colour of the complex is depend on the amount of lead in the
solution the colour produced in the sample solution should not be greater than the
standard solution if the colour produces in the 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 the interference and influence of other impurities have been
eliminated .
 Phenol red is used as indicator to develop the colour at the end of the process
Lead present as impurities in the substance gets separated by extracting an
alkaline solution with dithiazone extraction solution.

32. Procedure
Test Sample Standard Compound
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 6 ml of Ammonium citrate Add 6 ml 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 the solution alkaline by adding Ammonia solution Make the solution alkaline by adding Ammonia solution
Extract with 5 ml of diethizone until it becomes green Extract with 5 ml of diethizone until it becomes green
Combined dithizone extracts are shaken for 30 minutes
with 30 ml of nitric acid and chloroform layer is
discarded
Combined dithizone extracts are shaken for 30 minutes
with 30 ml of nitric acid and chloroform layer is discarded
To the acid solution add 5 ml of standard dithiazone
solution
To the acid solution add 5 ml of standard dithiazone
solution
Add 4 ml of ammonium cyanide shake 30 minutes Add 4 ml of ammonium cyanide shake 30 minutes
observe the colour observe the colour

33. EXERCISE
1.5.2 Multiple Choice Questions
1) Lead acetate cotton plug is used in limit test for Arsenic for which of the following purpose?
a) To promote release of phosphine gas.
b) To allow uniform passage of arsenious gas formed.
c) To avoid passage of hydrogen sulphide gas formed.
d) To improve the efficiency of reaction.
Answer is
c) To avoid passage of hydrogen sulphide gas formed.

34. 2) In the limit test for lead, lead present as impurity is extracted using.
a) Barium sulphate
b) Dithizone solution
c) Silver nitrate
d) Sodium phosphate
Answer is
b) Dithizone solution

35. 3) Limit test for sulphate has been based upon the precipitation of sulphate with which of the
following?
a) BaCl2, and AgCl
b) AgNO3 and Hcl
c) BaCl2, and Hcl
d) AgCl and H₂SO4
Answer is
c) BaCl2, and Hcl
4) The limit test for arsenic is based upon which of the following test?
a) Karl-Fisher test Nitrate test
b) Gutzeit test
c) Nitrate test
d) Goldbeaters test
Answer is
b) Gutzeit test

36. 5) In limit test for iron, purple color is due to formation of
a) Ferrous thioglycolate
b) Glycolate
d) Glycolic acid
c) Ferric thioglycolate
Answer is
a) Ferrous thioglycolate
6) Apparatus used for limit test is:
a) Measuring cylinder
b) Nessler cylinder
c) Conical flask
d) Test tube
Answer is
b) Nessler cylinder

37. 7) Limit test are ----- test designed to identify and control small quantities of impurities.
a) Quantitative
b) Qualitative
c) Physiochemical
d) Therapeutic
Answer is
a)Quantitative
8) Which of the following error is unpredictable
a)Determinate error
b) Indeterminate error
c) Gross error
d) None of the above
Answer is
b) Indeterminate error

38. 1.5.2. Very Short Answer Type Questions
1) Define Pharmaceutical chemistry and impurities.
2) Enlist environmental factors which impure the drug substances
3) Write down the principle of heavy metal limit test.
1.5.3. Short Answer Type Questions
1) Explain the limit test of arsenic with diagram, principle and procedure.
2) Write a note on limit test of sulphate
3) Discuss on limit test for chlorides.
1.5.4.Long Answer Type Questions.
1) Discuss various sources of impurities in pharmaceutical products. Discuss principle,
chemistry involved and method of limit test of iron.
2) Write a detail note on sources of impurities in pharmaceuticals.

39. Thank You