LAB MANUAL ANALYSIS Pharmaceutical Analysis BP 1ST SEMESTER

Lab Manual B. Pharm 1st year Pharmaceutical Analysis

1. RBMI
Rakshpal Bahadur College of Pharmacy,
Bareilly








B. PHARM 1
ST
SEM







PHARMACEUTICAL ANALYSIS LAB
MANUAL

2. LIST OF EXPERIMENTS




S. No. EXPERIMENTS
1 TO STUDY THE COMMO N GLASSWARE, EQUIPMENT, AND GOOD
LABORATORY PRACTICES (GLP)
2 TO PERFORM THE LIMIT TEST OF CHLORIDES
3 TO PERFORM THE LIMIT TEST OF SULPHATES
4 TO PREPARE AND STANDARDIZE 0.1N HCl SOLUTION
5 TO PREPARE AND STANDARDIZE 0.1N POTASSIUM PERMANGANATE

6 TO PREPARE AND STANDARDIZE 0.1N SULPHURIC ACID (H2SO4)
7 TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF AMMONIUM
CHLORIDE (NH4Cl)
8 TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF FERROUS
SULPHATE (FeSO4)
9 TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF SODIUM
CHLORIDE (NaCl) BY PRECIPITATION TITRATION
10 TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF HYDROGEN
PEROXIDE (H2O2) BY PERMANGANOMETERY

3. Experiment 01


In chemistry, a variety of glassware and techniques are used for the preparation, separation, and purification
of organic compounds.
1) FLASKS: These are the common type of glassware used for refluxing & distillation. The
different types of flasks are

1) Round-bottomed flask

2) Volumetric flask (measuring flask or graduated flask)




3). Conical flask (Erlenmeyer flask)



4). Iodine flask
AIM: TO STUDY THE COMMO N GLASSWARE, EQUIPMENT, AND
GOOD LABORATORY PRACTICES (GLP)

4. 5). Buchner’s flask

2) CONDENSERS: Air and water condensers are used for refluxing & also ordinary distillation.
The air condenser is employed if the liquid has a very high boiling point.






3) FUNNELS: Three types of funnels are generally used 1) Ordinary 2) Buchner and
3) Separatory funnel.





4). ADAPTORS: These are normally used to deliver distillate from the condenser to the receiver vacuum
and can also be applied to the adaptor if required.

5. 5). PIPETTE: It is a glass tube which indicates at the centre to bear the volume of liquid as marked on it may be
cylindrical with graduation.They may be of 1ml, 2ml, 5ml, 10ml, 25ml etc.






Bulb Pipette Graduated pipette
6). BURETTE: It is a long- graduated tube with a stop cork at one end. It is made up of glass or polyvinylchloride in
different volumes. It can be used to transfer or measure a desired volume of liquid. Each ml of the volume of liquid
can be read on the graduated surface of the burette.






7). MEASURING CYLINDER: It is a tall cylinder made up of thick glass and is graduated. It is available in the
capacity of 5ml, 10ml, 25ml, 50ml, 100ml, etc. It is used to measure a definite volume of a liquid.

8).BEAKERS: These are cylindrical, flat-bottomed containers available in the capacities of 25ml,
50ml,100ml etc the volume of the beaker is noted on the face of the beaker.

6. EQUIPMENT:
1). DESSICATOR: It is a covered glass container designed to store compounds in a dry atmosphere. It
usually contains a drying agent in the bottom part separated using a porcelain plate having holes in it through
the surface. The drying agents generally used are silica gel, calcium chloride, and activated aluminum. It
should be noted that a desiccator whose vapor pressure is greater than that of the substance itself. These are
of two types: ordinary and vacuum dessicator.







2). STIRRERS: These are generally made of glass but those made of stainless steel or Teflon are also used.
The stirrer is attached to a small electric motor with the aid of small pressure tubing & mechanical agitation
is achieved.
a). Magnetic stirrer: It is used for stirring small quantities of non-viscous reaction mixtures. The stirring is
achieved by a magnetic spinning bar which is added to the reaction mixture.






b). Mechanical stirrer: Large-scale mixtures &viscous reaction mixtures require great power for uniform
stirring this is achieved by an electric motor attached

7. GOOD LABORATORY PRACTICES
1. Wear neat and ironed laboratory apron while working

2. Come well prepared by reading the principle and procedure for the experiment concerned.

3. Read the procedure and/or consult the lab in charge to solve your problem or clear your doubts.
4. Do not consult your classmates for doubts which often do not give the benefit.

5. Maintain discipline and norms

6. Use clean glass wares.

7. Use strong acids, bases, and other corrosives carefully.

8. Do not displace the reagents from their respective places.

9. Do not interchange pipettes/ droppers from one reagent to the other without thorough cleaning.

10. Use the gas whenever necessary; close the knob when not required.

11. Prepare your reagents for correct results.

12. Submit laboratory records for correction in every practical class

13. Take the signature from your lecturer for all the observations.

14. Leave the laboratory only after cleaning your workplace

8. Experiment 02


Reference: 1. Dr. G.Devalo Rao. Practical pharmaceutical inorganic chemistry
2. Anees Ahamed Siddiqui and Mohammed Ali. Practical pharmaceutical chemistry.

Requirements: Apparatus: Nessler cylinder,
Chemicals: Chloride Sample, dil. Nitric

Principle
It is based upon the chemical reaction between silver nitrate and soluble chlorides in the presence
of dilute nitric acid to give opalescence of silver chloride. If the Opalescence produced is compared
with the standard solution. If the opalescence in the sample is less than the standard, it passes the test.
If it is more than the standard, it fails the test.
Dil. HNO3
Cl
-
+ AgNO3 AgCl + No3
Procedure
Two 50 ml Nessler Cylinders were taken and labeled as “Test” and the other as ‘Standard’.
Test Standard
1. The specified quantity of the
substance was dissolved in distilled
water and transferred to the Nessler
cylinder.
2. 10 ml of dilute nitric acid (HNO3)
was added to the above mixture.
3. The above mixture was diluted with
50 ml of water.
4. 1 ml of silver nitrate (AgNO3) was
added to the above solution.
5. The opalescence/ turbidity was
observed.
1. 1 ml of 0.05845% w/v solution of
NaCl was taken and transferred to
the Nessler cylinder.
2. 10 ml of dilute nitric acid (HNO3)
was added to the above mixture.
3. The above mixture was diluted with
50 ml of water.
4. 1 ml of silver nitrate (AgNO3) was
added to the above solution.
5. The opalescence/ turbidity was
observed.

Note: The extent of turbidity depends upon the amount of chloride present.


AIM: TO PERFORM THE LIMIT TEST OF CHLORIDES
Glass rod
acid, silver nitrate, Sodium chloride

9. Observation: The opalescence produced in the sample solution should not be greater than the standard
solution. Then, the sample will pass the limit test of chloride and vice versa.
Result: The given sample passes/fails the limit test of chlorides.

10. Experiment 03


Reference: 1. Dr. G.Devalo Rao. Practical pharmaceutical inorganic chemistry
2. Anees Ahamed Siddiqui and Mohammed Ali. Practical pharmaceutical chemistry.

Requirements: Nessler’s cylinder, beaker, pipette, measuring cylinder, glass rods.
Chemicals: BaCl2 solution, BaSO4 reagent, dil. HCl, standard solution of potassium sulphate, distilled water,
test sample,
Principle: This test is designed for the control of sulphate impurity in inorganic substances. It depends upon
the precipitation of the sulphate with barium chloride in the presence of HCl and traces of barium sulphate.
The turbidity obtained is compared with standard solution, which contains the known amount of sulphate
ions under the same experimental conditions.

SO
4
2-
+ BaCl
2
Dil.HCl
Procedure:

Preparation of BaSO4 reagent: This reagent must be freely prepared by mixing 10 ml of 25 % solution
of BaCl2 and 15 ml of ethanolic sulphate standard solution, which contains 0.10891% K2SO4 in 3%
ethanol. K2SO4 has been added to increase the sensitivity of the test. A very small amount of barium
sulphate present in the reagent acts as a seeding agent for the precipitation of barium sulphate. Alcohol
helps in preventing supersaturation of barium sulphate.
Preparation of test solution: Dissolve a specified quantity of substance in water or prepare solution as
directed by I.P. and transfer to Nessler’s cylinder. To this add 2 ml of dilute HCl and make the volume
to 45 ml with water. And add 5 ml of BaSO4 reagent. Stir and keep it aside for 5 min.
Preparation of standard solution: Take 1 ml of 0.1089% w/v of K2SO4 in Nessler’s cylinder add 2 ml
of HCl transfer to Nessler’s cylinder and make the volume to 45 ml with H2O. To this add 5 ml of
BaSO4 reagent. Stir and keep aside for 5 min. Finally, compare the turbidity of the two solutions
AIM: TO PERFORM THE LIMIT TEST OF SULPHATES
BaSO4 + 2Cl
-

11. STANDARD SOLUTION Observation Inference
1ml of 0.1089 % w/v solution of
potassium sulphate in Nessler’s
cylinder was taken + 2ml of dilute
hydrochloric acid + Dilute to 45 ml
in Nessler’s cylinder + 5ml of
barium sulphate reagent. Keep
aside for 5 min.







The opalescence produced
in the sample solution is
greater than / less than that
of the standard solution.







The sample passes/
doesn’t pass the limit
test for sulphates.
TEST SOLUTION

Take the specific weight(1gm) of
sample + 2ml of dilute
hydrochloric acid was taken and
diluted to 45 ml in Nessler’s
cylinder + 5ml of barium sulphate
reagent. Keep aside for 5 min.

Note: The opalescence produced in the test solution is compared with that produced in the
standard solution.

Result: The given sample passes/fails the limit test for sulphates.

12. Experiment 04


Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No-140

Requirements Glasswares : Conical flask, burette, pipette, burette stand, beaker.
Reagents: HCl, sodium carbonate, methyl orange & distilled water.

Principle: Standardization of hydrochloric acid is done by acid-base titration. When
hydrochloric acid is allowed to react with sodium carbonate, carbon dioxide and water is
produced. In this titration, to locate the endpoint methyl orange indicator is used. Appearance
of the pale-yellow colour is the endpoint.

H2SO4 + Na2CO3 Na2SO4 + CO2 + H2O
Procedure:
Preparation of 0.1 N HCl-
8.7 ml HCl was dissolved in 1 L of distilled water.

Preparation of 0.1 N Na2CO3-
1. The 5.3 g of Na2CO3 was dissolved in 1L of distilled water.
2. All the glassware such as the burette, pipette, beaker, and conical task was rinsed
properly before use.
3. The Na2CO3 solution was filled onto the burette using a funnel, and the reading was
set to zero.
4. The HCl solution (10 ml) was pipette out and poured into the conical flask.
5. 2-3 drops of methyl orange indicator were added and dropwise.
6. Finally, the titrant was added dropwise on the conical flask having HCl solution till
the endpoint was achieved.
7. The endpoint was visualized by changing of colour from faint pink to yellow.
8. The titration procedure was repeated three times to get accurate results
Observation table:
S. No. Volume of 0.1N
HCl (ml)
Burette reading Volume of Na2CO3
rundown (ml) Initial (ml) Final (ml)
1. 10
2. 10
3. 10
N 1V 1= N2V 2
N 2 = N 1V 1
V 2
AIM: TO PREPARE AND STANDARDIZE 0.1N H Cl SOLUTION

13. Result: The 0.1 N solution of HCl was prepared and standardized and its actual concentration
was found to be ……..N.

14. Experiment 05



Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No-145

Requirements Glasswares: Conical flask, burette, pipette, burette stand, beaker.
Reagents: KMnO4, oxalic acid, conc. H2SO4, distilled water.
Principle- The permanganate ion gets reduced to different products depending upon the
reaction conditions. In the basic medium, manganate ions are formed, in the neutral medium
the product is manganese dioxide is formed, whereas, in the acidic solution, manganous ions
are produced as shown by the following equation.


Preparation of Reagents:
1. Preparation of O.IN KMnO4- Dissolve about 3.16g of KMnO4 crystals in 1000ml
distilled water and boil the prepared solution, filter and cool.
2. Preparation of 0.1 N H2C2O4.2H2O- Accurately dissolve 6.3 g of oxalic acid crystals
in 1000 ml distilled water.
Procedure:
1. 25 ml of 0.1 N oxalic acid solution was pipetted out, 5 ml of conc. H2SO4 was added
to the flask, swirl the contents carefully and warmed to 70˚C.
2. Titrate the warmed solution against the KMnO4 solution from the burette till the pink
colour persists for about 30 seconds.
AIM: TO PREPARE AND STANDARDIZE 0.1N
POTASSIUM PERMANGANATE

15. 3. Repeat the experiment three or more times until two consecutive results are the same
or precise and tabulate the results.
4. Take the precise readings for the calculation of normality.
Observation table:
S. No. Volume of 0.1N
oxalic acid (ml)
Burette reading Volume of KMnO4
rundown (ml) Initial (ml) Final (ml)
1. 25
2. 25
3. 25

N 1V 1= N2V 2
N 2 = N 1V 1
V 2
Result: The 0.1N solution of potassium permanganate was prepared and standardized and its
actual concentration was found to be …….. N.

16. Experiment 06




Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No-157.

Requirements Glassware: Conical flask, burette, pipette, burette stand, beaker.
Reagents: H2SO4, sodium carbonate, methyl orange, distilled water.
Principle: Standardization of sulphuric acid is done by acid-base titration. When sulphuric acid
is allowed to react with sodium carbonate, carbon dioxide and water is produced. In this
titration, to locate the endpoint methyl orange indicator is used. Appearance of the pale yellow
colour is the endpoint.
H2SO4 + Na 2CO 3 → Na 2SO 4 + CO2 + H2 O
Procedure:
1. 0.15 g of anhydrous sodium carbonate was weighed accurately and previously
heated at about 270
o
C for 1 hour.
2. Dissolve it in 100 ml of water, and 2-3 drops of methyl orange indicator was added.
3. The solution was titrated until it became faintly pink which persisted for 30
seconds.

S. No. Volume of 0.1N
H2SO4 (ml)
Burette reading Volume of Na 2CO3
rundown (ml) Initial (ml) Final (ml)
1. 10
2. 10
3. 10
N 1V 1= N 2V 2
N 2 = N 1V 1
V 2

Result: The 0.1N solution of H2SO4 was prepared and standardized and its actual concentration
was found to be …….. N.






AIM: TO PREPARE AND STANDARDIZE 0.1N
SULPHURIC ACID (H2SO4)

17. Experiment 07


Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No- 160

Requirements Glassware: Conical flask, burette, pipette, burette stand, beaker and funnel.
Reagents: NH4Cl, 0.1N NaOH solution, phenolphthalein indicator,
formaldehyde, 0.1N oxalic acid solution
Principle- NH4Cl is an example of a diuretic system. The assay of NH4Cl is based on the
principle of formal titration. The formal titration is done in the presence of HCHO. When
NH4Cl is titrated with HCHO. HCl is liberated. The liberated HCl is titrated with a standard
solution of NaOH using phenolphthalein as an indicator.
NH4Cl + 6 HCHO (CH2)6H4 + 4 HCl + 6H2O
Procedure-
A). Standardization of 0.1 N NaOH Solution:
1. 10 ml of 0.1 N oxalic acid solution was taken into a conical flask and 2-3 drops of
phenolphthalein indicator was added to it.
2. The above solution was titrated against NaOH solution until a permanent pink colour
was observed.
3. Repeat the titration 3 times for precise readings. The results was tabulated.
B). For assay:
1. 0.1 gm of NH4Cl was weighed and dissolved in a mixture of 20 ml of water and 5 ml
of HCHO solution.
2. The contents of the flask was titrated with 0.1N NaOH using a phenolphthalein
indicator.
3. Repeat the titration for concordant values. The result was tabulated.
Equivalent or IP Factor: Each ml of 0.1 N NaOH is equivalent to 0.005349 gm of NH4Cl.
S. No. Volume of (ml)
NH4Cl
Burette reading Volume of KMnO4
rundown (ml) Initial (ml) Final (ml)
1. 25
2. 25
3. 25
N 1V 1= N2V 2
N 2 = N 1V 1
V 2
AIM: TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF
AMMONIUM CHLORIDE ( NH4Cl)

18. Calculation for % purity of NH4Cl = Vol. of NaOH X IP factor X Normality of NaOH (actual)
Weight of NH4Cl (g) X Normality of NaOH (assumed)

Result: The percentage purity of NH4Cl was found to be ………%

























X 100

19. Experiment 08


Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No- 173

Requirements Glassware: Conical flask, burette, pipette, burette stand, beaker and funnel.
Reagents: Dil. H2SO4, 0.1N KMnO4 solution, 0.1N oxalic acid solution,
ferrous sulphate (FeSO4).
Principle- This assay is based upon oxidation-reduction titrations in which oxidation involves
the loss of electrons whereas the reduction process involves the gain up electrons. When
KMnO4 is used as an oxidising agent in the titration it is known as permanganate titration. these
titrations are mainly conducted in an acidic solution
• The ability of KMnO4 solution to oxidize is due to the conversion of the MnO4
-

(permanganate ion) to Mn
2+
(Manganese ion) in an acidic solution.
a) Solutions Containing MnO4
-
ions are purple in colour.
b) Solutions of salts containing Mn
2+
ions are colourless. Therefore, a permanganate
solution is decolorised when added to a solution of reducing agents.
• The moment there is an excess KMnO4, the solution becomes purple.
• Thus, the permanganate ion can act as a self-indicator, especially in acidic conditions.
• The assay of FeSO4 depends upon the oxidation-reduction type of titration where Fe
2+
(ferrous ion) is readily oxidized by KMnO4 in an acidic solution (H2SO4) into Fe
3+

(ferric ion). Thus, FESO4 acts as a reducing agent.
Factors
A). Factors for standardization: Each ml of 0.1 N KMnO4
B). Factor for assay: Each ml of 0.1 N KMnO4
Procedure
Standardization of 0.1 N KMnO4
1. 25mL of the prepared 0. 1 N oxalic acid solution was pipetted out, 5mL of a conc.
H2SO4 was added and the flask was shaken properly and warmed up to 70˚C.
2. The warmed solution was titrated against KMnO4 solution from the burette till the
pink colour persisted for about 30 sec.
3. The experiment was repeated three or more times until two consecutive results were
the same or precise. Then the results were tabulated
Assay of ferrous sulphate
1. 1 gm of FeSO4 was accurately weighed, and 10 ml of dil. H2SO4 was added to it.
2. To the above reaction mixture, 20mL of distilled water was added and it was titrated
against 0.1N KMnO4 solution.

AIM: TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF FERROUS
SULPHATE (FeSO4)
0.0278 g of FeSO4. 7 H2O
0.0278 g of FeSO4. 7 H2O

20. Observation table:
S. No. Volume of
Oxalic acid
(ml)
Burette reading Volume of KMnO4
rundown (ml) Initial (ml) Final (ml)
1. 25
2. 25
3. 25
N 1V 1= N2V 2
N 2 = N 1V 1
V 2
Calculation for % purity of FeSO4 = Factor X Normality (assumed) X Burette reading of assay
Weight of FeSO4 (g) X Normality of NaOH (assumed)

Result: The percentage purity of a given sample of FeSO4was found to be ………%
















X 100

21. Experiment 09


Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No- 174

Requirements Glassware: Conical flask, burette, pipette, burette stand, beaker and funnel.
Reagents: AgNO3, NaCl, K2Cr2O4 and distilled water.
Principle- NaCl is an example of an electrolyte replenisher. It is assayed by Mohr's method.
In this method, the sample is dissolved in H2O and titrated against a standard sol" ay AgNO3
using potassium chromate (K2CrO4) as an indicator. At the endpoint brick red colour ppt is
obtained due to the formation of silver chromate.
This method is based on the fact that silver halide is more insoluble than AgCrO4. Hence as
long as there is any Cl
-
left in the solution no AgCrO4 is formed even though if formed, it will
immediately change to AgCl according to the equation.
AgNO3 + NaCl AgCl + NaNO3
Procedure:
Standardization of 0.1N AgNO3
1. 10 ml of 0.1 N NaCl solution was taken into a conical flask and 1 ml of K2CrO4
indicator was added to it.
2. The content of the flask was titrated against the AgNO3 solution until brick red ppt
was` formed.
3. Titration was repeated 3 times for precise readings.
Assay of NaCl
1. 0.25 g of NaCl was weighed and dissolved in 50 ml water and titrated against 0.1 N
AgNO3 using 5% K2CrO4 solution as an indicator.
2. The titration was repeated for precise reading.
Factor
Each ml of AgNO3
Observation table
S. No. Volume of
NaCl (ml)
Burette reading Volume of 0.1 N
AgNO3 rundown (ml)
Initial (ml) Final (ml)
1. 10
2. 10
3. 10

AIM: TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF SODIUM
CHLORIDE (NaCl) BY PRECIPITATION TITRATION
0.05845 g of NaCl

22. N 1V 1= N2V 2
N 2 = N 1V 1
V 2
Calculation for % purity of NaCl = Vol of AgNO3 X Normality (actual) X Factor
Weight of NaCl (g) X Normality (assumed)

Result: The percentage purity of a given sample of NaCl was found to be ………%























X 100

23. Experiment 10


Reference: A.H Beckett, et al., “Practical Pharmaceutical Chemistry” fourth edition, CBS
publishers and distributors,2005, Page No- 176

Requirements Glassware: Conical flask, burette, pipette, burette stand, beaker and funnel.
Reagents: KMnO4, glacial acetic acid, conc. H2O2, oxalic acid and distilled
water.
Principle- Hydrogen peroxide (H2O2) Can be analysed by many methods however, titration
with KMnO4 in an acidic medium is an easy and official method. This assay is based on the
redox reaction. H2O2 is an oxidising agent because it liberates oxygen rapidly. However, when
it is titrated in an acidic medium against KMnO4, it acts as on reducing agent.
2 KMnO4 + 3 H2SO4 + 5 H2O2 K2SO4 + MnSO4 + 8 H2O + 5 O2
Procedure-
Standardization of O.IN KMnO4
1. Pipette out a 10 mL conical flask of 0.1 N oxalic acid solution in a conical flask and
add 10 ml of dil. H2SO4.
2. Warm the contents of the flask at 70˚ C & titrate with KMnO4 solution.
3. Continue the titration until a faint pink colour is produced.
4. Repeat the titration for precise reading.
Assay of H2O2
1. 10 ml of H2O2 was diluted to 250 ml of distilled water. To 25 ml of this diluted solution,
5 ml of 1N H2SO4 solution was added.
2. The above titrating mixture was titrated against 0.1 N KMnO4 solution until a
permanent pink colour was obtained.
Factor
0.1 N KMnO4 solution
Observation table:
S. No. Volume of
oxalic acid (ml)
Burette reading Volume of KMnO4
rundown (ml) Initial (ml) Final (ml)
1. 10
2. 10
3. 10
N 1V 1= N2V 2
N 2 = N 1V 1
V 2

AIM: TO PERFORM THE ASSAY OF THE GIVEN SAMPLE OF
HYDROGEN PEROXIDE (H 2O2) BY PERMANGANOMETERY
0.001701g of H2O2

24. Calculation for % purity of NaCl = Vol of KMnO4 X Normality (actual) X Factor
Weight of H2O2 (g) X Normality (assumed)

Result: The percentage purity of a given sample of H2O2 was found to be ………%



X 100