Pharmaceutical Analysis-II Lab

Course Code: Phrm-3236

Name:
ID:
Sec:
th
Batch: 29
 Abstract

Titration is a common laboratory method of quantitative chemical analysis to determine

the concentration of an identified analyte (a substance to be analyzed). Direct titration is a
way to determine the contents of a substance quantitatively. Scientists may be aware of a
reactant, but not know the reactant's quantity. Direct titration is sometimes based on
indicators that respond to the analyzed material, called the analyte. A back titration is a
titration method where the concentration of an analyte is determined by reacting it with a
known amount of excess reagent. The remaining excess reagent is then titrated with
another, second reagent.
Aspirin, also known as acetylsalicylic acid (ASA), is a medication used to reduce pain,
fever, or inflammation. Specific inflammatory conditions which aspirin is used to treat
include Kawasaki disease, pericarditis, and rheumatic fever.
Vitamin C, also known as ascorbic acid and ascorbate, is a vitamin found in various foods
and sold as a dietary supplement. It is used to prevent and treat scurvy.
Calcium gluconate is a mineral supplement and medication. As a medication it is used by
injection into a vein to treat low blood calcium, high blood potassium, and magnesium
toxicity.
Iron (II) fumarate, also known as ferrous fumarate, is the iron (II) salt of fumaric acid,
occurring as a reddish-orange powder, used to supplement iron intake. It has the chemical
formula C4H2FeO4.
Nortriptyline hydrochloride is used to treat mental/mood problems such as depression. It
may help improve mood and feelings of well-being, relieve anxiety and tension, and
increase your energy level. This medication belongs to a class of medications called
tricyclic antidepressants.
 Index

Remarks
SI Experiment Name Date Page No
from
Course
No
Teacher
Estimation of Acetyl Salicylic
01 Acid in a Tablet dosage form.
Estimation of ascorbic acid
02 (vitamin C) in chewable tablet
dosage form.
Estimation of calcium in
03 calcium gluconate injection by
complexometric titration.
Estimation of elemental iron in
04 ferrous fumarate capsule dosage
form.
Estimation of Nortriptyline in
05 Nortriptyline hydrochloride
capsule dosage form.
 Date

Experiment No: 1
Experiment name: Estimation of Acetyl Salicylic Acid in a Tablet dosage form.

Principle
Acetyl Salicylic Acid (ASA), commonly known as Aspirin, is a non-steroidal anti-inflammatory
drug (NSAID). It has analgesic, anti-inflammatory, anti-pyretic and anti-thrombotic activity.
Nowadays its use as analgesic or anti-inflammatory drug has decreased but its use as anti-
thrombotic drug has increased.
Chemically Aspirin is the acetyl ester of salicylic acid. It is sparingly soluble in water, but soluble
in sodium hydroxide. Its structure is shown below:
O O

C C
OH HO CH3 O CH3
A c e tic a c id
COOH COOH

+ E s te r ific a tio n

S a lic y lic a c id A c e ty l s a lic y lic a c id

Now, ASA can be hydrolyzed in the presence of acid or base to produce salicylic acid and acetic
acid. If a base, such as sodium hydroxide (NaOH) is used then the acids produced will immediately
form salts. Thus, NaOH is consumed in the reaction.
O

C
O CH3 OH
O
COOH COOH
C

+ H 2O
N aO H
H y d r o ly s is
+ HO CH3
A c e tic a c id

A c e ty l s a lic y lic a c id S a lic y lic a c id

OH OH
O O
COOH COONa
C C
+ HO CH3 + 2 N aO H
A c id -b a s e r e a c tio n
+ NaO CH3 + 2 H 2O

A c e tic a c id A c e tic a c id

S a lic y lic a c id

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From the schematics shown above, we can say that 1 mole of acetylsalicylic acid reacts with 2
moles of sodium hydroxide and produces salt and water.
O

C
O CH3 OH
O
COOH COONa
H y d r o ly s is C
+ 2 N aO H
A c id -b a s e re a c tio n + N aO CH3 + H 2O
S o d iu m a c e ta te

A c e ty l s a lic y lic a c id S o d iu m s a lic y la t e

In the method of back titration, the amount of sodium hydroxide consumed in the above reaction
is measured by titrating the sodium hydroxide with a standardized acid. In this experiment 0.5M
Hydrochloric acid (HCl) is used.
In this experiment, a known but excess amount of 0.5M NaOH (say X mL) is added to the solution
containing ASA and heated to cause hydrolysis. Certain amount of the added NaOH will be
consumed. The remaining amount is titrated against 0.5M HCl. Now,
Volume of HCl used in titration  V1  Known from burette
Concentrat ion of HCl used in titration  S1  Known from standardiz ation
Concentrat ion of NaOH  S 2  0.5M
Volume of NaOH  V2  Unknown

V1S1  V2 S 2
V1S1
 V2 
S2

This V2 is the volume of remaining NaOH. So, we can determine the volume of NaOH consumed
in the reaction with ASA by subtracting this volume from the initially added volume (X-V 2 mL).
Then, since 1 mole of ASA reacts with 2 moles of NaOH:
2moles of NaOH  1 mole of ASA
 1000mL 2M NaOH  180.16gm of ASA
 1mL 2M NaOH  180.16mg of ASA
 1mL 0.5M NaOH  45.04mg of ASA

In reality, two experiments are performed. In one experiment, only the X mL of NaOH solution is
titrated. In the other experiment, X mL of NaOH is added to the ASA solution and then titrated.
From the difference of the volume of HCl consumed in the two experiments, we get the volume
of NaOH consumed.

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Required apparatus
1. Electronic balance
2. Burette
3. Pipette
4. Mortar & Pestle
5. Reflux condenser
6. Measuring cylinder (50mL preferred)
7. Conical flasks
8. Funnel

Required reagents
1. 0.5M (=0.5N) sodium hydroxide solution
2. 0.5M (=0.5N) hydrochloric acid solution (will be standardized in the experiment)
3. Sodium carbonate (solid)
4. 0.5% phenolphthalein solution
5. Distilled water

Procedure
1. Examination of the sample tablets
1. Sample tablets in their packaging will be given to you.
2. Examine the tablets to identify the manufacturer and the claimed strength.
3. Note down this information appropriately.

2. Preparation of ASA powder

1. Take 20 sample tablets out of their packaging and then accurately weigh them in an
electronic balance.
2. Calculate the average weight of one tablet.
3. Then take the 20 tablets in a clear and dry mortar.
4. Crush them into fine powder with the help of a pestle.

3. Preparation of ASA sample solution

1. First, calculate what amount of the powder contains 500mg of ASA.
2. Weigh that amount of powder in an electronic balance. NOTE: Do not worry if the
weighing is not precise. However, you must note down the exact weight that you took. For
example, if you were supposed to weigh down 680.54mg exactly, but you took 684.56mg;
this is not a problem. But you must note that you took 684.56mg.
3. Take the weighed powder in a conical flask.

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4. Measure 30mL of 0.5M (=0.5N) NaOH using a measuring cylinder and add it to the conical
flask. NOTE: 30mL must be precisely measured. Furthermore, the concentration must be
0.5M precisely, otherwise result will differ.
5. Place the conical flask in the reflux condenser and heat gently for 10 minutes. NOTE: Take
help from Lab officer/assistant to use the reflux condenser.

4. Titration of ASA solution

1. Take the conical flask out of the reflux condenser and allow it to cool. NOTE: Cooling is
not essential.
2. Titrate the contents of the flask with 0.5M (=0.5N) HCl using a burette.
3. Use phenolphthalein solution as the indicator. NOTE: Use no more than 2 drops.
4. Complete the titration when color changes from pink to colorless.

5. Blank titration
1. Take 30mL of 0.5M (=0.5N) sodium hydroxide in another conical flask.
2. Heat in the reflux condenser for 10 minutes. NOTE: This step is not essential. It can be
done to ensure both experiments are at same environment.
3. Titrate the solution with 0.5M (=0.5N) HCl using phenolphthalein solution as indicator.
NOTE: Add indicator to the flask.
4. Complete titration when color changes from pink to colorless.

6. Standardization of HCl (by Correction Factor method)

1. Weigh about 1gm of sodium carbonate (Na2CO3). NOTE: The weight doesn’t have to be
exactly 1gm, but you must note the weight you took.
2. Dissolve the weighed Na2CO3 in 20mL of distilled water in a conical flask.
3. Titrate this solution against the 0.5M (=0.5N) HCl solution (in the burette).
4. Use methyl orange as the indicator and add 1-2 drops in the conical flask.
5. Complete titration when color changes from yellow to orange. NOTE: Check the color
change carefully. Color should not disappear after shaking.
6. Theoretically 1mL of 0.5M HCl neutralizes 26.5mg sodium carbonate. Use this to calculate
the amount of sodium carbonate theoretically neutralized.
7. Determine the correction factor.

Data & Calculation

1. Product details
Manufacturer :

Product claim : Each tablet contains _______mg of ASA.

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2. Powder weight calculation

Weight of 20 tablets  _______gm

 Average weight of 1 tablet  gm  _______mg

20

Now,
_______mg of ASA is present in _______mg of tablet powder
500 
 500mg of ASA is present in mg of tablet powder

 ________mg of tablet powder

Actual weight of tablet powder taken  _________mg of tablet powder

3. Determination of correction factor

Actual weight of Na 2CO 3 taken  _________gm  __________mg

Now, the acid-base reaction here is:

Na 2CO 3  2HCl  2NaCl  H 2 O  CO 2

So,
1
1 mole of HCl 
mole of Na 2CO 3
2
1000mL 1M of HCl  53 gm of Na 2CO 3
53  0.5
 1mL, 0.5M of HCl  gm of Na 2CO 3
1000
26.5
 gm of Na 2CO 3
1000
 26.5mg of Na 2CO 3

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Now,

Volume of 0.5M HCl required to neutralize Na 2 CO 3  VC  _______mL

Amount of Na 2CO 3 practically neutralized by the volume of HCl  _______ mg
Amount of Na 2CO 3 theoretically neutralized by that volume  VC  26.5mg

 _____  26.5mg

 _______mg

Now,
Amount of Na 2 CO 3 practically neutralized
Correction factor, f  
Amount of Na 2CO 3 theoretically neutralized

4. Titration data
Material Initial burette Final burette Difference Corrected
reading (mL) reading (mL) = Titre volume, volume
A (mL) A  f (mL)

Sample

Blank

5. Calculation of ASA in tablet

From the difference of volume of HCl required in Sample and Blank experiments, we can
determine the volume of NaOH consumed in acid-base reaction involving ASA.
So,

Volume of NaOH consumed  (______ - _______)mL  _______mL

So, ______mL of sodium hydroxide is required to completely titrate the ASA present in

_______mg of tablet powder.

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Now,
1mL 0.5M of NaOH  45.04mg of ASA

 ______mL 0.5M of NaOH  _______  45.04 mg of ASA

 _________mg of ASA

Now,
________mg of tablet powder contains ______mg of ASA

 ________mg of tablet powder contains mg of ASA

 _____________ mg of ASA

 Each tablet contains  _______mg ASA

6. Potency Determination

Obtained value
%Potency   100   100  _________%
Claimed value

Results

Each tablet contains __________mg of ASA and the potency is _______%. This is

_____________________ The ________ specified range.

Precautions
1. Take all weights carefully and note the weights after the window of the balance is closed.
2. Handle the acid and the base with care. Wash with large amount of flowing water if spilt
on skin. Seek assistance from the lab officer/assistant.
3. Observe all the color changes very carefully. Shake the conical flask after every drop of
acid is added to the conical flask. Only note volume when the color change is permanent.
4. Handle reflux condenser with care. Seek help from lab officer/assistant.

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Experiment No: 2
Experiment name: Estimation of ascorbic acid (vitamin C) in chewable tablet
dosage form.

Principle
Ascorbic acid, commonly known as Vitamin C is one of the essential micronutrients for humans.
It is required for collagen synthesis and therefore a deficiency leads to a disease called scurvy.
Furthermore, it is now known that ascorbic acid is a reducing agent and therefore acts as an
antioxidant and may be protective against various forms of cancer. Ascorbic acid is available as
over-the-counter dietary supplement in the form of tablets.

Since ascorbic acid is a reducing agent, it can be estimated in a dosage form by oxidation-reduction
titration. According to the method prescribed by British Pharmacopoeia (BP), Ammonium Cerium
(IV) Sulfate (ACS), formula Ce(SO4)2.2(NH4)2SO4.2H2O is used as the oxidizing agent. In the
reaction, in an acidic media Ce4+ takes electron from ascorbic acid and reduces to Ce3+. The
reaction is:
OH OH

O O O O
HOH2C HOH2C
+ 2 Ce4+ + 2 H+ + 2 Ce3+

HO OH O O

Ascorbic acid

Now, we can see that:

2 moles of ACS  1 mole of Ascorbic acid
 1000mL 2M ACS  176.12gm of Ascorbic acid
 1mL 2M ACS  176.12mg of Ascorbic acid
176.12
 1mL 0.1M ACS  mg  8.806mg of Ascorbic acid
20

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So, if 0.1M ACS solution is used as the titrant, then each mL of the reagent is equivalent to
8.806mg of ascorbic acid. So, if we know the volume of titrant used to titrate a sample of ascorbic
acid, we can determine the amount of ascorbic acid in the sample.
Now, ACS is not a primary standard substance. Hence, we can use iodometry to standardize ACS.
Here, potassium iodide acts as a reducing agent, which is oxidized by ACS into iodine. The iodine
is violet in color, upon addition of sodium thiosulfate the iodine is again reverted back to colorless
iodide. When all iodine is consumed by sodium thiosulfate, the solution will become colorless.
The reaction is:

From the above reaction, we can see that:

2 moles of Sodium thiosulfat e  2 moles of ACS
 1000mL 2M Sodium thiosulfat e  2  632.55gm of ACS
 1mL 2M Sodium thiosulfat e 1265.1mg of ACS
1265.1  0.1
 1mL 0.1M Sodium thiosulfat e  mg  63.26mg of ACS
2
So, by determining the volume of sodium thiosulfate a required to neutralize a specific volume of
ACS solution, we can standardize the ACS solution and determine the correction factor.
Required apparatus
1. Electronic balance
2. Burette
3. Pipette
4. Mortar & Pestle
5. Measuring cylinder (50mL preferred)
6. Conical flasks
7. Beaker
8. Funnel

Required reagents
1. 1M (=0.5N) Sulfuric acid (H2SO4) solution
2. 0.1M Ammonium Cerium (IV) sulfate solution (will be standardized in the experiment)
3. Potassium iodide (KI)
4. 0.1M sodium thiosulfate solution
5. Ferroin solution
6. Starch solution
7. Distilled water

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Procedure
1. Examination of the sample tablets
1. Sample tablets in their packaging will be given to you.
2. Examine the tablets to identify the manufacturer and the claimed strength.
3. Note down this information appropriately.

2. Preparation of ascorbic acid powder

1. Take 20 sample tablets out of their packaging and then accurately weigh them in an
electronic balance.
2. Calculate the average weight of one tablet.
3. Then take the 20 tablets in a clear and dry mortar.
4. Crush them into fine powder with the help of a pestle.

3. Preparation of ascorbic acid sample solution

1. First, calculate what amount of the powder contains 150mg of ASA.
2. Weigh that amount of powder in an electronic balance. NOTE: Do not worry if the
weighing is not precise. However, you must note down the exact weight that you took.
3. In a beaker, prepare a solution of 3 part water and 2 part 1M H2SO4. Use measuring
cylinder to add appropriate amounts of water and sulfuric acid.
4. Take 50mL of this solution in a conical flask and dissolve the weighed powder in the
conical flask.

4. Titration of ascorbic acid solution

1. Allow the conical flask to cool.
2. Then add 25mL of distilled water.
3. Immediately Titrate the contents of the flask with 0.1M Ammonium Cerium (IV)
sulfate from a burette.
4. Use ferroin solution as the indicator. NOTE: Use no more than 2 drops.
5. Complete the titration when color changes from red to light blue.

5. Standardization of Ammonium Cerium sulfate

1. Take 25mL of our 0.1M ammonium cerium sulfate in a conical flask.
2. Add 2gm of potassium iodide and 150mL of distilled water.
3. Titrate immediately with freshly prepared 0.1M sodium thiosulfate from the burette.
4. Use 1mL of starch solution as the indicator and add to the conical flask.
5. Complete titration when color changes from light blue to colorless. NOTE: Check the
color change carefully. Color should not disappear after shaking.
6. Determine the correction factor. 1mL of 0.1M sodium thiosulfate is equivalent to
63.26mg of ammonium cerium sulfate.

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Data & Calculation

1. Product details
Manufacturer :

Product claim : Each tablet contains _______mg of ascorbic acid.

2. Powder weight calculation

Weight of 20 tablets  _______gm

 Average weight of 1 tablet  gm  _______mg

20

Now,
_______mg of Ascorbic acid is present in _______mg of tablet powder
150 
150mg of Ascorbic acid is present in mg of tablet powder

 ________mg of tablet powder

Actual weight of tablet powder taken  _________mg of tablet powder

3. Standardization of ammonium cerium sulfate

Now,
1mL 0.1M ACS  63.26mg of ACS
25mL 0.1M ACS  25  63.26mg of ACS
1581.5mg of ACS

So, amount of ACS theoretica lly neutralize d  1581.5mg

Again,
1mL 0.1M sodium thiosulfat e  63.26mg of ACS
 ________mL 0.1M sodium thiosulfat e   63.26mg of ACS
 mg of ACS
So, amount of ACS actually neutralize d  mg

So,

Amount of ACS neutralize d practically

Correction factor, f  
Amount of ACS neutralize d theoretica lly


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4. Titration data
Material Initial burette Final burette Difference Corrected
reading (mL) reading (mL) = Titre volume, volume
A (mL) A  f (mL)
Sample

5. Determination of ascorbic acid in tablet

1mL 0.1M ammonium cerium sulfate  8.806mg of ascorbic acid

 ______mL of 0.1M ACS  ________  8.806mg of ascorbic acid
 mg of ascorbic acid

Now,

________mg of tablet powder contains ______mg of Ascorbic acid


 ________mg of tablet powder contains mg of Ascorbic acid

 _____________ mg of Ascorbic acid

 Each tablet contains  _______mg Ascorbic acid

6. Potency Determination

Obtained value
%Potency   100   100  _________%
Claimed value

Results

Each tablet contains __________mg of Ascorbic acid and the potency is _______%. This is
_____________________ the ________ specified range.

Precautions
1. Take all weights carefully and note the weights after the window of the balance is
closed.
2. Handle the acid with care. Wash with large amount of flowing water if spilt on skin.
Seek assistance from the lab officer/assistant.
3. Observe all the color changes very carefully. Shake the conical flask after every drop
of titrant is added to the conical flask. Note the volume only when the color change is
permanent.

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Experiment No: 3
Experiment name: Estimation of calcium in calcium gluconate injection by
complexometric titration.

Principle
Calcium gluconate is a salt of calcium used to supply calcium to the body in cases of hypocalcemia
and some other complications. Calcium is a common ion in the human body; it is directly involved
with muscle contraction (including heart muscle) and nerve signal conduction. So, hypocalcemia
can have serious consequences. So, calcium gluconate is used in those cases. It is given in the form
of intravenous injection.

The amount of calcium in calcium gluconate injectable preparation can be easily determined by
complexometric titration. Usually, disodium edetate (Na 2EDTA) is used as the complexing agent
i.e. titrant. The reaction preferentially occurs at a basic pH as follows:

Now, according to the reaction:

1 mole of disodium edetate  1 mole of Ca 2

 1000mL 1M disodium edetate  40.08gm of Ca 2
 1mL 1M disodium edetate  40.08mg of Ca 2
 1mL 0.1M disodium edetate  40.08  0.1mg  4.008mg of Ca 2

So, by determining the volume of titrant (0.1M disodium edetate) required to complete the titration
of a sample, we can determine the amount of Ca2+ present in the sample.
Now, disodium edetate is not a primary standard substance. To standardize the substance, we have
to use Zinc powder which is a primary standard. The reaction proceeds as:

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In the reaction:

1 mole of disodium edetate  1 mole of Zn 2

 1000mL 1M disodium edetate  65.4gm of Zn 2
 1mL 1M disodium edetate  65.4mg of Zn 2
 1mL 0.1M disodium edetate  65.4  0.1mg  6.54mg of Zn 2

In the correction factor method, a specific amount of Zinc will be taken and titrated against
disodium edetate. The volume of disodium edetate used will be determined and the theoretical
amount of zinc neutralized (from above equation) will be determined. The correction factor is the
ratio of actual amount (amount actually taken) and the theoretical amount (calculated amount).
Required apparatus
1. Electronic balance
2. Burette
3. Pipette
4. Measuring cylinder (50mL & 100mL)
5. Conical flasks
6. Funnel

Required reagents
1. 10M sodium hydroxide (NaOH) solution
2. 2M sodium hydroxide solution
3. 7M hydrochloric acid
4. 0.1M disodium edetate solution
5. Calcon carboxylic triturate
6. Zinc granules
7. Bromine water
8. Xylenol orange triturate
9. Hexamethylenetetramine
10. Litmus paper
11. Distilled water

Procedure
1. Examination of the sample product
1. Sample product in its packaging will be given to you.
2. Examine the injectable to identify the manufacturer and the claimed strength.
3. Note down this information appropriately.

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2. Preparation of calcium gluconate sample solution

1. First, calculate what volume of the injectable contains 500mg of calcium.
2. Take out this volume of injectable from the ampoule using a pipette. NOTE: Do not
worry if the volume is not precise. However, you must note down the exact volume
that you took. NOTE2: Break the ampoule carefully.
3. Take a conical flask and add the measured volume of calcium gluconate from the
pipette.
4. Add 200mL of water to the conical flask and mix.

3. Titration of ascorbic acid solution

1. Add 6mL of 10M sodium hydroxide.
2. Then add 15mg of calcon carboxylic triturate. NOTE: Precise 15mg is not necessary.
3. Titrate the solution with 0.1M disodium edetate until the color of the solution changes
from violet to full blue.

4. Standardization of disodium edetate (correction factor method)

1. Weigh 120mg of Zinc granules in an electronic balance.
2. Dissolve the weighed zinc in 4mL of 7M hydrochloric acid. NOTE: Be very careful
when handling 7M hydrochloric acid.
3. Add 0.1mL of bromine water to the solution. Heat to boiling to remove excess bromine
from the solution.
4. Add 2M sodium hydroxide until the solution is weakly acidic or neutral. NOTE: Use
litmus paper to check acidity of the solution. Alternatively pH meter can be used.
5. Add 200mL of distilled water.
6. Then add 50mg of xylenol orange triturate. Add Hexamethylenetetramine until the
solution is violet-pink.
7. Add 2gm more of hexamethylenetetramine.
8. Titrate with 0.1M disodium edetate until the color changes from violet-pink to yellow.
9. Determine the correction factor. 1mL of 0.1M disodium edetate is equivalent to 6.54mg
of disodium edetate.

Data & Calculation

1. Product details
Manufacturer :

Product claim : Each 5mL contains _______mg of calcium as calcium gluconate.

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2. Volume calculation

______mg of calcium is present in _______mL of injectable

500 
 500mg of calcium is present in mL of injectable

 ________mL of injectable

Actual volume of injectable taken  _________mL

3. Standardization of disodium edetate

Now,
1mL 0.1M disodium edetate  6.54mg of Zinc
______mL 0.1M disodium edetate  _____  6.54mg of Zinc
 _________ of Zinc

So, amount of Zinc theoretica lly neutralize d  __________mg

Amount of Zinc actually neutralize d  mg

So,

Amount of Zinc neutralize d practicall y

Correction factor, f  
Amount of Zinc neutralize d theoretica lly


4. Titration data
Material Initial burette Final burette Difference Corrected
reading (mL) reading (mL) = Titre volume, volume
A (mL) A  f (mL)
Sample

5. Determination of calcium in the injection

1mL 0.1M disodium edetate  4.008mg of calcium

 ______mL of 0.1M disodium edetate  ________  4.008mg of calcium
 mg of calcium

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Now,

________mL of injectable contains ______mg of calcium


 ________mL of injectable contains mg of calcium

 _____________mg of calcium

 Each ______mL of injectable contains  _______mg of calcium

6. Potency Determination
Obtained value
%Potency   100   100  _________%
Claimed value

Results

Each _____mL of injectable contains __________mg of calcium as calcium gluconate and the
potency is _______%. This is _____________________ the ________ specified range.
Precautions
1. Take all weights carefully and note the weights after the window of the balance is
closed.
2. Handle the base with care. Wash with large amount of flowing water if spilt on skin.
Seek assistance from the lab officer/assistant.
3. Observe all the color changes very carefully. Shake the conical flask after every drop
of titrant is added to the conical flask. Only note volume when the color change is
permanent.
4. Do not try to prepare bromine water yourself, seek help from lab officer.

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Experiment No: 4
Experiment name: Estimation of elemental iron in ferrous fumarate capsule
dosage form.

Principle
Ferrous fumarate is a salt of Iron (II). It is used to supply iron, one of the essential micronutrients
to the body. Iron is necessary for many functions of the body, especially for maintaining RBC
level in the body. So, in iron-deficiency anemia, iron supplement is required. Since the body can
only absorb iron in the ferrous form i.e. Iron (II) form, only ferrous salts such as ferrous fumarate
is administered.

Iron can be estimated from a ferrous salt by oxidation-reduction titration. According to the method
described by BP, ammonium cerium (IV) sulfate is used as the oxidizing agent. Here, in acidic
media the reaction occurs as follows:

In the reaction:
1 mole of ACS  1 mole of Iron
 1000mL 1M ACS  55.85gm of Iron
 1mL 1M ACS  55.85mg of Iron
 1mL 0.1M ACS  55.85  0.1mg  5.585mg of Iron

So, by determining the volume of ACS required to complete the titration of a sample, we can
determine the amount of Iron present in the sample.
Now, ACS is not a primary standard substance. Hence, we can use iodometry to standardize ACS.
Here, potassium iodide acts as a reducing agent, which is oxidized by ACS into iodine. The iodine
is violet in color, upon addition of sodium thiosulfate the iodine is again reverted back to colorless
iodide. When all iodine is consumed by sodium thiosulfate, the solution will become colorless.
The reaction is:

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From the above reaction, we can see that:

2 moles of Sodium thiosulfat e  2 moles of ACS
 1000mL 2M Sodium thiosulfat e  2  632.55gm of ACS
 1mL 2M Sodium thiosulfat e 1265.1mg of ACS
1265.1  0.1
 1mL 0.1M Sodium thiosulfat e  mg  63.26mg of ACS
2
Now, 1mL 0f 0.1M ACS is equal to 63.26mg ACS. So by taking 25mL of the ACS solution and
titrating against sodium thiosulfate we can determine the correction factor. Here the theoretical
amount of ACS is 63.26 × 25 = 1581.5mg (because we don’t know if the solution is actually 0.1M
ACS) and the actual amount is the volume of sodium thiosulfate required multiplied by 63.26. The
correction factor is the ratio of actual amount and theoretical amount.
Required apparatus
1. Electronic balance
2. Burette
3. Pipette
4. Mortar & Pestle (may not be required)
5. Measuring cylinder (50mL preferred)
6. Conical flasks
7. Beaker
8. Funnel

Required reagents
1. 1M (=0.5N) Sulfuric acid (H2SO4) solution
2. 0.1M Ammonium Cerium (IV) sulfate solution (will be standardized in the experiment)
3. Potassium iodide (KI)
4. 0.1M sodium thiosulfate solution
5. Ferroin solution
6. Starch solution
7. Distilled water

Procedure
1. Examination of the sample capsules
1. Sample capsules in their packaging will be given to you.
2. Examine the capsule packaging to identify the manufacturer and the claimed strength.
3. Note down this information appropriately.

2. Preparation of ferrous fumarate powder

1. Take 20 sample capsules out of their packaging.

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2. Open the capsule shell of those 20 capsules and pour the content (should be powder
form) inside the capsule shell into a weighed paper. Accurately weigh the powder in an
electronic balance.
3. Calculate the average weight of powder present inside one capsule.
4. If the content is not present as fine powder, take the content in a mortar and crush using
a pestle.

3. Preparation of ferrous fumarate sample solution

1. First, calculate what amount of the powder contains 300mg of ferrous fumarate.
2. Weigh that amount of powder in an electronic balance. NOTE: Do not worry if the
weighing is not precise. However, you must note down the exact weight that you took.
3. Take the weighed powder in a conical flask and add 75mL of 1M H 2SO4 solution.
Dissolve ferrous fumarate by shaking. Use measuring cylinder to add appropriate
sulfuric acid.

4. Titration of ferrous fumarate solution

1. Allow the conical flask to cool.
2. Then add 25mL of distilled water.
3. Immediately Titrate the contents of the flask with 0.1M Ammonium Cerium (IV)
sulfate from a burette.
4. Use ferroin solution as the indicator. NOTE: Use no more than 2 drops.
5. Complete the titration when color changes from red to light blue.

5. Standardization of Ammonium Cerium sulfate

1. Take 25mL of our 0.1M ammonium cerium sulfate in a conical flask.
2. Add 2gm of potassium iodide and 150mL of distilled water.
3. Titrate immediately with freshly prepared 0.1M sodium thiosulfate from the burette.
4. Use 1mL of starch solution as the indicator and add to the conical flask.
5. Complete titration when color changes from light blue to colorless. NOTE: Check the
color change carefully. Color should not disappear after shaking.
6. Determine the correction factor. 1mL of 0.1M sodium thiosulfate is equivalent to
63.26mg of ammonium cerium sulfate.

Data & Calculation

1. Product details
Manufacturer :

Product claim : Each capsule contains _______mg of elemental iron as

ferrous fumarate.

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2. Powder weight calculation

Weight of powder inside 20 capsules  _____________mg

 Average weight of powder inside 1 capsule  mg  _______mg

20

Now,
_______mg of iron is present in _______mg of capsule powder
150 
 300mg of iron is present in mg of capsule powder

 ________mg of capsule powder

Actual weight of capsule powder taken  _________mg of capsule powder

3. Standardization of ammonium cerium sulfate

Now,
1mL 0.1M ACS  63.26mg of ACS
25mL 0.1M ACS  25  63.26mg of ACS
1581.5mg of ACS

So, amount of ACS theoretica lly neutralize d  1581.5mg

Again,
1mL 0.1M sodium thiosulfat e  63.26mg of ACS
 ________mL 0.1M sodium thiosulfat e   63.26mg of ACS
 mg of ACS
So, amount of ACS actually neutralize d  mg

So,

Amount of ACS neutralize d practically

Correction factor, f  
Amount of ACS neutralize d theoretica lly


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4. Titration data
Material Initial burette Final burette Difference Corrected
reading (mL) reading (mL) = Titre volume, volume
A (mL) A  f (mL)
Sample

5. Determination of ferrous fumarate in capsule

1mL 0.1M ammonium cerium sulfate  5.585mg of Iron

 ______mL of 0.1M ACS  ________  5.585mg of Iron
 mg of Iron

Now,

________mg of capsule powder contains ______mg of Iron


 ________mg of capsule powder contains mg of Iron

 _____________mg of Iron

 Each capsule contains  _______mg of Iron

6. Potency Determination

Obtained value
%Potency   100   100  _________%
Claimed value

Results

Each capsule contains __________mg of elemental iron as ferrous fumarate and the potency is
_______%. This is _____________________ the ________ specified range.

Precautions
1. Take all weights carefully and note the weights after the window of the balance is
closed.
2. Handle the acid with care. Wash with large amount of flowing water if spilt on skin.
Seek assistance from the lab officer/assistant.
3. Observe all the color changes very carefully. Shake the conical flask after every drop
of titrant is added to the conical flask. Only note volume when the color change is
permanent.
4. Only add 1-2 drops of indicator.

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Experiment No: 5
Experiment name: Estimation of Nortriptyline in Nortriptyline hydrochloride
capsule dosage form.

Principle
Nortriptyline is a tricyclic antidepressant (TCA) drug used for the treatment of depressive
disorders. It is sold as the hydrochloride salt and may be dispensed in tablet or capsule form.
Chemically, Nortriptyline is a weak base but it is sparingly soluble in water.

NH

N o r tr ip t y lin e

Since it is a weakly basic substance it can be estimated by acid-base titration with a suitable acid
titrant. However, for various reasons, aqueous acid-base titration is not feasible for nortriptyline.
Hence we estimate Nortriptyline using non-aqueous acid-base titration method.
In this method, all reagents and apparatus are kept water free. Glacial acetic acid is used as the
solvent, as a result the basicity of nortriptyline is enhanced. Perchloric acid is used as the acidic
titrant. The reaction occurs as follows:

+ H C lO 4 + C lO 4 

NH
NH2

From the reaction we can say that:

1 mole of Perchloric acid  1 mole of Nortriptyl ine
 1000mL 1M Perchloric acid  263.38gm of Nortriptyl ine
 1mL 1M Perchloric acid  263.38mg of Nortriptyl ine
 1mL 0.02M Perchloric acid  263.38  0.02mg  5.268mg of Nortriptyl ine

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So, by titrating nortriptyline sample against perchloric acid and determining the volume of
perchloric acid required to complete the titration, we can determine the amount of nortriptyline in
the sample.
However, perchloric acid is not a primary standard. Therefore, potassium hydrogen phthalate
(KHP) is used to standardize the perchloric acid solution via correction factor method. Here the
reaction is:
COOH COOH

+ H C lO 4 + K C lO 4

COOK COOH

P o t a s s iu m h y d r o g e n
p h th a la t e (K H P )

Here,
1 mole of Perchloric acid  1 mole of KHP
 1000mL 1M Perchloric acid  204.22gm of KHP
 1mL 1M Perchloric acid  204.22mg of KHP
 1mL 0.02M Perchloric acid  204.22  0.02mg  4.084mg of KHP

The correction factor can be determined by taking a specific amount of KHP and determining the
volume of perchloric acid required to completely titrate it. Then the theoretical amount is
calculated from the above equation. The correction factor is the ratio of actual amount (the amount
of KHP actually taken) to the theoretical amount.
Required apparatus
1. Electronic balance
2. Completely dry Burette
3. Completely dry Pipette
4. Completely dry Mortar & Pestle
5. Completely dry Measuring cylinder (50mL preferred)
6. Completely dry conical flasks
7. Completely dry Beaker
8. Completely dry Funnel

Required reagents
1. Anhydrous acetic acid
2. 0.02M perchloric acid solution (prepared in anhydrous acetic acid)
3. 0.5% Crystal violet solution
4. Potassium hydrogen phthalate

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Procedure
1. Examination of the sample capsules
1. Sample capsules in their packaging will be given to you.
2. Examine the capsule packaging to identify the manufacturer and the claimed strength.
3. Note down this information appropriately.

2. Preparation of nortriptyline powder

1. Take 20 sample capsules out of their packaging.
2. Open the capsule shell of those 20 capsules and pour the content (should be powder
form) inside the capsule shell into a weighed paper. Accurately weigh the powder in an
electronic balance.
3. Calculate the average weight of powder present inside one capsule.
4. If the content is not present as fine powder, take the content in a mortar and crush using
a pestle.

3. Preparation of nortriptyline sample solution

1. First, calculate what amount of the powder contains 100mg of nortriptyline.
2. Weigh that amount of powder in an electronic balance. NOTE: Do not worry if the
weighing is not precise. However, you must note down the exact weight that you took.
3. In a conical flask, take 50mL of anhydrous acetic acid and add 3mL of mercuric (II)
acetate. Then add the weighed powder in the conical flask and shake. Use measuring
cylinder to add appropriate of acetic acid.

4. Titration of nortriptyline solution

1. Allow the conical flask to cool.
2. Immediately titrate the contents of the flask with 0.02M perchloric acid.
3. Use 0.5% crystal violet solution as the indicator. NOTE: Use no more than 2 drops.
4. Complete the titration when color changes from purple to green.

5. Standardization of 0.02M perchloric acid

1. Weigh 70mg of potassium hydrogen phthalate (precisely note the amount you took)
and dissolve in 50mL of anhydrous acetic acid in a conical flask.
2. Titrate immediately with 0.02M perchloric acid solution (from burette) using 0.05mL
crystal violet solution as the indicator.
3. Complete titration when color changes from purple to green. NOTE: Check the color
change carefully. Color should not disappear after shaking.
4. Determine the correction factor. 1mL of 0.02M perchloric acid is equivalent to
4.084mg of potassium hydrogen phthalate (KHP).

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Data & Calculation

1. Product details
Manufacturer :

Product claim : Each capsule contains _______mg of nortriptyline as

nortriptyline hydrochloride.
2. Powder weight calculation
Weight of powder inside 20 capsules  _____________mg

 Average weight of powder inside1 capsule  mg  _______mg

20

Now,
_______mg of Nortriptyline is present in _______mg of capsule powder
100 
100mg of Nortriptyline is present in mg of capsule powder

 ________mg of capsule powder

Actual weight of capsule powder taken  _________mg of capsule powder

3. Standardization of 0.02M perchloric acid

Now,
1mL 0.02M perchloric acid  4.084mg of KHP
________ 0.02M perchloric acid  ______  4.084mg of KHP
 ___________mg of KHP

So, amount of KHP theoretically neutralize d  __________mg

But, amount of KHP actually neutralize d  mg

So,

Amount of KHP neutralize d practically

Correction factor, f  
Amount of KHP neutralize d theoretica lly


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4. Titration data
Material Initial burette Final burette Difference Corrected
reading (mL) reading (mL) = Titre volume, volume
A (mL) A  f (mL)
Sample

5. Determination of nortriptyline in capsule

1mL 0.02M perchloric acid  5.268mg of Nortriptyline

 ______mL of 0.02M perchloric acid  ________  5.268mg of Nortriptyline
 mg of Nortriptyline

Now,

________mg of capsule powder contains ______mg of Nortriptyline


 ________mg of capsule powder contains mg of Nortriptyline

 _____________mg of Nortriptyline

 Each capsule contains  _______mg of Nortriptyline

6. Potency Determination

Obtained value
%Potency   100   100  _________%
Claimed value

Results

Each capsule contains __________mg of nortriptyline as nortriptyline hydrochloride and the

potency is _______%. This is _____________________ the ________ specified range.

Precautions
1. Take all weights carefully and note the weights after the window of the balance is closed.
2. Ensure that all apparatus used in the experiment and free of water. Do not wash anything
with water.
3. Handle the acid with care. Wash with large amount of flowing water if spilt on skin. Seek
assistance from the lab officer/assistant.
4. Perchloric acid has a very pungent and obnoxious odor. So, be careful about handling the
acid.
5. When disposing the acid, seek help from lab officer.
6. Observe all the color changes very carefully. Shake the conical flask after every drop of
acid is added to the conical flask. Only note volume when the color change is permanent.

27