Instrumental Analysis Yb

Laboratory Manual

of

Instrumental Analysis

B. Pharm IVth Sem Lab Manual of Pharmaceutical Analysis-II

Index

Exp. No. Object Page No.

1 To perform calibration of various apparatus used in laboratory 4

2 To prepare and standardize 0.1N Perchloric acid solution 4

3 To perform the assay of the given sample of Metronidazole tablets 5

4 To perform the separation and identification the given mixture of amino

acids by using paper chromatography

5

5 To prepare and standardize 0.05M EDTA solution 6

6 To perform the assay of the given sample of Calcium Gluconate 7

7 To determine hardness of tap water by complexometric method 8

8 To perform the separation and identification the given mixture of drugs by

TLC

8

9 To determination of moisture content in the given sample by Karl Fischer

titration

9

10 To estimate the amount of Sulphadiazine present in the given tablets by

Potentiometry

10

Experiment 1

Sanjay Pharmacy College, Mathura 2 | P a g e


Object

To perform calibration of various apparatus used in laboratory

Reference

Rao G.D.”Practical Pharmaceutical Analysis”, Ist edition, Birla Publication, Delhi, 2007, pp. 141-

143.

Procedure

A. Weigh and Balance

1. Never exceed the stated maximum load of the balance.

2. Keep the balance clean. Remove dust from the pan and from the floor of the pan

compartment with a camel-hair brush.

3. Objects to be weighed should never4 be handled with the fingers; always use tongs or a loop

of clean paper.

4. Objects to be weighed should be slowed to attain the temperature of the balance before

weighing, and if the object has been heated, sufficient time must be allowed for cooling. The

time required to attain the temperature of the balance varies with the size.

5. No chemicals which might injure the balance pan should ever be placed directly on it.

6. Addition of chemicals to the receptacle must be done out side the balance case.

7. Nothing must be left on the pan when the weighing has been completed.

8. Avoid exposing the balance to corrosive atmospheres.

A. Pipette

Weigh an empty weighing bottle or a small conical flask. Fill the pipette to the mark with

distilled water. Deliver the contents into the weighing into the weighing vessel and reweigh.

Repeat the process 3 times. Find out the temperature of water and with the help of observed

weights of water.

A. Burette



Fill a clean vertically clamped with distilled water. Record the temperature of this water which is

same as that of room temperature. Adjust the level of the water to zero and carefully run out a

certain apparent volume into the flask which has been weighed earlier. Weight the flask with

water and find out the weight of water by difference.

A. Graduated Flask

The procedure of standardizing a measuring flask is practically same as that applied to pipette. It

is only necessary to weigh the water delivered by or contained in the flask, as the case may be.

Sometimes, the measuring flasks are also standardized in reference to a standardized flask

knowing exactly the difference of volumes with the help of other standard apparatus such as a

burette.

Result

All apparatus used in laboratory have been calibrated.

Experiment 2



Object

To prepare and standardize 0.1N Perchloric acid solution.

Referance


77.

Procedure

Preparation of 0.1N Percloric acid

Dissolve 8.5 ml of 72% HClO4 in about 900 ml glacial acetic acid with constant stirring, add

about 30 ml acetic anhydride and make up the volume (1000 ml) with glacial acetic acid and

keep the mixture for 24 hour. Acetic anhydride absorbed all the water from HClO4 and glacial

acetic acid and renders the solution virtually anhydrous. HClO4 must be well diluted with glacial

acetic acid before adding acetic anhydride because reaction between HClO4 and acetic anhydride

is explosive.

Preparation of 0.1N Potassium Hydrogen Phthalate

Weight accurately 2.04 gm of Potassium Hydrogen Phthalate and dissolve in glacial acetic acid

and adjust the volume to 100ml with the same solvent.

Standardization

Pipette out 10ml of Potassium Hydrogen Phthalate solution into dry conical flask and add few

drops of 5% w/v crystal violet in glacial acetic acid as indicator. This solution is titrated with 0.1

N HClO4. The colour changes from blue to blue green. Repeat the titration for concordant values.

1 ml of 0.1N HClO4 = 0.020414 gms of potassium acid Phthalate.

Observation table

S. No. Volume of 0.1 Potassium Hydrogen Burette volume reading (ml) Volume of HClO4 Average Volume



Phthalate (ml) rundown (ml) of HClO4

rundown (ml)

Initial Final

1

2

3

Calculation

The normality of HClO4 calculated by following formula

N1V1 = N2V2

Where

N1 = Normality of Potassium Hydrogen Phthalate

V1 = Volume of Potassium Hydrogen Phthalate

N2 = Normality of HClO4

V2 = Volume of HClO4

Result

The exact normality of the given sample of Percloric acid was found to be……………..

Experiment 3



Object

To perform the assay of the given sample of Metronidazole tablets.

Referance

1. Rao G.D.”Practical Pharmaceutical Analysis”, Ist edition, Birla Publication, Delhi, 2007,

pp. 82-83.

2. Indian Pharmacopoeia, Volume IInd, VIth edition, 2007, pp. 766-767

Procedure

1. For Standardization of Perchloric Acid

Similar to as given under Experiment No. 2 of this manual.

1. For Assay of Metronidazole tablets

Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.2 g

of Metronidazole, transfer to a sintered-glass crucible and extract with six quantities, each of 10

ml, of hot acetone. Cool, add to the combined extracts 50 ml of acetic anhydride. Titrate with 0.1

M perchloric acid, using 0.1 ml of a 1 per cent w/v solution of brilliant green in anhydrous

glacial acetic acid as indicator to a yellowish-green end-point.

1 ml of 0.1 M perchloric acid is equivalent to 0.01712 g of Metronidazole

Observation table




S. No. Volume of Metronidazole tablet

solution (ml)

Burette volume reading (ml) Volume of HClO4

rundown (ml)

Average Volume

of HClO4

rundown (ml) Initial Final

1

2

3

Calculation






Result

The % purity of the given sample of Metronidazole tablets was found to be………………

Experiment 4

Object



To perform the separation and identification the given mixture of amino acids by using paper

chromatography.

Referance


131

Procedure

1. Cut the Whatman paper in such a way that it does not touch the beaker.

2. Mark three spots on the paper.

3. Prepare a mobile phase (n-butanol: acetic acid: water = 4:1:5) & keep it for some time for

saturation and cover the beaker with watch glass.

4. Mark the paper with sample and keep it in the developing chamber.

5. Dry the paper and spray the visualizing agent (ninhydrine) and find Rf value.

Calculation

Results

The Rf value of amino acids in a given mixture was found to be.............................

Experiment 5

Object



To prepare and standardize 0.05M EDTA solution.

Referance


65.

Procedure

Preparation of 0.05 M EDTA

18.6 gm of disodium edentate dehydrate dissolve in 1 liter freshly prepared dionized distilled

water, it form approximately 0.05 M solution.

Preparation of x M Calcium Chloride

Solutions of any molarity xM may be prepared by dissolving 147x g of calcium chloride in

efficient water to produce 1000 ml.

Standardization

Pipette out 10ml of 0.05M CaCl2 solution into dry conical flask and add 5 ml of pH 10 buffer

solution. Add few drops of SBT indicator. This solution is titrated with 0.05 M EDTA solution.

Repeat the titration for concordant values.

Observation table

S. No. Volume of 0.05M CaCl2 solution

(ml)

Burette volume reading (ml) Volume of 0.05 M

EDTA rundown (ml)

Average Volume

of 0.05 M EDTA


1

2

3

Calculation

The normality of HClO4 calculated by following formula

M1V1 = M2V2

Where



M1 = Molarity of Potassium CaCl2

V1 = Volume of Potassium CaCl2

M2 = Molarity of EDTA

V2 = Volume of EDTA

Result

The exact normality of the given sample of EDTA was found to be……………..

Experiment 6

Object

To perform the assay of the given sample of Calcium Gluconate.



Referance

1. Rao G.D.”Practical Pharmaceutical Analysis”, Ist edition, Birla Publication, Delhi, 2007,

pp. 68-69.

2. Indian Pharmacopoeia, Volume IInd, VIth edition, 2007, p. 223.

Procedure

1. For Standardization of EDTA


1. For Assay of Calcium Gluconate

Weigh accurately about 0.5 gm Calcium Gluconate and dissolve in 50 ml of warm water; cool,

add 5.0 ml of 0.05 M magnesium sulphate and 10 ml of strong ammonia solution and titrate with

0.05 M disodium edetate using mordant black II mixture as indicator. From the volume of 0.05

M disodium edetate required subtract the volume of the magnesium sulphate solution added.

1 ml of the remainder of 0.05 M disodium edetate is equivalent to 0.02242 g of Calcium

Gluconate.

Observation table




S. No. Volume of Calcium Gluconate

solution (ml)

Burette volume reading (ml) Volume of EDTA

rundown (ml)

Average Volume

of EDTA


1

2

3

Calculation






Result

The % purity of the given sample of Calcium Gluconate was found to be………………

Experiment 7

Object

To determine hardness of tap water by complexometric method.



Referance

1. Rao G.D.”Practical Pharmaceutical Analysis”, Ist edition, Birla Publication, Delhi, 2007, pp.

137-138.

2. Vogel's - Textbook of quantitative chemical analysis, 5th Edition, 1989, pp. 332-333.

Procedure

Preparation of 0.01 M EDTA

Solutions of any molarity xM may be prepared by dissolving 372.2x g of disodium edetate in

sufficient water to produce 1000 ml.

For the determination of total hardness of water

For the determination of permanent hardness of water

Observation table



2. For determination hardness of tap water

S. No. Volume of Water (ml) Burette volume reading (ml) Volume of EDTA Average Volume



rundown (ml) of EDTA

rundown (ml)

Initial Final

1

2

3

Calculation



2. For determination hardness of tap water

Temporary hardness = Total hardness – Permanent hardness

Result

1. The Total Hardness of water was found to be ……….

2. The Permanent Hardness of water was found to be ……….

3. The Temporary Hardness of water was found to be ……….

Experiment 8

Object

To perform the separation and identification the given mixture of drugs by TLC.



Referance

Rao G.D.”Practical Pharmaceutical Analysis”, Ist edition, Birla Publication, Delhi, 2007, pp.

134-135.

Procedure

1. Prepare silica gel-G slurry by mixing silica gel- G with water till slurry of desired

viscosity and uniform.

2. TLC plates were then prepared by dipping method.

3. After air drying plates are activated in oven at 105°C for 30 minutes.

4. Standard solution of iodohydroxyquinoline and tinidazole were prepared by dissolving in

minimum amount of methanol in appropriate amount.

5. Tablet/sample solution was prepared by crushing tablet and then solubilizing in methanol.

6. Mobile phases were prepared by mixing two solutions of methanol and ammonia

(200:03)

7. Cover all the four chambers by lid and then saturate the chamber by putting filter paper in

the chamber.

8. Place spots on TLC plate (activated) with the help of capillary tube.

9. Prepare with three plates each for iodohydroxyquinoline, tinidazole and sample solution.

10. Put all the plates in a chamber.

11. After the ascending of solvent front, take out the plates and then dry in for 5-10 minutes.

12. Put the TLC plates into iodine vapour chamber.

13. Note down the distance travelled by solvent front and also distance travelled by each

spot.

14. By measuring distances we can calculate Rf value.

Calculation



Result

Rf value for iodohydroxyquinoline was found to be…………

Rf value for tinidazole was found to be…………

Rf value for sample was found to be…………

Experiment 9

Object

To determination of moisture content in the given sample by Karl Fischer titration.

Referance


128-129.

Procedure



1. Fill the titration beaker with 20 ml of methanol (27°C).

2. Connect the main plug to 230 volts A.C and switch “On” the mains switch.

3. Switch “On” the burette stop switch on the main unit.

4. Switch “On” the stirrers switch and regulate the speed of the stirring capsule with

magnetic stirrer regulator.

5. Red neon light on the instrument indicates the instrument is “ON”.

6. Set the timer knob to 20 second on the dial.

7. Keep the stop-cock closed and now press momentarily the “start titration button”.

Immediately the yellow neon will go off and there will be a click sound of solenoid value

indicating that the solenoid value is energized.

8. Now slowly open the stop cock of the automatic KF burette. So that KF regret from the

glan bent tube is added dropwise to the methanol in the beaker.

9. Now add 100 mg of sodium tartrate.

10. Re-fill the KF burette to its zero and press start titration buttons momentially .the

solenoid value will operate as above and as soon as the titration is over, the yellow neon

will above and as soon as on the KF burette and calculate the KF factor.

11. Weigh a specified quantity of sample and add it to the methanol in the beaker.

12. Press the start titration button and then titrate the sample on the above.

Calculation

Results

The moisture content in the given sample was found to be..........%



Experiment 10

Object

To estimate the amount of Sulphadiazine present in the given tablets by Potentiometry.

Referance


132-133.

Procedure

Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.5 g

of Sulphadiazine and dissolve in 100 ml of a mixture of 2 volumes of water and 1 volume of



hydrochloric acid and carry out the titration vessel of potentiometer. Immerse the platinum

electrodes in the solution and apply a voltage of about 50 mv across the electrodes, when

polarization of the electrodes takes place. Place the burettes tip just above the surface of the

solution and stir the solution gently, maintaining the temperature at about 15 °C. Add 0.1M

NaNO2 slowly and when the titration is within 1ml of the end point, add the titrant in 0.1 ml

portions, allowing not less than 1 minute between additions. At the end point, when a slight

excess of NaNO2 is present, the electrodes are depolarized, current flow and a permanent

deflection of the needle is obtained. Repeat the process to get concurrent values.

1 ml of 0.1 M sodium nitrite is equivalent to 0.02503 g of Sulphadiazine.

ResultThe amount of Sulphadiazine present in the given sample was found to be…………………

Viva


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Instrumental Analysis Yb