INDEX

Sr. Aim of the Practical Page Date of Signature

No. No. Practical
1 To perform the assay of Paracetamol Tablets
as per IP-2007.
2 To determine % w/w of Paracetamol in
Paracetamol tablets by colorimetry.

3 To perform Calibration of UV-visible

spectrophotometer.

4 To perform TLC of tablets containing PCM +

diclofenac.

5 Estimation of Diclofenac + Paracetamol in

pharmaceutical dosage form by Simultaneous
equation method.
6 To determine the percentage purity of CuSO4 in
the given unknown solutionof CuSO4 using UV-
Visible Spectrophotometer.
7 To determine content of Quinine sulphate in tablet
dosage form using fluorimetry.

8 To Demonstrate the HPLC.

SSSPC,ZUNDALS
 EXPERIMENT NO: 01

AIM: To perform the assay of Paracetamol Tablets as per IP-2007.

REFERENCE:

1. IP-1996
2. Dr. A.V. Kasture – A Textbook of Pharmaceutical Analysis Vol-Ⅱ, Nirali Prakashan, Page no: 156 to
158
.
REQUIREMENTS:

Volumetric flask, UV- Spectrophotometer

PRINCIPLE:

The assay of paracetamol tablets is based on the UV spectroscopy.

This UV spectrophotometry method depends upon the Lambert’s Beer Law. It states that when a
monochromatic light passes through an absorbing medium or solution, the rate of intensity of
incident light decreased exponentially with the increase in the concentration of absorbing species.

Equation for Lambert’s Beer’s Law is A = εbc

PROCEDURE:

Weigh 20 tablets individually, and find average weight.

Powder 20 tablets.
Weigh accurately the quantity of power equivalent to about 0.15gm of PCM. Add 50ml of0.1 M NaOH
solution, dilute it by adding 100ml of distilled water. Shake for 15min, and add sufficient distilled water
to produce 200ml.

Mix, filter and dilute 10ml of solution up to 100ml with water. To 10ml of above solution add 10ml
0.1M NaOH and dilute to 100ml with water.

Mix well and measure absorbance at 257nm.

RESULT:
The given paracetamol tablets contain %w/w of paracetamol.

CONCLUSION:
The assay limit is 95-105%, so given tablets pass /fail as per the standards of IP-1996.
The batch of PCM tablets pass/fail.
 EXPERIMENT NO: 02

AIM:To determine % w/w of Paracetamol in Paracetamol tablets by colorimetry.

REQUIREMENTS:
PCM tablets
1N NaOH solution
1N HCl solution
3% NaNO2 solution

PRINCIPLE:
Sodium nitrite in presence of dilute hydrochloric acid give nitrous acid (HNO2) This nitrous acid
reacts with the acetaminophen (paracetamol) to form 3-nitrosoacetaminophene. In alkaline medium,
3-nitrosoacetaminophen produces yellow colored 3-nitroso acetaminophen anion. The intensity of
developed yellow color is directly proportional to the concentration of the paracetamol present in
the sample.

PROCEDURE:

FOR STANDARD:
Weigh 250 mg of fine PCM powder. Dissolve it in water and dilute it to produce 250 ml with
H₂O. Transfer and dilute 20 ml of this solution up to 100ml with water. Transfer 5,10,15,20 & 25
ml solution in different 100 ml volumetric flasks, add sufficient water to make 25ml. To this
solution add 1 ml of HCl solution & 2ml of 3% NaNO2 solution, stand for 5 minutes. Add 3ml
of NaOH solution & dilute it with H₂O up to 100 ml.

FOR TEST SOLUTION:

Weigh & powder 20 tablets. Calculate average weight. Take quantity of powder equivalent to
100 mg. Dissolve it in distilled water and filter. Make volume 100 ml with H₂O. Take 10 ml of
the solution & dilute up to 100 ml with water.
Take appropriate amount of this solution to produce (20 mcg/ml & 40 mcg/ml). Add 1ml HCl
solution, 2 ml NaNO2 Solution, and stand for 5 mins. Add 3 ml of NaOH solution. Dilute it up to
100ml with water. Measure the absorbance at 430 nm. Plot the graph of conc. vs absorbance.
Find out the concentration of drug in unknown sample from the graph.
RESULTS:

Average weight of Paracetamol Tablets= mg

% w/w of Paracetamol in PCM tablets by colorimetry = ……….
The % stated value of Paracetamol in PCM tablets is = 95-105% (IP1996) = %

CONCLUSION:
The PCM tablets comply the limits for Paracetamol assay according to specification of IP1996.
 EXPERIMENT NO:03

AIM: To perform calibration of UV visible spectrophotometer.

REFERENCE:

IP, Volume 1.
UV visible spectrophotometer manual, Shimadzu.

REQUIREMENTS:

Chemicals/Reagents: K2Cr2O7,0.005 m Sulphuric acid,1.2% w/v solution of potassium

chloride,0.02% v/v solution of toluene in hexane.

APPARATUS/INSTRUMENTS: Volumetric flask, UV visible spectrophotometer,Holmium oxide

filter.

OPERATING PROCEDURE:

1. Turn ON the power, the spectrophotometer is checked and initialized.

2. The time required for this initialization is approximately 4 min (5 min. when the multi cell is used), if
all of the items are properly completed.

3. As each time enters its initialization operation, it is highlighted. When the initialization for that item is
properly completed, the star next te it is also highlighted.

4. Relation between the screen of the UV-1700 and the sheet keys.

5. The modes and settings in the various screens can be selected using the number keys '0' through '9' or
the function keys 'F1' through 'F4'.

6. When selecting modes or settings, it is not necessary to press the ENTER key after you have pressed
the number keys or function key.

7. On the other hand, when entering numeric values, such as wavelength setting on display mode, press
the ENTER key to confirm that value.

8. Now wash the cuvette properly by water or specified solution.

9. For PHOTOMETRIC MODE (measure the absorbance or %T of a sample at a fixed wavelength).

10. Fill the cuvette % th of the capacity with blank solution and wipe the outer surface of the cuvette by
using tissue paper.
11. Open the compartment, keep the cuvettes in the cell holder, the side of the clear surface should be
towards the beam of light pass through it.

12. Press the number key 1.

13. Enter the wavelength by GO TO WL. key fused to change the current wavelength Eg. for
wavelength 257mm press the 2.5 and 7 numeric key.

14. Press AUTO ZERO key (the current wavelength will automatically be set to "Absorbance (100%).
Wait till beep sound.

15. Remove the cuvette towards the analyst and wash with specified solution, if not specified then use
water. Fill the cuvette with sample solution and keep in the cell holder (follow the steps 10 and 11).

16. Observed the absorbance and note down.

17. Return to the mode selection screen by pressing MODE key or RETURN key

18. Remove the cuvettes from the compartment and wash the cuvettes

19. Switch off the UV-VISIBLE SPECTROPHOTOMETER.

NOTE: while switch off the instrument, it should be in mode selection screen.

20. For SPECTRUM MODE, follow the steps 10 and 11. Select the SPECTRUM MODE by pressing 2
on mode selection screen.

21. Adjust the parameters on display as per requirement by using numeric keys, eg. for Meas. mode
press 1 and select absorbance or %T.

22. Do the baseline correction by pressing Flkey. Wait till beep sound.

23. Follow the step 15.

24. Press START/STOP key. Wait till full spectrum display in the scanning range.

25. See the spectrum by using cursor keys ((-),). Note down the result.

26. Follow the steps 17 and 18.

27. Switch off the UV-VISIBLE SPECTROPHOTOMETER.

NOTE: while switch off the instrument, it should be in mode selection screen.

28. For QUANTITATION MODE (create a calibration curve from a std. sample and quantitates an
unknown sample). Follow steps 10 and 11.

29. Select the QUANTITATION MODE by pressing 3 from mode selection screen.
30. Enter the various parameters value by pressing numeric keys. eg. Enter the measurement wavelength
or enter measurement wavelengths 21,32 and 13 by pressing 1. For wavelength 420nm press 4, 2 and 0.

31. Follow steps 14 and 15.

32. Observed the result and note down.

33. Repeat the steps 17 and 18.

34. Switch off the UV-VISIBLE SPECTROPHOTOMETER.

NOTE: white switch off the instrument, it should be in mode selection screen.

CALIBRATION FREQUENCY:

Every month with tolerance of 13 days or after breakdown / service.

CALIBRATION PARAMETERS:

A. CONTROL OF WAVELENGTHS

Select a scan mode of instrument and enter the following parameters in scan mode wavelength.
Maximum 700 mm, minimum-250 nm, speed 30 nm/minute, graphic plot- yes, ordinate maximum-2.6,
minimum 0.000, scale 50 nm/cm., Print data yes, threshold 0,100 abs.

Autozero the instrument by air as a blank. Keep the holmium oxide filter in a cell holder and run the
instrument. After completion of the rus time compare the maximum absorbance obtained against
particular nm. 1.e. 536.4, 460.0, 360.9, 279.3. The permitted tolerance is 4 nm for the range 200 nm to
400 nm and 3 nm for the range 400 nm to 600 nm.

B. CONTROL OF ABSORBANCE

Check the absorbance using suitable filters or a solution of potassium dichromate uv at the wavelengths
indicated in the following table, which gives for each wavelength the exact values and permitted limits
of the specific absorbance. The tolerance for the absorbance is + 0.01.

Use solution of potassium dichromate uv which has been previously dried to constant weight at 130°.
For the control of absorbance at 235 nm, 257 nm, 313 nm, and 350 nm, dissolve 57.0-63.0 mg of
potassium dichromate uv in 0.005 m sulphuric acid and dilute to 1000.0 ml with the same acid. For the
control of absorbance at 430 nm, dissolve 57.063.0 mg of potassium dichromate uv in 0.005 m sulphuric
acid and dilute to 100.0 ml with the same acid.

Preparation of 0.005m sulphuric acid: take 0.27 ml in 1000.0 ml volumetric flask and make volume with
distilled water.
C. LIMIT OF STRAY LIGHT

Stray light may be detected at a given wavelength with suitable filters or solutions, for example,
absorbance of a 1.2 per cent w/v solution of Potassium chloride in a 1 cm cell should be greater than 2.0
at about 200 nm when compared with water as reference liquid.

D. RESOLUTION POWER

When stated in a monograph, record the spectrum of 0.02 per cent v / v solution of toluene in hexane
UV. The ratio of the absorbance at the maximum at about 269 nm to that at the maximum at about 266
nm is not less than 1.5 unless otherwise specified in the monograph.
 EXPERIMENT: 04

AIM:To perform TLC of tablets containing Paracetamol and Diclofenac.

REFERENCE:The Clerk`s Isolation and Identification of Drugs

REQUIREMENTS:

System IG:Gives good separation

Plates: Silica gel G, 250 micrometers thick
Mobile Phase: Ethyl acetate: Methanol: Strong ammonia solution (80:10:10, V/V/V)
Location reagents.

1.Chromic acid solution

DRUG COLOR

Diclofenac Red

Paracetamol Purple

2.Acidic Fecl3 solution

Blue OR Violate spots are given by phenols. This solution may be used to over spray a plate which
has been previously sprayed with Vanurk's reagent.

DRUG COLOR

Diclofenac Purple

Paracetamol Purple
PROCEDURE:

1. PREPARATION OF PLATES:
Silica gel plates: Mix 30 gm of silica gel G sufficient for 5 (20×20 cm) plates. Mix well
with 60ml of water. Stir well to remove air bubble & quickly apply, in a layer of 250
micrometers thickness, on the plates which have been previously cleaned with acetone to
remove grease. Allow the plates to dry in air.

2. APPLICATION OF SAMPLE:
The line is drawn with a pencil parallel to & 2 cm from, bottom of the plates. The
samples originated on to this line, called the origin, keeping distance 2cm from the side of
the plates and the least 1 cm from each other. The sample, normally 1-10 micrograms of
material depending on the thickness of the plate, is applied in as small as volume of
solvent as possible. It may be applied by a micropipette, such that the spot is no more
than 4mm in diameter, or else resolution will be lost. The solvent used to apply the spot
should be volatile and have low polarity so that the spots do not diffuse too much. It is
essential that the spot should dry at the end of application.

3. RUNNING THE PLATES:

A saturated developing chamber is normally used, with the glass lid. The tank is lined
with filter paper on 3 sides & the Mobile phase (usually 100ml for 20×20cm plates) is
added. The tank is then allowed to equilibrated. TLC plate is placed in a vertical position
in the tank so that the application line must be above the level of Mobile phase.

4. VISUALIZATION:
After running let the plates dry sometime & then spray plates with location reagents (here
FeCl3 solution & Chromic acid solution on the different plates).
Then keep plates in oven at 100°-150° C to obtain spots with both standard & samples
(diclofenac sodium & paracetamol).

5. Measure the distance travelled by the solvent & both solute (in test) & that of standard in
both the location reagents & find out the Rf values.
 OBSERVATIONS:

Rf value = distance travelled by the substance from origin

distance travelled by the solvent front from origin

LOCATION FeCl3 Chromic acid

REAGENTS

Distance 12.3 cm 14.5 cm

travelled
by solvent

Sample STD TEST STD TEST

(PCM pwd) (PCM tablet) (PCM pwd) (PCM tablet)

Distance
travelled …... (diclofenac) ….. (diclofenac)
by solute 7.2 cm 8.8 cm

…… (PCM) …... (PCM)

CALCULATION:
RESULT:

LOCATION FeCl3 Chromic acid

REAGENTS

STD TEST STD TEST

(PCM pwd) (PCM pwd) (PCM pwd) (PCM pwd)

Rf values
0.59 …... (diclofenac) 0.61 …… (diclofenac)

….. (PCM) ……. (PCM)

NOTE: Rf value should lie within 0 - 1 range.

 EXPERIMENT NO: 05
AIM: Estimation of Diclofenac and Paracetamol in pharmaceutical dosage form by simultaneous
equation method.

REFERENCES:

CHEMICALS/REAGENTS: PCM pure drug, diclofenac pure drug, 0.1 N NaOH, distilled water,
Methanol.

APPARATUS/INSTRUMENTS: Beakers, volumetric flask, UV visible spectrophotometer, filter

paper.

PROCEDURE:

 Prepare the solutions of 10 ppm of each drug of PCM and diclofenac.

 Scan the solutions to find out the lambda max of each solution as λ1 for PCM and overline λ2 for
diclofenac.

 Prepare the stock solutions of PCM as 100 ppm and diclofenac as 10ppm.

 Make solutions of concentration of 10,20,30,40 and 50ppm of PCM drug solution and concentration
of 1,2,3,4 and 5ppm of diclofenac drug solutions from the respective drug stock solutions.

 Find out absorbance of each of the solutions of both the drugs at λ1 and λ2

 calculate the absorptivity of each of the solutions at both the λmax and take an average.

 Prepare mixture of PCM and diclofenac by mixing 4 ml from PCM stock solution and 4 ml from
diclofenac stock solutions and dilute upto 10ml with distilled water.

 Find out absorbance of mixture as λ1 and λ2 for PCM and diclofenac respectively.

CALCULATION;
RESULT

 Concentration of paracetamol (Cx) found to be = ppm.

 Concentration of diclofenac(Cy) found to be = ppm.
 EXPERIMENT NO: 06

AIM:To determine % purity ofCuSO4 in the given unknown solution of CuSO4 using UV-
Visible Spectrophotometer.

REFERENCE:International Journal of Pharmacy and Pharmaceutical Science, Volume 2, Issue 03,

2010, Page no. 71-73

PRINCIPLE:

 Copper forms an intense blue colored complex with ammonium ion & this complex formation is
used as the basis for determination of copper content in given unknown solution at λmax 638nm.

REQUIREMENTS:

Apparatus: UV-Visible Spectrophotometer, Weighing balance, Butter paper, Volumetric

flasks

Reagent:

1) CuSO4 solution
2) NH4OH
3) Distilled water

PROCEDURE:

 In a series of 5 test-tube add 1,2,3,4 ml of 1% CuSO4 solution.

 Then add 5ml of 2% NH4OH solution is in each test tube similarly in next 2 test tube’s pipette out
1.5, 3.5ml of unknown CuSO4 solution & add 5ml 2% NH4OH in both Test tube.
 Mix well all test tubes & dilute upto 10ml with distilled water.
 Measure absorbance of all solutions at 638nm on spectrophotometer.
 Plot the graph of concentration of CuSO4 vs absorbance & find out unknown concentration of
CuSO4 from graph.

RESULT:

 Concentration of unknown = ________ mg/ml.

 EXPERIMENT NO: 07

AIM: To determine content of Quinine Sulphate in tablet dosage form by using fluorimetry.

REFERENCE:

Practical Pharmaceutical Chemistry by: A. H. Becket & J. B. Stenlake, 4th Edition, Part- 2,
page no., 369-373.

REQUIREMENTS:

Quinine sulphate, potassium iodide, 0.5 M H2SO4, Distilled water, volumetric flask, pipette

THEORY:

Chemical quenching occurs here. Chemical quenching is reduction in intensity by ray of chemical
interaction and such compounds/ chemicals are called quenchers (KI)

Quechua fluorometry: This method is mainly used in analysis

The drug or analyte serves as quencher. Here decrease in fluorescence intensity is measured with
increase in concentration of quencher i.e., drug
Eg., BMRC (fluorescing compound) Diazotized sulpha drug (Primary aromatic amine) act as
quencher

Prepare same concentration of BMR (7-8) and to that add different concentration of drug and
measure fluorescence

PROCEDURE:

Prepare 100 µg/ml standard solution of Quinine sulphate.

Prepare 0.1M potassium iodide (KI) solution, for that weigh 1.66 gm of KI and dissolve in 100 ml
volumetric flask with distilled water and dilute up to 100 ml with water.
Take fix quantity of quinine sulphate (30 ml) into six volumetric flask. In each flask add 0 ml, 1ml, 2 ml,
3 ml, 4ml, 5ml 0.1M KI solution.
Measure fluorescence intensity of this and plot the graph of fluorescence intensity against concentration
of KI.
RESULT & CONCLUSION:

The fluorescence intensity decreases as a concentration of quencher is increased.

 EXPERIMENT NO: 08

AIM:To Demonstrate the HPLC

THEORY:

HPLC: The technique of HPLC is High Performance Liquid Chromatography. It is so called

because of improved performance when compared to classical column chromatography. It is also
called as High-Pressure Liquid Chromatography since high pressure is used when compared to
classical column chromatography.

Types of HPLC techniques:

A. Based on model of chromatography:

There are two models- Normal phase mode

- Reverse phase mode

1. Normal phase mode: In normal phase mode stationary phase is polar in nature and the
mobile phase is non-polar.
2. Reverse phase mode: In reverse phase mode stationary phase is non-polar in nature and
mobile phase is polar.

B. Based on Principle of separation:

1. Adsorption chromatography: The principle of separation is adsorption.

2. Ion exchange chromatography: The principle of separation is ion exchange which is
reversible exchange of functional group.
3. Ion-pair chromatography: In this reverse phase column is converted temporarily into ion
exchange column by using ion pairing agent.
4. Size exclusion: A mixture of component is mixed with different molecular sizes are
separated by using gels. The mechanism of separation is by stearic and diffusion effect.
5. Affinity chromatography: It is used for the affinity of the sample with specific stationary
phase.
6. Chiral phase chromatography: Separation of optical isomers can be done by using chiral
stationary phase.

C. Based on elusion technique:

1. Isocratic separation: In this technique same polarity and same mobile phase combination is
used.
2. Gradient separation: In this technique lower polarity strength of mobile phase is used
moreover the composition of the mobile phase can also be changed.
 D. Based on scale of operation:
1. Analytical HPLC: Only analysis of sample is done.
2. Preparative HPLC: Separation of few grams of mixture by HPLC.

E. Based on type of analysis:

1. Qualitative analysis: Used to identify the drug/ impurity.
2. Quantitative analysis: To determine the quantity of individual component.

Principle of separation in HPLC:

The principle of separation in normal phase mode and reverse phase mode is adsorption when a
mixture of components is introduced in HPLC column they travel according to their relative
affinities towards the stationary phase. The component which is having more affinity towards the
adsorbent travels slower and the component which has less affinity towards the stationary phase
travels faster. Since two components may never have the same affinity towards the stationary phase,
the components are separated.

Fig: HPLC

Instrumental requirements:

1. Pumps:
● Solvent delivery system
● Pressure: 1000-3000 psi
● Particle size: 5-10 mu
● Resistance- high
● Pressure- high
● Pump- mechanical pneumatic pump

2. Check values:
● These are present to control the flow rate of solvent and block pressure.

3. Pulse dampener:
● Used for dampening the pulse observed from the baseline called by the pumps.

4. Mixing units, gradient controller and solvent degassing:

● Using the mix solvent in different preparations.
● Solvent degassing:
o Vacuum filtration
o Helium purging
o Ultra-sonication

● Injector:
o Manual or auto Injector
o Septum injector
o Stop flow injector
o Rheodyne injector

● Guard column:
Very small quantity of adsorbent and remove the impurities and help in improving the life of
the analytical column.

5. Analytical column:
● Column material: S.S, glass, polyethylene etc
● Column length: varies from 5cm-30cm
● Column diameter: Ranges from 2mm-50mm
● Practical size: 1-20mum
● Practical nature: Spherical uniform sized, porous material
● Surface area: 1gm of stationary phase provide surface area ranging from 100-861.59m with
average of 400-590m
 6. Detectors.
Fluorometric index detector: Based on fluorescent radiation emitted by some class of compounds
Conductivity detector: Based upon electrical conductivity
Amphoteric detector: Based on the oxidation of the compound/ reduction when potential is
applied
Photodiode array detector: Similar to UV detector but wider selection range of wavelength

7.Recorder:
● Used to record the response

8.Integrator:
● Improved version of recorder with same data processing

Applications of HPLC:

1) Qualitative analysis and quantitative analysis

2) Checking the purity of compound
3) Presence of impurities
4) Qualitative analysis
a) Direct comparison method
b) Calibration curve method
c) Internal standard method
5) Multi component analysis for determination of mixture of drugs
6) Isolation and identification of drugs
7) Isolation and identification of mixture
8) Biopharmaceutical and pharmacokinetic studies
9) Stability study
10) Purification

Comparison of classical column chromatography with HPLC:

Parameter Classical Column HPLC

Chromatography

Stationary phase Large Small

Particle size 60-20 µ 3-20

Column size Large Small

Length * diameter 0.5-50m * 0.5-5cm 5-50cm * 1-10mm

Column material Glass Mostly metal

Column packing Slurry packed at Slurry packed at high pressure

pressure
law pressure-often gravity 500psi
law
(<20psi)
law(<20psi)

Operating pressure Law (<20psi) High (500-3000psi)

Flow rate Law to very law Medium high

Sample lead Law to medium Law to very law

Sample load Law to medium Often >1,000,000

(law <500 theoretical plates per meter

plate per meter)

Cost Law few hundreds High few lakhs

Detector flow Large 300-1000 Law 2-10

Cell volume
Types of vailable Limited range Wide range

Scale of operation Preparative scale Analytical scale