Parul Institute of Pharmacy and Research

Industrial Pharmacy Practical – 1

(MIP 105P)

NAME :-

ENROLLMENT NO. :-

CLASS :-

YEAR :-
 LABORATORY CERTIFICATE
Parul Institute of Pharmacy and Research

This is to certify that Mr./Ms. _____________________________________.

Student of PARUL INSTITUTE OF PHARMACY AND RESEARCH
studying in M. Pharma Program in 1st Semester/ First Year during the academic
session 2023-2024 has satisfactory performed the practical course work in the
subject Industrial Pharmacy PRACTICAL - I.

The Marks scored by the student based on his/her performance are

Principal Teacher in charge

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External Examiner Internal Examiner

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 INDEX
SR PAGE
NO. TITLE NO. DATE SIGNATURE
To perform the analysis of Paracetamol in
1.
bulk and in tablet dosage form by UV VIS
spectrophotometer.
To determine the concentration of
2. Paracetamol and Ibuprofen using
simultaneous equation method.
Demonstration of HPLC (High Performance
3. Liquid Chromatography).

To estimate concentration of Riboflavin in

4. the given unknown solution byfluorimetry.

To study the effect of quenching of

5. Potassium iodide on Fluorescence intensity
of Quinine Sulphate.
To determine the concentration of Na+ and
6. K+ in unknown sample of ORS using flame
photometer.
To study the effect of surfactants on the
7. solubility of Aspirin.

To study the effect of pH on solubility of

8. Aspirin.

To perform stability testing of Paracetamol

9. solution for photodegradation.

Stability studies of drugs in dosage forms

10. at 25°C, 60% RH and 40°C , 75% RH .

Compatibility study need to be performed

11. to access any possible integration drug and
excipients, before designing the dosage
form.
To prepare and evaluate different polymeric
12. membranes.

Formulation and evaluation of sustained

13.
release matrix tablet of Aceclofenac.
 Formulation and evaluation of
14. microspheres/ microcapsules.

Formulation and Evaluation of

15. Transdermal patch of Diclofenac Sodium.

Design and evaluation of a) face wash b)

16. creams c) lotions d) shampoo e) toothpaste
f) lipstick.
Demonstration of Gel electrophoresis.
17.

Preparation & evaluation of liposome

18. delivery system.
 INDUSTRIAL PHARMACY -1(MIP105P)

PRACTICAL-1 Date: / /202_

Aim: To perform the analysis of Paracetamol in bulk and in tablet dosage form by UV
VIS spectrophotometer.

Reference: Pharmacopoeia I. The Indian pharmacopoeia commission. Central Indian

Pharmacopoeia Laboratory, Ministry of Health and Family Welfare, Govt of India, Sector.
2007;23.

Requirements:
Apparatus:-Beaker, funnel, measuring cylinder, pipette, glass cuvette, glass rod, filter paper.
Chemicals:-Paracetamol, NaOH, water.

Theory: UV-Visible spectrophotometry is one of the most frequently employed techniques

in pharmaceutical analysis. It involves measuring the amount of ultraviolet or visible radiation
1cm
absorbed by a substanc3e in solution. In qualitative analysis, organic compounds can be
identified by use of spectrophotometer, if any recorded data is available, and quantitative
spectrophotometric analysis is used to ascertain the quantity of molecular species absorbing the
radiation. Spectrophotometric technique is simple, rapid, moderately specific and applicable
to small quantities of compounds. The fundamental law that governs the quantitative
spectrophotometric analysis is the Beer -Lambert law. Beer's law: It states that the intensity
of a beam of parallel monochromatic radiation decreases exponentially with the number
of absorbing molecules. In other words, absorbance is proportional to the concentration.
Lambert's law. It states that the intensity of a beam of parallel monochromatic radiation
decreases exponentially as it passes through a medium of homogeneous thickness. A
combination of these two laws yields the Beer-Lambert law. Beer-Lambert law: When beam
of light is passed through a transparent cell containing a solution of an absorbing substance,
reduction of the intensity of light may occur. Mathematically, Beer Lambert law is
expressed as:
(i) A = a b c
Where,
A= absorbance or optical density.
a = absorptivity or extinction coefficient.
b = path length of radiation through sample (cm)
c = concentration of solute in solution.
(ii) using A= abc, A= A1% bc
Procedure:
Bulk Assay:
1. Weigh accurately 100 mg of paracetamol powder and dissolve in sufficient vol. of 0.1
M NaOH and make up the volume up to 100 ml with 0.1 M NaOH.
2. From above solution take 10 ml and dilute with 0.1 M NaOH to make volume 100 ml.
3. From the resultant solution, pipette out 1 ml, 2 ml, 3 ml, 4 ml and 5 ml and transfer in
10 ml volumetric flask separately.
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4. Measure the absorbance of solution against blank at 257 nm.

5. Prepare the unknown sample of Paracetamol and measure the absorbance at 257 nm
against blank.

Observation table:

Concentration Absorbance

Calibration curve:

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Calculation:

Tablet Assay Procedure:

• Weigh accurately 20 tablets and powder it.
• Weigh tablet powder equivalent to 0.15gm of Paracetamol.
• Add 50 ml 0.1M NaOH. Dilute upto 100 ml of water. Shake for 15 minutes and add
sufficient water to produce 200 ml.
• Mix well and filter. From this filtrate, pipette out 10 ml and transfer in 100 ml
volumetric flask. Dilute upto 100 ml with water.
• Again pipette out 10 ml from the resultant solution and add 10 ml of 0.1M NaOH.
Dilute it upto 100 ml with water and mix well.
• Measure absorbance at 257 nm against blank.
• Calculate % purity of drug using (i) standard calibration curve (ii) A (1%, 1 cm) value
of 715.

Observation:

Weight of 20 tablets powder (A) = g

Average weight of one tablet powder (B) = g
Label claim of tablet (C) = g of Paracetamol

C g of Paracetamol is present in B g of Tablet powder

So, g of Paracetamol is present in (?) g of Tablet powder

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g of Paracetamol is present in g of Tablet powder

Observation Table:

Concentration Absorbance
(µg/ml) (at 257 nm)
2
4
6
8
10
Test

Calibration Curve:

Calculation:

Calculation for serial dilution of pure drug:

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Calculation for test solution of tablet (dilution scheme):

150 mg in 200 ml 75 mg/100 ml 0.75 mg/ml

0.75 mg/100 ml 0.075 mg/ml 10 ml (7.5 mg/100 ml)

0.0075 mg (7.5 µg/ml)

Calculation of % Purity:

7.5 µg/ml 100%

µg/ml ?

= ×100/7.5

= %

Result:

The % purity of Paracetamol tablet was found to be %.

IP 2018 Limit: Paracetamol Tablets contain not less than 95.0 percent and not more than 105.0
percent of the stated amount of Paracetamol C8H9NO2.

Conclusion:

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Parul Institute of Pharmacy & Research, M. Pharm- Pharmaceutics Semester I Page No.
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PRACTICAL-2 Date: / /202_

Aim: To determine the concentration of Paracetamol and Ibuprofen using simultaneous

equation method.

Reference: Issa YM, Zayed SI, Habib IH. Simultaneous determination of ibuprofen and
paracetamol using derivatives of the ratio spectra method. Arabian Journal of Chemistry.
2011;4(3):259-63.

Requirements:
Apparatus:-Beaker, funnel, measuring cylinder, pipette, glass cuvette, glass rod, filter paper.
Chemicals: - Paracetamol standard powder, Ibuprofen standard powder, Market sample
containing Paracetamol and Ibuprofen, Methanol.

Principle and Theory: The concentration of given mixture can be determined by simultaneous
equation, if following criteria are satisfied.

A) The λmax of both the compound should be distinct.

B) Both compound should absorb at λmax of one-another.

C) There is no reaction between solutes.

Absorbance is an additive property.

Overlain Spectra of Drugs X and Y

Now, simultaneous equation,

A1ax2 − A2ax1
Cy =
ax2ay1 − ax1ay2

A1ax2 − A2ax1
Cx =
ax2ay1 − ax1ay2

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Cy = Concentration of component y in the mixture.

Cx = Concentration of component x in the mixture.

ax1 = Absorptivity of compound x at first wavelength.

ax2 = Absorptivity of compound x at second wave-length.

ay1 = Absorptivity of compound y at first wavelength.

ay2 = Absorptivity of compound y at second wavelength.

A1 = Absorption of mixture at first wave-length.
A2 =Absorption of mixture at second wave-length.

Procedure:-

 Make the stock solution of Ibuprofen and Paracetamol of 1000 µg/ml. From that
solution take 1 ml solution and dilute it to 10 ml. This solution is having concentration
of 100 µg/ml.

 From this solutions make 5 µg/ml and 10 µg/ml of both Paracetamol and Ibuprofen.
Take the absorbance of these solutions and also find out λmax for both compounds.

 Design the concentration in such a way that absorbance comes between 0.1–1.

 Now make the solution of 2 µg/ml, 4 µg/ml, 6 µg/ml, 8 µg/ml and 10 µg/ml
concentration of Paracetamol. And make the solution of 2 µg/ml, 4 µg/ml, 6µg/ml,
8µg/ml and 10 µg/ml concentration of Ibuprofen. Take absorbance of all above
solutions at both λmax.

 Now make the solution containing of both Paracetamol and Ibuprofen. Measure the
absorbance of that solution at both wave-lengths.

Observations & Calculations:

λmax of Paracetamol is nm. (λ1)

λmax of Ibuprofen is nm. (λ2)

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For Paracetamol
Concentration Absorbance at Absorptivity Absorbance at Absorptivity
(μg/ml) λ1 ( nm) λ2 ( nm)
2
4
6
8
10
Avg.= ax1= Avg.= ax2=

For Ibuprofen
Concentration Absorbance at Absorptivity Absorbance at Absorptivity
(μg/ml) λ1 ( nm) λ2 ( nm)
2
4
6
8
10
Avg.= ay1= Avg.= ay2=

For Mixture:-

Sample name Absorbance at λ1( nm) Absorbance at λ2 ( nm)

= A1 = A2
Sample 1(Paracetamol of 6
µg/ml)
Sample 2 (Ibuprofen 6 µg/ml)
Sample 3 (Paracetamol of 6
µg/ml + (Ibuprofen of 6
µg/ml)

1) For 6 µg/ml Paracetamol:-

A1ax2 − A2ax1
Cy =
ax2ay1 − ax1ay2

A1ax2 − A2ax1
Cx =
ax2ay1 − ax1ay2

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2) For 6 µg/ml Ibuprofen:-

A1ax2 − A2ax1
Cy =
ax2ay1 − ax1ay2
A1ax2 − A2ax1
Cx =
ax2ay1 − ax1ay2
3) For final sample 5 µg/ml Paracetamol +5 µg/ml Ibuprofen:-

ax1 = ay1 = A1 =
ax2 = ay2 = A2 =

A1ax2 − A2ax1
Cy =
ax2ay1 − ax1ay2
A1ax2 − A2ax1
Cx =
ax2ay1 − ax1ay2
For sample 1:-

% of Paracetamol:

6 µg/ml 100%

µg/ml ?

= ×100/6

= %
% of Ibuprofen:

6 µg/ml 100%

µg/ml ?

= ×100/6

= %
For sample 2:-
% of Paracetamol:

6 µg/ml 100%
µg/ml ?
= ×100/6

= %
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% of Ibuprofen:

6 µg/ml 100%

µg/ml ?

= ×100/6

= %

For sample 3:-

% of Paracetamol:

6 µg/ml 100%
µg/ml ?
= ×100/6

= %

% of Ibuprofen:

6 µg/ml 100%

µg/ml ?

= ×100/6

= %

Results:-
Sample 1 contains % Paracetamol and % Ibuprofen.
Sample 2 contains % Paracetamol and % Ibuprofen.
Sample 3 contains % of Paracetamol and % of Ibuprofen.

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Parul Institute of Pharmacy & Research, M. Pharm- Pharmaceutics Semester I Page No.
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PRACTICAL-3 Date: / /202_

Aim: Demonstration of HPLC (High Performance Liquid Chromatography).

Reference: Lopez-Sanchez RD, Lara-Diaz VJ, Aranda-Gutierrez A, Martinez-Cardona JA,

Hernandez JA. HPLC method for quantification of caffeine and its three major metabolites in
human plasma using fetal bovine serum matrix to evaluate prenatal drug exposure. Journal of
Analytical Methods in Chemistry. 2018;

◼ HPLC INTRUMENTATION CONSIST OF

➢ Solvent Reservoir (HPLC solvent reservoir systems)

➢ Pumps

➢ Pre Guard Column

➢ Sample injection system

➢ Columns

➢ Detector

➢ Recorder and integrators

FIGURE5.1: HPLC DIAGRAM

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1) HPLC SOLVENT RESERVOIR SYSTEMS

➢ The glass bottles can be used to store the mobile phase.

➢ The mobile phase is pumped under pressure from one or several reservoirs and flows
through the column at a constant rate.

➢ Desirable feature in the solvent delivery system is the capability for generating a solvent
gradient.

➢ Degasser is needed to remove dissolved air by subjecting the mobile phase under
vacuum, distillation, spurging with fine spray of an inert gas at lower solubility or by
heating and ultrasonic stirring.

2) PUMPS

◼ Pass mobile phase through column at high pressure and at controlled flow rate.

◼ Performance of pump directly affects the Rt, reproducibility, detector sensitivity.

Ideal characteristic of a pump

• Non corrosive and compatible with solvent.
• Provide high pressure to push mobile phase
• Provide constant flow rate to mobile phase.
• Easy to change for one mobile phase to another.

Type of pump used in HPLC

• Reciprocating pump
• Displacement pump
• Pneumatic pump

3) SAMPLE INJECTION SYSTEM

(A) Septum injectors
(B) Stop flow septumless injection.
(C) Rheodyne injector / loop valve type.

(A) Septum injection port.

Syringe is used to inject the sample through a self sealing inert septum directly into
the mobile phase.
Drawback: - leaching effect of the mobile phase with the septum resulting in the
formation of ghost peaks.

(B) Stop flow septumless injection.

Flow of mobile phase through the column is stopped for a while.
Syringe is used to inject the sample.

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Drawback: formation of ghost peak.

(C) Rheodyne injector / loop valve type.

Sophisticated modern method with good precision.
Sample is introduced in the column without causing interruption to mobile phase
flow.
Volume of sample ranges between 2 µl to over 100 µl.

Operation of sample loop.

Sampling mode
Injection mode.

Sample is loaded at atmospheric pressure into an external loop in the micro volume sampling
valve, and subsequently injected into the mobile phase by suitable rotation of the valve.

Fig: Micro volume sampling valve operation of a Sampling loop

Column
Made up of stainless steel or heavy glass to withstand the pressure.
The columns are usually long (10 – 30 cm) narrow tubes.
It contains stationary phase at particle diameters of 25 µm or less.
The interior of column should be smooth and uniform.
Column end fitting are designed to have a zero void volume.
Classification of Column Based On Application

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Classification of column on the basis of components:

BONDED PHASE COLUMN

Internal diameter 4 – 5 mm and length 10 – 30 cm.

Size of stationary phase is 3 – 5 µm in diameter.
Used for the estimation of drugs, metabolites, pharmaceutical preparation and body fluids like
plasma.

NARROW BORE COLUMN

Internal diameter is 2 – 4 mm. (signal is increased 4 times)
Require high pressure to propel mobile phase.
Used for the high resolution analytical work of compounds with very high Rt.

SHORT FAST COLUMN

Length of column is 3 – 6 cm.
Used for the substances which have good affinity towards the stationary phase.
Analysis time is also less (1- 4 min for gradient elusion & 15 – 120 sec for isocratic elusion).

PREPARATIVE COLUMN
Used for analytical separation i.e. to isolate or purify sample in the range of 10-100 mg from
complex mixture.
Length – 25 - 100 cm
Internal diameter – 6 mm or more.

Preparative column are of three types:

1) Micro preparative or semi preparative column
Modified version of analytical column
Uses same packaging and meant for purifying sample less than 100 mg.

2) Preparative column
Inner diameter – 25 mm.
Stationary phase diameter – 15- 100 µm

3) Macro Preparative Column

Column length – 20 – 30 cm
Inner diameter – 600 mm

GUARD COLUMN
They are placed before the separating column.
Serve as a protective factor that prolongs the life and usefulness of the column.
They are dependable column designed to filter or remove.
Particles that clog the separation column.
Compounds and ions that could ultimately cause baseline drift, decrease resolution, decrease
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sensitivity and create false peaks.

Advantages
Can withstand high pressure exerted by mobile phase.
Life of column is more.
No bleeding effect.
Disadvantages
Very expensive
Manually cannot be fabricated

DETECTORS
Based on the application, the detectors can be classified into
Bulk property detectors
Solute property detectors.

A) BULK PROPERTY DETECTORS

➢ Compare an overall change in physical property of mobile phase with or without an
eluting solute.
These types of detectors tend to be relatively insensitive and require temperature
control.
e.g., Refractive index detector.

B) SOLUTE PROPERTY DETECTORS

➢ They respond to a physical property of the solute that is not exhibited by the pure mobile
phase.
These detectors are more sensitive; detect the sample in nanograms quantity.
e.g., UV visible detector, electrochemical detector, fluorescence detector.

1) ULTRAVIOLET VISIBLE DETECTOR

➢ UV detectors are the most commonly used detector.
They measure the ability of a sample to absorb light. This can be accomplished at oneor
several wavelengths.
➢ A variable wavelength UV detector, capable of monitoring from 190 to 460-600 nm
will be found suitable for the detection of the majority of samples.
➢ Mobile phase from the column is passed through a flow cell held in the radiation beam
of UV / visible spectrophotometer.
➢ Selective in nature, detect only those solutes that absorb UV/ visible radiation
e.g., alkenes, aromatic compounds and compound having multiple bonds between C and
O, N or S.

BASICALLY TWO TYPES OF ABSORBANCE DETECTORS ARE AVAILABLE

• Fixed wavelength
detector

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• Variable wavelength
detector

2)FLOURIMETRIC DETECTORS
➢ Very sensitive, but very selective.
➢ It is possible to detect even a presence of a single analyte molecule in the flow-cell.
➢ Fluorescence occurs when compounds having specific functional groups are excited by
shorter wavelength energy and emit higher wavelength radiation.
➢ Fluorescence is often collected at right angle to excitation beam.
➢ Only one sixth of fluorescence is collected. If concave mirror is placed around the
sample cell about 75 % of the emission is collected.
➢ With all sample cells, scattered radiation from the excitation source is selectively
removed.
3) Refractive Index Detector/ Differential refractometer
➢ The detection principle involves measuring of the change in refractive index of the
column effluent passing through the flow-cell.
➢ It responds to any solute whose refractive index is significantly different from that of
the mobile phase.
➢ Principle: It is based on two principles.

Deflection (deflection type refractometer)

Reflection (reflection type refractometer)

4) Electrochemical Detector/ Amperometric detector

➢ It is based on the measurements of the current resulting from an oxidation/reduction
reaction of the analyte at a suitable electrode.
➢ The level of the current is directly proportional to the analyte concentration

Recorder and integrators

Recorders are used to record the response obtained from the detector after amplification. They
record the baseline and all the peaks obtained, with respect to time. Retention time for all the
peaks can be calculated.

Chromatography condition:

• Composition of mobile phase and its pH:

• Flow rate (isocratic): ml/min.

• Volume injected: 100 μL

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• Type of detector: UV detector

• Detector mode: Variable

• Wavelength: _nm

• Column: C18 Octadecylsilane, Phenomenex

• Temperature: Ambient
Observation Table:

Sr. No. Concentration (μg/ml)

Area

1 5
2 10
3 15
4 20
5 25
6 Unknown
Standard Curve:

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Calculation:

Result: The concentration of given drug Paracetamol was found to be µg/ml using
HPLC method.

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Parul Institute of Pharmacy & Research, M. Pharm- Pharmaceutics Semester I Page No.
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PRACTICAL-4 Date: / /202_

Aim: To estimate concentration of Riboflavin in the given unknown solution by

fluorimetry.

Reference: - Zhou T, Li H, Shang M, Sun D, Liu C, Che G. Recent analytical methodologies

and analytical trends for riboflavin (vitamin B2) analysis in food, biological and pharmaceutical
samples. TrAC Trends in Analytical Chemistry. 2021.

Requirements:

Apparatus: -Beaker, Fluorimeter, funnel, measuring cylinder, Volumetric flask, pipette, glass
cuvette, glass rod, filter paper.

Chemicals:- Glacial acetic acid, Riboflavin.

Theory:

From the Beer-Lambert law following equation is obtained for fluorescent radiation,
F α I0 – I or F = K (I0 - I ) ------------ (1)
Where F= intensity of fluorescent radiation,
K= proportionality constant,
I0= intensity of incident light,
I= intensity of transmitted light.

We know that the Beer-Lambert law is

I = I0 10 – abc
Or I0 - I = I0 – I0 10- abc
= I0 (1 – 10– abc) --------------------- (2)

On substituting (2) in (1), we get,

F = KI0 (1 – 10– abc) ---------------------------- (3)

On rearranging (3), we get,

log KI0 = abc

KI0 – F (4)

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On simplifying (4), we get,

F = 2.303 KI0.abc
F = K’c

Measurement of fluorescence:-
F = Kc.
Fs = Kcs for standard
Fu = Kcu for unknown
Cu/Cs = Fu/Fs
Application of Fluorimetry:
Fluorimetry has assumed a major role in analysis particularly in determination of trace
contamination in our environment, industries and bodies, because fluorimetry gives high
sensitivity (in low parts per trillion) for applicable compounds.

Fluorimetry have high specificity because it depends on two spectra, one is excitation spectra
and another is emission spectra.

By use of fluorimetry we can measure life times of the fluorescent state.

When a fluorescent compound present with one or more non fluorescent compounds then we
can also readily analyzed by use of fluorimetry.

We can analyze the compounds which have overlapping absorption spectra by use of
fluorimetry.

Many drugs have high quantum efficiencies for fluorescence, so it can be easily analyzed by
fluorimetry. For example, Quinine and Lysergic acid diethylamide. (If 1 ng/ml drug present in
5 ml sample of blood plasma or urine then also analyzed by fluorimetry)

Fluorescence spectroscopy is used in biochemical, medical and chemical research fields for
analyzing organic compounds.

Atomic Fluorescence Spectroscopy (AFS) techniques are useful in other kinds of analysis
/measurement of a compound present in air or water, or other media, it is also used for heavy
metals detection, such as mercury.

Fluorescence can also be used to redirect photons.

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Procedure:
✓ Weigh accurately 10 mg standard powder of Riboflavin and transfer it in 100 ml
volumetric flask.
✓ Add 5 ml of Glacial acetic acid and 30 ml of distilled water.
✓ Dissolve contents and make up the volume up to 100 ml with distilled water.
✓ From this solution; prepare appropriate dilution to get concentration of 0.5, 1.0, 1.5, 2.0
& 2.5 μg/ml and measure fluorescence intensity of solution.
✓ Prepare calibration curve of fluorescence intensity vs. concentration.
✓ From calibration curve calculate concentration of Riboflavin in unkown solution.

Observation table:

Concentration Fluorescence
(μg/ml) Intensity
0.5

1.0

1.5

2.0

2.5

Test

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Observation:

From the graph, at the fluorescence intensity of the concentration is around

μg/ml.

Result: The concentration of given test Riboflavin sample was found to be μg/ml,
byfluorimetry.

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Parul Institute of Pharmacy & Research, M. Pharm- Pharmaceutics Semester I Page No.
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PRACTICAL-5 Date: / /202_

Aim: To study the effect of quenching of Potassium iodide on Fluorescence intensity of

Quinine Sulphate.

Reference: - Guo Y, Cao F, Qiu P, Wang Z. Studies of the effect of halide ions on the
fluorescence of quinine sulfate. Luminescence. 2019;34(4):450-455.
Requirements: -
Apparatus:-Beaker, Fluorimeter, funnel, measuring cylinder, Volumetric flask, pipette, glass
cuvette, glass rod, filter paper.
Chemicals: - Quinine sulphate, 0.05M sulphuric acid, Potassium iodide.

Principle:-
Quenching refers to any process which decreases the fluorescence intensity of a substance. A
variety of processes can result in quenching, such as excited state reaction, energy transfer,
complex- formation and coalitional quenching. As a consequence, quenching is often heavily
dependent on pressure and temperature. Molecular oxygen, iodide ions and acrylamide are
common quenchers.
The quenching depends upon interaction or reaction between the analyte and the quencher and
the rate of quenching is a function of the temperature and viscosity of the solution, as well as
the concentration of the quencher Q. The rate constant of external conversion kec, is related to
the second order quenching rate constant kq given by following formula:

kev = kq [ Q ]

Mainly the quenchers are of two types:

1. Collisional (dynamic) quencher: Decreases fluorescence by dissipating absorbed energy as

heat due to collision with quenching species. E.g quinine in 0.05 M sulphuric acid is highly
fluorescent but nonfluorescent in 0.1 M hydrochloric acid because of collisional quenching
by halide ion.
2. Static quencher: It forms a complex with fluorescent substance and changes its fluorescence
characteristics. Eg. Caffeine (xanthine derivative) decreases fluorescence of riboflavin.
The halide ion is a well-known quencher for quinine fluorescence. The principle behind this
experiment is the presence of halide results in quenching due to collisions.

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Procedure:
1. Preparation of quinine sulphate solution :
Weigh accurately 0.1 g quinine sulphate and dissolve in 100 ml of 0.05 M sulphuric
acid. Dilute 5 ml of the above solution to 50.0 ml with 0.05 M sulphuricacid (100.0
g/ml)
Again dilute 5.0 ml of this solution to 50.0 ml with 0.05M sulphuric acid. Further
dilute 5.0 ml of the above solution to 50.0 ml with 0.05 M sulphuric acid. The
resulting solution contains 1 g/ml quinine sulphate (standard solution).
2. Preparation of potassium iodide solution:
Dissolve 100 mg of potassium iodide in 100 ml of 0.05M sulphuric acid (1mg/ml).
Dilute 10 ml of above solution to 100 ml with 0.05 M sulphuric acid (100 g/ml).
3. Transfer 1ml of 1 g/ml solution of quinine sulphatein each six 10 ml volumetric
flasks.
4. Add 0 ,1,2,3,4 and 5 ml of potassium iodide (100 g/ml) in each flask.
5. Make up the volume to 10 ml with 0.05M Sulphuric acid.
6. Measure fluorescence of each solution using 0.05 M sulphuric acid as blank and
100 % using standard solution.
7. Plot the graph of % fluorescence intensity vs. volume of KI added.

Observation table:

Sr. no Volume of KI Fluorescence

added (ml) intensity (%)

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Graph:

Conclusion:
A decrease in fluorescence intensity was observed for quinine sulphate with the increase in
concentration potassium iodide solution.

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PRACTICAL-6 Date: / /202_

Aim: To determine the concentration of Na+ and K+ in unknown sample of ORS using
flamephotometer.

Reference:- Modern pharmaceutical analytical Techniques experiment Estimation of Sodium

(Na) and Potassium (K) Ions by Flame Photometry. 2023, 10.13140/RG.2.2.34285.87522.
Requirement:
Apparatus: -Beaker,funnel, measuring cylinder, volumetric flask, pipette, glass rod, filter paper,
Flame –photometer.
Chemicals:- KCl, distilled water, NaCl.

Principle:

• When solution of metal in form of salt dispensed in the form of fine spray with help of
an atmosphere into hot flame

• The solution is evacuated and evaporated to have dry metal due to higher energy
transformed into gaseous state to give separate atom or radical, while returning to
ground state they emit energy so line spectra obtained

• The wavelength of these spectra are characteristic of the metal

• Flame-photometry is used for analysis of biological fluids, solid plant materials and in
determination of K+, Na+, Li+, Ca++, Mg+, Zn+2 .

Preparation of standard solution:

Molecular weight of NaCl = 58 gm/mol Molecular weight of KCl = 74 gm/mol

i.e.,
1000 mmol NaCl ≈ 58 gms NaCl 1000 mmol KCl ≈ 74 gms KCl
4 mmol NaCl = X gms NaCl 2 mmol KCl = Y gms KCl

58 ∗ 4 74 ∗ 2
So, x = = 0.232 gm/lit NaCl So, Y = = 0.148 gm/lit KCl
1000 1000

• Take 0.0232 gms NaCl and 0.0148 gms KCl in 100 ml volumetric flask.
• Dissolve them with distilled water and then dilute upto 100 ml in it.
• It gives the 4 mmol/lt Na+ and 2 mmol/lt K+.

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Preparation of test solution:

The ORS contain 75 mmol/lit of Na+ and 20 mmol/lit of K+ and total weight of the ORS is 21gms.
So, simply it meant that,
75 mmol of Na+ is present in 21 gms ORS
2 mmol of Na+ is present in A gms ORS

21∗2
So, A= = 0.56𝑔𝑚𝑠ORS (which correspond to 0.53 mmol K+ also)
75

Observation: The concentration of Na+and K+ was found to be mmol/lit and

mmol/lit, respectively.

Calculation: (to find out %)For Na+:

If, 2 mmol/lit of Na+ is ≈ 100%
mmol/lit of Na+is =100× /2 = %

For K+:
If, 0.53 mmol/lit of K+ is ≈ 100%
mmol/lit of K+ is = 100× /0.53= %

Result:
The % of Na+ and K+ was found to be % and %, respectively.

Conclusion:
The % of Na+ ( %) and K+ ( %) falls/does not falls in the range as per IP whichis 90% -
110%, so we can conclude that the given ORS sample passes/fails the test.

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PRACTICAL-7 Date: / /202_

Aim: To study the effect of surfactants on the solubility of Aspirin.

Reference:-The effects of surfactants on the solubility and dissolution profiles of a poorly water-soluble
basic drug, carvedilol. Die Pharmazie-An International Journal of Pharmaceutical Sciences.
2015;70(12):784-90.

Requirements:
Apparatus: -Beaker, funnel, measuring cylinder, volumetric flask, pipette, glass rod, filter paper.
Chemical:- Aspirin, Sodium lauryl sulphate, Tween 80, cetrimide, methanol.

Principle:
The solubility of drugs, including Diclofenac Sodium, can be significantly influenced by the presence
of surfactants. Surfactants are amphiphilic molecules, meaning they have both hydrophobic (water-
repelling) and hydrophilic (water-attracting) parts. Their ability to alter the interfacial properties of
substances in solution can impact the solubility of poorly soluble drugs.

Micelle Formation:
Micelles are aggregates of surfactant molecules that assemble in a way that their hydrophobic tails
cluster together in the core, shielding them from water, while the hydrophilic heads are exposed to the
aqueous environment. Diclofenac Sodium, being a hydrophobic drug, can be encapsulated within the
hydrophobic core of these micelles, increasing its solubility in the aqueous medium. The hydrophilic
outer shell of the micelle makes the entire assembly water-soluble.

Wetting and Dispersion:

Surfactants can improve wetting, which is the ability of a liquid to spread over a solid surface. Diclofenac
Sodium may have poor wetting properties on its own due to its hydrophobic nature. By reducing the
surface tension of the solvent, surfactants enhance the wetting of Diclofenac Sodium on surfaces,
promoting its dispersion and dissolution.

Emulsification:
Surfactants can form emulsions when mixed with immiscible liquids. This is particularly relevant if
Diclofenac Sodium is poorly soluble in water but needs to be administered in an aqueous medium. The
surfactant can stabilize the dispersion of the hydrophobic drug in the aqueous solution, forming an
emulsion and facilitating its administration.

Increased Surface Area:

Surfactants can increase the surface area available for dissolution by breaking down aggregates of the
drug into smaller particles. This increased surface area enhances the interaction between the drug and
the solvent, leading to improved solubility.

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Reduction of Interfacial Tension:

Surfactants can reduce the interfacial tension between the drug and the solvent. This reduction allows
for easier penetration of the solvent into the drug particles, aiding in the solubilization process.

Procedure:
1. For standard of Sodium lauryl sulphate: Weigh accurately 100 mg of Aspirin powder and
dissolve in 50 ml of methanol.
2. From the resultant solution, pipette out 1ml and make up to 100 ml with methanol.
3. From the resultant solution, make the dilution of 1.0ml, 1.2ml, 1.4ml, 1.6ml, 1.8ml, and 2.0ml
and make up them with methanol upto 10ml each.
4. For test: Take 50mg of SLS in 10 ml of methanol, and add the drug until the saturation is
achieved.
5. Repeat the same procedure as mentioned above with different surfactants such as Tween 80
(TNT) and Cetrimide.
Observation table:

Calibration curve:

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Calculation:

Result:

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PRACTICAL-8 Date: / /202_

Aim: To study the effect of pH on solubility of Aspirin.

Reference: Joshi HA, Dyade GK, Patil RN. Effect of solubility enhancers on the solubility of
paracetamol. Indo American Journal of Pharmaceutical Research, 2012; 3 (1). 2012;1387.

Requirements:
Apparatus: Beaker, funnel, measuring cylinder, volumetric flask, pipette, glass rod, filter paper.
Chemicals:- Aspirin, 6.8 pH Phosphate buffer, 1.2 HCL acid buffer, 7.4 phosphate buffer, ethanol,
water.

Theory: The study of the effect of pH on the solubility of aspirin is essential for optimizing drug
formulations, improving drug delivery systems, enhancing therapeutic efficacy, and ensuring the safety
and bioavailability of the medication. This knowledge is crucial for pharmaceutical scientists,
formulators, and healthcare professionals involved in drug development and patient care.

1. Pharmaceutical Formulation:
- Aspirin is commonly used as a medication, and its pharmaceutical formulations may involve different
pH conditions. Understanding the solubility of aspirin under varying pH levels is crucial for formulating
drugs and designing dosage forms. This knowledge helps in creating drug formulations that maximize
the absorption and effectiveness of the medication.

2. Bioavailability:
- The bioavailability of a drug is influenced by its solubility. Bioavailability refers to the proportion
of the administered drug that reaches the systemic circulation and is available for the intended
therapeutic effect. The solubility of aspirin in different pH environments can affect its absorption in the
gastrointestinal tract, impacting bioavailability.

3. Drug Delivery Systems:

- PH-sensitive drug delivery systems are designed to release drugs at specific pH levels in the body.
Understanding the pH-dependent solubility of aspirin is essential for designing controlled-release
formulations that release the drug at the desired rate and site in the body.
4. Optimizing Drug Administration:
- Knowledge of the pH-dependent solubility of aspirin is important for optimizing drug administration
protocols. For instance, it helps determine whether the drug is better absorbed in the stomach or the
small intestine and whether it should be administered with or without food.
5. Chemical Stability:
- The stability of aspirin can be influenced by pH. Understanding the pH conditions that promote or
degrade the stability of aspirin is essential for ensuring the quality and shelf-life of pharmaceutical
products.

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6. Therapeutic Efficacy:
- The therapeutic efficacy of aspirin is closely related to its absorption and distribution in the body.
PH conditions can affect the solubility of aspirin, which in turn can impact its therapeutic effectiveness.
Therefore, understanding the pH-dependent solubility is crucial for predicting and optimizing the drug's
therapeutic outcomes.

7. Safety Considerations:
- Changes in pH conditions can influence the physical and chemical properties of aspirin. This includes
the potential for the formation of insoluble precipitates or the degradation of the drug under certain pH
conditions. Understanding these factors is important for ensuring the safety of the drug when
administered to patients.

Procedure:
1. Weight accurately 25 mg of Aspirin powder and dissolve in 25 ml of Ethanol and make up the volume
up to 100 ml with 6.8 pH Phosphate buffer.
2. From the resultant solution, pipette out 0.8 ml, 1 ml, 1.2 ml, 1.4 ml, 1.6 ml and 1.8 ml and transfer in
10 ml volumetric flask separately and make up the volume up to 10 ml with 6.8 pH Phosphate buffer.
3. Measure the absorbance of solution against blank at 235 nm.
4. Prepare the known saturated solution of Aspirin in the 6.8 pH Phosphate buffer and measure the
absorbance at 235 nm against blank.
5. Repeat the same procedure as mentioned above with different pH such as 1.2 HCL acid buffer, 7.4
phosphate buffer.
Observation table:

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Calibration curve:

Calculation:

Result:

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PRACTICAL-9 Date: / /202_

Aim: To perform stability testing of Paracetamol solution for photo degradation.

Reference: Welankiwar A, Saudagar S, Kumar J, Barabde A. Photostability testing of

pharmaceutical products. Int. Res. J. Pharm. 2013,2:11-5.

Requirement:
Apparatus: Beaker, funnel, measuring cylinder, pipette, glass rod, filter paper.
Chemicals:- Paracetamol, PEG 6000, Glycerin, Sucrose, Sweetener, Sodium Methyl paraben,
Sodium Propyl paraben, Sodium Benzoate, Citric acid monohydrate, Coloring agent, Flavoring
agent, water.

Theory:
Stability testing is an essential part of the drug development process, as it ensures that a drug
product maintains its safety, efficacy, and quality throughout its shelf life. Photo degradation is a
common concern during stability testing of drug products, particularly for those that are
susceptible to light-induced chemical reactions.
When conducting stability testing for photo degradation, it is essential to evaluate both solution and
solid dosage forms separately, as the degradation mechanisms and factors affecting each can
differ significantly. Here are some general considerations for stability testing of solution and
solid dosage forms for photo degradation:
Solution Dosage Forms:
Exposure Conditions: Testing should be conducted using accelerated stability testing conditions that
simulate the effect of light exposure on drug products. Conditions should include exposure to both
natural and artificial light sources (such as UV light), as well as varying temperatures and
humidity levels.
Sample Preparation: The drug product should be prepared according to the manufacturer's
instructions and protected from light during preparation.
Testing Methodology: The testing methodology should be appropriate for the specific drug
product, with appropriate analytical techniques used to detect and quantify any degradation
products formed.
Packaging Considerations: The stability of the drug product in its final packaging should also
be evaluated, as the packaging can provide additional protection against light-induced
degradation.
Overall, stability testing for photo degradation requires careful consideration of the specific
drug product, its formulation, and the testing methodology used. It is essential to follow industry
guidelines and regulations to ensure that the testing is conducted accurately and thoroughly.

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MATERIALS USED FOR THE PREPARATION

Ingredients Weight Function

Part I
Paracetamol 2.5g Active ingredient
Polyethylene glycol 6000 (PEG 6000) 10.0g Solubilizer
Glycerin 2.5g Diluent and sweetener
Water 30.0ml Diluent
Part II
Sucrose 30.0g Sweetening agent
Water 20.0g Diluent
Sweetener 0.220g Sweetener
Sodium Methyl paraben 0.150g Preservative
Sodium Propyl paraben 0.030g Preservative
Sodium Benzoate 0.150g Preservative
Citric acid monohydrate 0.070g pH modifier
Coloring agent 2.50mg Coloring agent
Flavoring agent 0.25ml Flavoring agent

Procedure of formulation of Paracetamol syrup :

Part I
1. Heat (PEG 6000) at 50°C and add Paracetamol in it. Stir the solution for 30 minutes.
2. Heat Glycerin at 50°C and then add in step 1 solution ((PEG 6000) + Paracetamol) under continues
stirring. Stir the solution for 20 minutes. Transparent solution will be obtained.
3. Heat water at 50°C and put it under continuous stirring.
4. Add step 1 solution ((PEG 6000)+ Paracetamol+Glycerin) slowly in to distilled water under continuous
stirring. Transparent solution will be obtained.

Part II
1. Weigh accurately sucrose. Add sucrose in hot (65°C) distilled water under continuous stirring till it
dissolved.
2. Filter above solution through filter press. Keep filtrate under stirring.
3. Add citric acid acid monohydrate, sodium methyl paraben, sodium propyl paraben, sweetener and
sodium benzoate in it with continuous stirring for 10 minutes.

Mixing of Part I and Part II

1. Slowly add part I in part II under continuous stirring. Stir it till clear solution is obtained.
2. Check pH above solution. If pH is not between 3.80-6.10 than add accordingly citric acid solution to
adjust Ph.
3. Add colour solution in above solution under stirring. Now, add flavor under continuous stirring.
4. Make volume 100 ml by distilled water if required.

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5. Clear transparent Paracetamol syrup is obtained.

Result:

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PRACTICAL-10 Date: / /202_

Aim: Stability studies of drugs in dosage forms at 25°C, 60% RH and 40°C, 75% RH.

Reference: González-González O, Ramirez IO, Ramirez BI, O’Connell P, Ballesteros MP, Torrado JJ,
Serrano DR. Drug stability: ich versus accelerated predictive stability studies. Pharmaceutics. 2022.

Requirements:
Apparatus: Beaker, funnel, measuring cylinder, pipette, glass rod , filter paper, pettri dish.
Chemicals: - Aspirin tablets.

Theory:
Stability studies of drugs in dosage forms are an important aspect of pharmaceutical
development and regulatory approval. These studies are designed to evaluate the effect of
various environmental factors, such as temperature and humidity, on the stability of drugs over
time.
In general, stability studies are conducted at three different temperatures and humidity
conditions, including 25°C/60% RH, 30°C/65% RH, and 40°C/75% RH. These conditions are
based on the International Conference on Harmonization (ICH) guidelines, which provide a
standardized approach to stability testing.
At 25°C/60% RH, stability studies are typically conducted over a period of 12 months. This
condition represents a moderate temperature and humidity environment that is commonly
encountered in the pharmaceutical industry. At this condition, the drug is exposed to a
temperature of 25°C and a relative humidity of 60%.
At 40°C/75% RH, stability studies are typically conducted over a period of 6 months. This
condition represents a high-temperature and high-humidity environment that may accelerate the
degradation of drugs. At this condition, the drug is exposed to a temperature of 40°C and a
relative humidity of 75%.
The purpose of stability studies at these conditions is to evaluate the effect of temperature and
humidity on the stability of drugs in dosage forms. The results of these studies are used to
establish the shelf-life of drugs and to determine appropriate storage conditions. In addition,
stability studies are also used to monitor the quality of drugs over time and to ensure that they
remain safe and effective for use by patients.
Procedure:
1. Perform calibration curve of aspirin pure drug.
2. Bring marketed tablets of aspirin and place the tablets –
a) Intact with package
b) Opened tablet from the package
c) Pure drug
3. Keep them in stability chamber for long term condition.
4. Another set must be kept at accelerated conditions.
5. Evaluate the tablets at 0,15 and 30 days.

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Result:

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PRACTICAL-11 Date: / /202_

Aim: Compatibility study need to be performed to access any possible integration drug and
excipients, before designing the dosage form.

For that the drug Diclofenac sodium is tested with for following excipients for possibility.
1) Diclofenac sodium + HPMC + Propylene Glycol

Put all these four samples in stability chamber for 1 month.

Evaluate this samples for FTIR and assay study to check purity of the drug at 0, 30 days.

Reference: Rojek B, Gazda M, Wesolowski M. Quantification of Compatibility Between

Polymeric Excipients and Atenolol Using Principal Component Analysis and Hierarchical
Cluster Analysis. AAPS Pharm SciTech. 2022 Jan;23:1-6.

Requirements:
Apparatus: FTIR, micro centrifuge tube
Chemicals: - Diclofenac sodium, HPMC, Propylene Glycol

Theory:
It is important to perform compatibility studies between drugs and excipients before
designinga dosage form to ensure that they are compatible and will not interact negatively
with each other. Compatibility studies can identify any potential physical, chemical, or
biologicalinteractions that may occur between the drug and excipients.
These studies can include tests such as Fourier-transform infrared spectroscopy (FTIR),
differential scanning calorimetry (DSC), X-ray diffraction (XRD), and high-performance liquid
chromatography (HPLC).
If incompatibilities are identified, appropriate measures can be taken to modify the formulation
or choose different excipients. This is critical to ensure the safety and efficacy of the final
product.

Procedure:
1. Sample Preparation:
- For solids, mix a small amount of the sample with a suitable matrix (e.g., KBr) and press the mixture
into a transparent disc. For liquids, place a drop of the sample on the ATR crystal.
2. Baseline Correction:
- Measure a baseline spectrum with an empty sample holder or the matrix alone to correct for
background interference.
3. Sample Measurement:
- Place the prepared sample in the FTIR spectrometer and ensure that it is properly aligned.
4. Instrument Setup:
- Choose the appropriate measurement parameters such as the scanning range, resolution, and
number of scans. Common scanning ranges are in the mid-infrared region (4000-400 cm⁻¹).

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5. Baseline Measurement:
- Measure the baseline again with the sample in place to account for any changes due to the sample.
6. Data Acquisition:
- Initiate the data acquisition process, and the instrument will collect the infrared spectrum of the
sample.
7. Analysis:
- Examine the obtained spectrum for peaks and troughs. Each peak corresponds to a specific
vibrational mode of the functional groups in the sample.
8. Interpretation:
- Compare the obtained spectrum with known spectra or databases to identify functional groups
present in the sample.
9. Cleaning:
- After analysis, clean the sample holder thoroughly to avoid contamination.

Result:-

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PRACTICAL-12 Date: / /202_

AIM:- To prepare and evaluate different polymeric membranes.

Reference:
Sonawane S, Thakur P, Sonawane SH, Bhanvase BA. Nanomaterials for membrane synthesis:
Introduction, mechanism, and challenges for wastewater treatment. In Handbook of Nanomaterials for
Wastewater Treatment 2021; 537-553.
Requirements:
Apparatus:-Beaker, Pipette, Glass rod.
Chemicals:-HPMC, Polyvinyl Alcohol, Chitosan, Sodium Alginate, Ethylcellulose, Water,
Ethanol, TWEEN 80.

Theory: Polymeric membranes play a crucial role in various fields, including chemistry, biology, and
industry, particularly in separation processes such as filtration and gas permeation. The theory of
polymeric membranes involves understanding the principles and mechanisms underlying their
structure, function, and performance. Here are key aspects of the theory of polymeric membranes:

1. Polymer Structure:
- Polymer Type: The choice of polymer significantly influences membrane properties. Common
polymers include polysulfone, polyethersulfone, polyvinylidene fluoride, and polyamide.
- Chain Architecture: The arrangement of polymer chains, whether linear or branched, affects
membrane permeability and selectivity.

2. Membrane Formation:
- Phase Inversion: Most polymeric membranes are prepared using a phase inversion process,
involving the dissolution of a polymer in a solvent followed by precipitation in a non-solvent.
- Membrane Morphology: The casting conditions and choice of solvents influence the membrane's
structure, including pore size, porosity, and thickness.

3. Membrane Transport Mechanisms:

- Solution-Diffusion Model: This model explains the permeation of gases and small molecules
through polymeric membranes. It involves the dissolution of the permeant into the polymer, diffusion
through the polymer matrix, and desorption on the other side.
- Knudsen Diffusion: For gas separation, especially in microporous membranes, the mean free path
of gas molecules becomes significant, and Knudsen diffusion plays a role.

4. Permeability and Selectivity:

- Permeability: It refers to the ease with which a substance can pass through the membrane. It
depends on factors like membrane thickness, polymer properties, and operating conditions.
- Selectivity: Membranes can selectively allow certain species to pass through based on factors like
size, shape, and chemical affinity.

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5. Membrane Characterization:
- Pore Size Distribution: Understanding the distribution of pore sizes is critical for predicting
membrane performance.
- Surface Morphology: Scanning electron microscopy (SEM) and atomic force microscopy (AFM)
are used to analyze the surface structure of membranes.

6. Transport Models:
- Fick's Law: Describes the diffusion of solutes through a membrane.
- Solution-Diffusion Model: Applied to gas permeation through polymeric membranes.

7. Applications:
- Reverse Osmosis (RO): Membranes selectively allow water molecules to pass through, removing
contaminants.
- Ultrafiltration (UF): Used for separation of macromolecules and colloids.
- Gas Separation: Membranes are employed for separating gases based on their molecular size and
affinity.
Method of preparation:-
Procedure:-
• Different Polymeric membranes composed of HPMC, Polyvinyl Alcohol, Chitosan,
Sodium Alginate, Ethylcellulose using water or ethanol were prepared by solvent casting
technique in a Petri dish.
• The bottom of Petridis lubricate by Tween 80.
• After mixing the above ingridients properly it was poured into the petri dish and an
inverted funnel was placed on the petri dish to facilitate the evaporation of solvent at a
controlled rate over the drying period of 24 hours at room temp.
• The dried polymeric membranes were removed and cut into 1 cm2 area and kept in
desiccators until used.

Evaluation parameter:
1. Thickness: It was measured at three different points using a screw gauge.
2. Percentage moisture content: The membranes were weighed individually and kept
in a desiccators containing activated silica at room temperature for 24 hours. Individual
membrane were weighed repeatedly until they showed a constant weight. The percentage
of moisture content was calculated as the difference between initial and final weight with
respect to final weight. % moisture content= (w2-w1/w2)*100.
3. Folding endurance: It was determined by repeatedly folding the membrane at the
same place untilit broke. The number of times of membrane could be folded at the same
place without breaking/cracking gave the value of folding endurance.
4. Weight variation: Five membranes were weighed individually and the average
weight was calculated.

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EVALUTION:
Thickness :1 mm
%Moisture content :
Initial weight of strip = __ mg
Final weight of strip = __ mg

% Moisture content = − − − − − × 100

= ___ _ %
Folding Endurance : 220 folds

Weight Variation
Table Weight variation of polymeric membranes

Sr. No. Weight of Patch (mg)

Average

RESULT:

Sr. No. Parameters Result

1 Thickness
2 % moisture content
3 Folding endurance
5 Weight variation

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EXPERIMENT NO: 13 Date:

Aim: Formulation and evaluation of sustained release matrix tablet of Aceclofenac.

Reference:-
A. Indian Pharmacopoeia 1996’, Vol-1 Govt. of India, Ministry of Health and
Familywelfare. Page no: 69
B. Brahmankar DM, Jaiswal SB Bio pharmaceutics and pharmacokinetics,. Page no:
216-219
Requirement

Apparatus:-Beaker, Pipette, Glass rod.

Chemicals:-Aceclofenac, HPMC K100M, Ethyl cellulose, Dicalcium stearate, MagnesiumStearate,
Talc, IPA.

Theory:-
Diffusion controlled systems also known as matrix systems are very popular forsustained
release formulations (Colombo et al. 2000). They can be divided up into different types of
mechanisms by which they prolong drug release, these includes reservoir matrix systems,
monolithic matrix systems and osmotic pumpsystems.

Reservoir matrix systems

This system involves a membrane which controls the release of drugs from the matrix
system. The drug will eventually diffuse through the membrane and its release is kept
constant by the diffusion distance that the drug particles have to cover (Fig. 1).

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Osmotic pump systems

Osmotic systems operate on osmotic pressure. They contain a core tablet that issurrounded by
a semi permeable membrane coating which has an orifice. The core tablet has two layers to it,
one containing the active ingredient/drug known as the active layer and the second containing
the osmotic agent which is also known as the push layer. Water enters the tablet through the
semi permeable membrane causing the drug to dissolve and suspend.

Monolithic matrix systems

These systems involve drug to be encapsulated or dispersed in a matrix. These systems can be
employed by forming hydrophobic matrices and/or hydrophilic matrices to allow for control
or prediction of drug release. They can be divided into soluble/hydrophilic matrix systems
which swell on hydration and dissolve to release drug and insoluble/hydrophobic matrix
systems which release drug after being dissolved by a solvent.

Polymers in sustained release formulations

Polymers are chains of covalently bound monomers. They are used throughout the
pharmaceutical industry and in relation to oral drug delivery systems they are used as carriers
for the drug. Polymers are used as a backbone in conventional and controlled release
formulations. For sustained release formulations the polymers need to have certain
characteristicsto control and maintain the rigidity of the matrix over a prolonged period (Kim
2000). There are a large number of polymers that are used in sustained release drug delivery.

Examples of a few polymers used in formulation of controlled release dosage forms

For the purpose of this study, the polymers that will be discussed are hydroxypropyl-
methylcellulose, sodium carboxymethylcellulose and sodium alginate.

Hydroxypropylmethyl cellulose (HPMC)

HPMC is a non-ionic derivative of cellulose ether (Kamel et al. 2008), it is stableover pH range
3.0-11. Hydroxypropylmethlcellulose (HPMC)is a semi-synthetic polymer. It is used as first
choice for the formulation of hydrophilic matrix systems as it provides a robust mechanism for
controlled release of drugs and choice of viscosity grades. Its nonionic nature minimizes
interaction problems when used in acidic, basic or electrolytic systems and provides
reproducible release profiles. It is also cost effective. Matrices containing HPMC are not
affected by the pH of fluid. It was found that the best grades to use for sustained release
formulations are K4M and K100M due to their high tensile strength. When hydrated the
polymer chains disentangle fromthe matrix.

Rationale behind the practical:-

Aceclofenac is a newer derivative of the diclofenac group of non-steroidal anti-inflammatory
drug (NSAID) that exhibits analgesic and anti-inflammatory activities. It directly blocks the
prostaglandin synthesis. It is considered to be thefirst-line drug in the symptomatic treatment
of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The aim of this work was to
prepare and evaluate the aceclofenac once daily sustained release tablets. It has less
gastrointestinal complications.In this study, SR aceclofenac tablet with differentproportion of
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Drug and polymers (HPMC, Ethyl Cellulose) were prepared by direct compression. They were
then studied with regard to their release behaviour. Acidic substances like aspirin, can cause
irritation to stomach wall hence HBS formulations are particularly useful in this case.

METHEDOLOGY:-

(1) Analytical Methods of Calibration curve of Acyclofenac at 256nm:-

Take 100mg of aceclofenac dissolved in 100ml phosphate buffer Ph 7.4 andmethanol
respectively (1000 g/ml).
Take 1ml of the above prepared solution , diluted to 10ml (100 g/ml)
Now pipette out 0.5, 1.0, 2.0, 3.0 and 4.0 ml from standard solution to produce
final concentration of 5 g/ml, 10 g/ml, 20 g/ml, 30 g/ml and 40 g/ml for
phosphate buffer. Now pipette out 0.1, 0.2, 0.3, 0.4 and 0.5 ml from standard
solution to produce final concentration of 1 g/ml, 2 g/ml, 3 g/ml, 4 g/ml and
5 g/ml for methanol.
Measure the absorbance at 256 nm using UV/Visible spectrophotometer and
plot the graph of concentration ( g/ml) versus absorbance.
CONCENTRATION ABSORBANCE (PHOSPHATE
SR. NO. (µg/ml) BUFFER pH 7.4)

1
2
3
4
5

TABLE 10.1: Calibration curve of aceclofenac in phosphate buffer 7.4

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CONCENTRATION ABSORBANCE
SR.NO. (g/ml) (METHANOL)
1
2
3
4
5

TABLE 10.2: Calibration curve of aceclofenac in methanol

(2) Method of preparation:

Procedure: -
 Prepare sustained release matrix tablets containing aceclofenac by directcompression
method.
 Pass all the excipient through sieve number 40.
 Weigh the required amount of all excipients and transfer them into mortar.
 Mix aceclofenc, HPMC, EC, dicalcium phosphate, magnesium stearate, and talcwith the
help of pestle for 10 minutes.
 Evaluate these lubricated granules for pre compression parameters and after that
compress into tablets at an average weight of 350mg by rotary tablet punchingmachine
with 9mm concave shape tablet punches.
 Preserve the compressed tablets in polyethylene bag with appropriate label till its
further use.
Formula:-

QTY/TAB
INGREDIENTS FOR 30 TABLETS (gm)
(mg)
Aceclofenac 100
HPMC k100 M 105
Ethyl cellulose 70
Dicalcium phosphate 68
Magnesium stearate 3.5
Talc 3.5
IPA q.s.
Total 350

TABLE 10.3: Formula of sustained release matrix tablet of aceclofenac

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- Evaluations parameter: -
Thickness & Diameter: -Thickness and diameter were measured byusing
Vernier callipers.
Friability: - Friability was measured by using Roche Friabilator.
Hardness: - The hardness of the floating tablets was measured using Monsanto
hardness tester.
Weight Variation: -Using electronic balance, weight variation test was done by
weighing 20 tablets individually using a digital balance.The average weight was
taken as the original weight.
Drug content estimation: -In vitro drug release studies from the prepared matrixtablets were
conducted on 1 tablet of each series by USP apparatus 2 at a speedof 50 rpm and a temperature
f 37 ± 0.5°C in 900ml distilled water as the dissolution medium. Samples were withdrawn after
1, 2, 3, 4, 5 and 6 hrs. The amount of Aceclofenac released then determined by UV
spectrophotometer at 256 nm in 0.1N HCl and in phosphate buffer 7.4, it is 259 nm. The release
in any time was obtained by calculating the mean cumulative% release. These test conditions
were according to test 2 in the monograph of extended-release tabletin USP 24.

Determination of Swelling Index

The swelling behavior of the dosage unit was measured by studying its weight gain. The
swelling index of tablets was determined by placing the tablets in Petridish contain distilled
water at 37 ± 0.5°C. After 0.5,1, 2, 3, 4, 5 and 6 hours eachtablets was taken out and blotted
with tissue paper to remove the excess of waterand weighed on the analytical balance. The
swelling index was calculated by using the following formula.

❖ % Swelling Index = × 100

In vitro dissolution studies: -The in vitro dissolution studies were carried out in0.1N HCl
using USP dissolution test apparatus employing paddle stirrer. One tablet was placed inside the
dissolution medium and the paddle was rotated at 75rpm. Around 5 ml samples were withdraw
at specific time intervals and the samevolume was replaced to maintain sink conditions. The
sample was analyzed fordrug content spectrophotometer at 278 nm.

Results: -

(1) Thickness: -
Sr.No Thickness (mm)
1.
2.
3.
Average

TABLE 10.4: Thickness Study

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(2) Diameter:-

SR.NO:- Diameter (mm)

1.
2.
3.
Average

TABLE 10.5: Diameter study

(3) Hardness:-
SR.NO:- Hardness (kg/cm2)
1.
2.
3.
Average
TABLE 10.6: Hardness study
(4) Weight variation:-
SR.NO:- Weight (mg)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Avg

TABLE 10.7: Weight variation study

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Here ± 5% variation is allowed according to Indian

Pharmacopeia

Hence limit is ± 5% = mg

(5) Friability Test:-

Initial weight of 10 tablet, W1= gm
Final weight of 10 tablet, W2 = gm
% Friability =

(6) Drug
content:

Absorbance

(Y) =
Y =
X=
X= * 10 (Dilution Factor)
Drug Content = mg
% Drug Content = * 100 /

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(7) Dissolution study: -

%Cummu

Release
lative
Drug
Cumm
ulative

Releas
Drug
Concent

mg/900
ration

ml)
Concent
ration
mg/5
ml)
µg/5 ml)
Concent
ration
Concent

µg/ml)
ration
=10
DF
Concentr

µg/ml)
ation
Absorbanc
ee
Time
no
Sr

TABLE 10.8: Dissolution study of sustained release matrix tablet of aceclofenac

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%Cummu

Release
lative
Drug
Cumm
ulative

Releas
Drug
Concent

mg/900
ration

ml)
Concent
ration
mg/5
ml)
µg/5 ml)
Concent
ration
Concent

µg/ml)
ration =10
Concentr DF

µg/ml)
ation
Absorbanc
ee
Time
no
Sr

TABLE 10.8.2: in vitro dissolution studies for Aceclofenac in 7.4 Phosphate Buffer

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RESULT:-

Sr. No Parameters Result

1. Colour Brown
2. Texture Smooth
3. Weight variation
4. Friability
5. Hardness kg/ cm2
6. Thickness mm
7. Diameter mm
8. Drug content

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Parul Institute of Pharmacy & Research, M. Pharm- Pharmaceutics Semester I Page No.
 INDUSTRIAL PHARMACY - 1(MIP105P)

PRACTICAL-14 Date: / /202_

Aim: Formulation and evaluation of microspheres/ microcapsules.

Reference: Belwal SD, Joshi D, Rautela A, Kumar P. Formulation and evaluation of Microsphere of
Aceclofenac. Journal of Advancement in Pharmacology. 2020;1(1):65-70.

Requirement:
Apparatus: -Beaker, funnel, measuring cylinder, pipette, glass rod, filter paper, mechanical stirrer.
Chemicals:- Diclofenac sodium, ethyl cellulose, dichloromethane, methanol, PVP K30, water.

Theory:
Microspheres and microcapsules are tiny particles that are designed to encapsulate drugs or
other bioactive compounds. They can be used to improve drug delivery and reduce side
effectsby targeting specific tissues or organs.
Formulation of Microspheres/Microcapsules:
Selection of polymer: The choice of polymer is critical in microsphere/microcapsule
formulation as it determines the release profile and biocompatibility of the final product.
Common polymers used include alginate, chitosan, gelatin, and poly(lactic-co-glycolic acid)
(PLGA).
Preparation of polymer solution: The polymer is dissolved in a suitable solvent to form
asolution. The solvent must be compatible with the polymer and should evaporate quickly.
Addition of drug: The drug is added to the polymer solution and mixed thoroughly to ensure
homogeneity.
Emulsification: The polymer solution containing the drug is emulsified to form droplets. This
can be achieved by mechanical agitation or sonication.
Solidification: The droplets are solidified to form microspheres/microcapsules. This can be
achieved by various methods such as chemical crosslinking, coacervation, or solvent
evaporation.
Evaluation of Microspheres/Microcapsules:
Particle size and size distribution: The size and size distribution of microspheres/microcapsules
are critical as they can affect the release profile and efficacy of the final product. Particle
size can be determined using various techniques such as laser diffraction, dynamic light
scattering,and scanning electron microscopy.
Encapsulation efficiency: Encapsulation efficiency refers to the amount of drug
encapsulated within the microsphere/microcapsule. It can be determined by separating the
drug-loaded microspheres/microcapsules from the unencapsulated drug and measuring the
amount of drugin each fraction.
Drug release profile: The release profile of the drug from the microsphere/microcapsule can
be determined using various techniques such as UV spectrophotometry or HPLC. The
release profile is affected by various factors such as polymer type, particle size, and drug
solubility.

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Biocompatibility: The biocompatibility of the microspheres/microcapsules is critical as it

determines their safety and efficacy. Biocompatibility can be determined using various in vitro
and in vivo assays such as cell viability assays, hemocompatibility assays, and histological
analysis.
Overall, the formulation and evaluation of microspheres/microcapsules require a thorough
understanding of the physicochemical properties of the polymer, drug, and solvent, as well
asthe appropriate analytical techniques for characterization.

Procedure: -
1. Weigh the diclofenac sodium 2gm and ethyl cellulose 2gm. Dissolve diclofenac sodium in a
mixture of methanol and dichloromethane.
2. Add ethyl cellulose to the diclofenac sodium solution while stirring continuously with a magnetic
stirrer at 500 rpm. Continue stirring the solution until ethyl cellulose is completely dissolved.
3. Prepare the continuous phase by dissolving PVP K30 5% in 200 ml distilled water using a
mechanical stirrer. Once the PVP K30 solution is prepared, adjust the stirring speed to ensure thorough
mixing and homogenization.
4. Once the both phases prepared, we use injection method. Use a syringe to inject the diclofenac
sodium/ethyl cellulose solution into the continuous phase of PVP K30 solution dropwise. During
injection, maintain stirring of the continuous phase to promote uniform distribution.
5. After complete injection, continue stirring the mixture allow for solvent evaporation and
solidification of the microspheres & stop the stirring and allow the microspheres to settle.
6. Collect the microspheres by filter by using Whatman filter paper & allot to dry the microspheres
under hot air oven.

Observation Table:-

Result:

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 INDUSTRIAL PHARMACY - 1(MIP105P)

PRACTICAL-15 Date: / /202_

AIM: - Formulation and Evaluation of Transdermal patch of Diclofenac Sodium.

Reference: Kriplani P, Sharma A, Aman PP, Chopra B, Dhingra A, Deswal G. Formulation and
evaluation of transdermal patch of diclofenac sodium. Global Journal of Pharmacy and
Pharmaceutical Sciences. 2018;4(5):001-4.

Requirements: -
Apparatus: -Beaker, Pipette, Glass rod, magnetic stirrer, magnetic bead, pettri dish, funnel.
Chemicals:-Diclofenac Sodium, Hydroxypropyl Methylcellulose, Propylene Glycol,
Tween 80, alcohol and water.

THEORY: • Transdermal therapeutic systems are defined as self-contained, discrete

dosage forms when applied to the intact skin, deliver the drug through the skin at a
controlled rate tothe systemic circulation.

• One of the approaches of Transdermal therapeutic systems is the maintenance of the

blood concentration of drug at therapeutic level by means of controlled permeation
throughout the skin during a long period of time and using only one administration.

• There is an increasing recognition that the skin can also serve as the port of administration for
systemically active drugs.

• In this case, the drug applied topically will be absorbed first into the blood circulation
and then be transported to target tissue, which could be rather remote from the site of drug
application, to achieve is its therapeutics purposes.

• The advantages of delivering drugs across the skin for systemic therapy are well documented.

• Some of the main advantages of Transdermal drug delivery system are to deliver steady
infusion of drug over an extended period of time, to increase the therapeutic value of many
drugs by avoiding specific problems associated with the drug e.g. GI irritation, low absorption,
decomposition due to hepatic first pass effect, formation of metabolites that cause side
effects,short half-life necessitating frequent dosing etc. the drug input can be terminated at
any point of time by removing Transdermal patch because selfadministration is possible
with these system.

Estimation of Diclofenac Sodium:

• Accurately weighed 10 mg Diclofenac Sodium was dissolved in 100ml Methanol to produce

concentration of 1000 g/ml.
• 10 ml of above solution was withdrawn and diluted to 100 ml to produce concentration
of 100 g/ml.

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• Then 0.2, 0.4, 0.6, 0.8, 1, 1.2 ml of aliquots was withdrawn and diluted up to 10ml and
theabsorbance was measured at 266 nm.
• Calibration curve shown in table.
• Calibration Curve of Diclofenac Sodium

Sr. No. Concentration (µg/ml) Absorbance

• Calibration Curve of Diclofenac Sodium at 266nm

• Ingredients for preparation of Transdermal Patch of Diclofenac Sodium

Sr. no Ingredients Quantity Use

1 Diclofenac 10 mg API
Sodium
2 HPMC K4 M 300 mg Polymer
3 Propylene glycol 3 % w/w Plasticizer
4 Water 20 ml Solvent

Method of preparation: -
Procedure:-
• Matrix type Transdermal Patches composed of HPMC K4M, Propylene glycol, water and
drug was prepared by solvent casting technique in a Petri dish.
• The bottom of Petridis lubricates by Tween 80.
• A fixed volume of drug dissolved in 10 ml Alcohol. (sol A)
• HPMC K4 M dissolved in 10 ml of water via magnetic stirrer. (sol B)
• Above sol A and sol B mixed via magnetic stirrer and then add propylene glycol .mix it
properly and poured it into the petri dish and an inverted funnel was placed on the petri
dish to facilitate the evaporation of solvent at a controlled rate over the drying period of
24 hours at room temp.
• The dried patch was removed and cut into 1.00cm2 area and kept in desiccators until used.

Evaluation parameter:
1. Thickness: It was measured at three different points using a screw gauge.
2. Weight variation: Five films from each batch were weight individually and the average
weight was calculated.Percentage moisture content: The films were weighed individually
and kept in a desiccators containing activated silica at room temperature for 24 hours.
Individual films were weighed repeatedly until they showed a constant weight. The

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percentage of moisture content was calculated as the difference between initial and final
weight with respect to final weight. % moisture content= (w2-w1/w2)*100.
3. Folding endurance: It was determined by repeatedly folding the film at the same place
until it broke. The number of times of film could be folded at the same place without
breaking/cracking gave the value of folding endurance.
4. Drug content: A film of 1.00cm2 area was cut and taken in a 100ml volumetric flask and
dissolved in 25 ml methanol and make up volume up to 100ml with methanol. The solution was
filtered and drug was determined spectroscopically at λmax266 nm after suitable dilution.
5. In vitro drug release studies: A modified Franz diffusion cell was used to study the in
vitro release profile as well as the permeation of Diclofenac from patch. For this study the
patches were stuck to an aluminum foil which was slightly larger than patch fixed using
water impermeable adhesive to ensure that the receptor fluid could not come in contact with
the sides of the film. The faces with lower drug concentration were placed in contact with
the receptor fluid methanol for maintaining sink condition. Before placing the patch fixed on
aluminum foil on to the diffusion cell, the mouth of the cell was coated with a thin layer of
silicon grease to prevent leakage of the receptor fluid. 1 ml of the receptor fluid was withdrawn
at an interval of 1h. It was immediately replaced with 1ml of fresh drug free methanol solution
maintain constant volume. The removed fluid, after suitable dilution with methanol was
analyzed spectrophotometrically at λmax 266nm and concentration was observed from the
calibration curve.

EVALUTION:
Thickness :1 mm
%Moisture content:
Initial weight of strip = ____ mg
Final weight of strip = ____mg

% Moisture content = − − − − − × 100

= ___ _ %
Folding Endurance : 220 folds
➢ Drug Content :Total wt. of patch = mg
Wt. of 1cm2 piece of patch = ____ mg
Absorbance = ____ ___ Y = m
x + c y = _____ x + _______
_______=_____x + ______ _
X = _____ µg/ml
Drug content = X × dilution factor
= ___ __ × 100
= ___ _ µg / 1000
= __ ____ mg

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Thus,
10 mg patch contain __ mg drug
So,_____ mg patch contain= = ____ mg
____ mg patch contains ____mg of drug
% drug content = =______ %
Weight Variation
Sr. No. Weight of Patch (mg)

Average
Table Weight variation of patch
In-vitro Diffusion study Table in-vitro diffusion study of Transdermal Patch of Diclofenac
Sodium

Time Absorbance Con.(µg/ml) DF Con.(__µg/ml) Con.(___µg/ml) (c) %

(min) (a) (a×100) (b) CDR CDR
(b+c)

RESULT:
Sr. No. Parameters Result
1 Thickness
2 % moisture content
3 Folding endurance
4 Drug Content
5 Weight variation
6 Dissolution study

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 INDUSTRIAL PHARMACY - 1(MIP105P)

PRACTICAL-16 Date: / /202_

Aim: Design and evaluation of a) face wash b) creams c) lotions d) shampoo e) toothpaste
and f) lipstick.

Reference: a) Jaseer JJ, Ajay M, Jasmal M, George N, Vaishna V. Formulation and Evaluation of
Liquid Based Face Wash. International Journal of Pharmaceutical Research and Applications 2022, pp:
1533-1547
b) Chauhan L, Gupta S. Creams: A review on classification, preparation methods, evaluation and its
applications. Journal of drug delivery and therapeutics. 2020 ;10(5-s):281-9.
c) Saptarini NM, Hadisoebroto G. Formulation and evaluation of lotion and cream of nanosized
chitosan-mangosteen (Garcinia mangostana L.) pericarp extract. Rasayan J. Chem. 2020,:789-95.
d) Reddy VS. Formulation and evaluation of synthetic anti-dandruff shampoo. Asian Journal of
Pharmaceutics (AJP). 2018.
e) Vranic E, Lacevic A, Mehmedagic A, Uzunovic A. Formulation ingredients for toothpastes and
mouthwashes. Bosnian journal of basic medical sciences. 2004;4(4):51.
f) Bono A, Mun HC, Rajin M. Effect of various formulation on viscosity and melting point of natural
ingredient based lipstick. InStudies in surface science and catalysis 2006 (Vol. 159, pp. 693-696).

Requirements:
Apparatus:-Beaker , funnel, measuring cylinder , pipette, glass rod , filter paper, mortar pestle,
hot water bath.
Chemicals: Propylene glycol, Ka Hydroxide, SLS, Sorbitol, Methylparaben, Charcoal, White
bee wax, Liquid paraffin, Borax, Rose oil, Stearic acid, Calcium Carbonate, Mg Carbonate, Mg
Hydroxide, Cream tragacanth, Glycerin, Peppermint oil, Saccharine, Cetylalcohol.

Theory:
Design and evaluation of personal care products such as face wash, body wash, creams, lotions,
shampoo, toothpaste, and lipstick involve a thorough understanding of the ingredients, their
functionality, and the user’s expectations. In this response, I will outline the general steps
involved in designing and evaluating each of these personal care products.

Face Wash
Design: The design of a face wash involves selecting the active and inactive ingredients,
determining their concentrations, and creating a formula that cleanses the skin effectively while
also being gentle and non-irritating. Common active ingredients in face washes include salicylic
acid, benzoyl peroxide, and glycolic acid, while inactive ingredients can include
surfactants, emollients, and humectants.

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MATERIALS USED FOR THE PREPARATION OF FACE WASH

Ingredients Uses of ingredients Quantity
Stearic acid Emulsifying agent 12 g
Sodium phosphate Emulsifying agent 3g
Liquid paraffin (light) Emollient 3 ml
Propyl paraben Preservative 0.1 g
Propylene glycol Humectant 6.5 ml
Potassium hydroxide Emulsifier 0.06 g
Sodium lauryl Conditioning agent 15 g
sulphate
Sorbitol Moisturing agent 5g
Methyl paraben Preservative 0.1 g
Water Vehicle 55 ml
Charcoal Skin whitening agent 2g
Peppermint oil Fragrance q.s

METHOD OF PREPARATION
Liquid based facewash was prepared by the following ways:
A. Preparation of Mixture A
• Take a clean and dried mortar and pestle
• Add stearic acid and sodium phosphate in to the mortar and pestle and triturate properly
• To this add gram of propyl paraben and continue trituration.
B. Preparation of Mixture B
• Take another clean and dried mortar and pestle and add propylene glycol, potassium
hydroxide and triturate
• sodium lauryl sulphate, sorbitol, methyl paraben was added to the above mixture and
triturate well.
• Mixture A and Mixture B was mixed together and triturate until a proper consistency is
obtained
• To this add of water and charcoal and mix well.
• To this perfume was added. Properly mix all the above ingredients.
Evaluation: The evaluation of a face wash typically involves testing its cleansing efficacy,
irritation potential, and sensory attributes such as scent and texture. Consumer panels and
clinical studies can be used to evaluate a face wash’s performance and user acceptance.
Creams and Lotions
Design: The design of creams and lotions involves selecting the active and inactive ingredients,
determining their concentrations, and creating a formula that provides the desired benefits,
such as hydration, skin barrier protection, or anti-aging properties. Common active
ingredients include retinol, vitamin C, hyaluronic acid, and peptides, while inactive

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ingredients include emollients, humectants, and thickeners.

MATERIALS USED FOR THE PREPARATION OF COLD CREAM
Ingredients Quantity
White Bee’s Wax 4.4 g
Liquid Paraffin 13.2 g
Borax 0.22 g
Rose oil 0.044 ml
Rose Water 4.4 ml
METHOD OF PREPARATION
Formulation can be prepared by adding two different phases which are as follows.
Phase 1: Melt the solid ingredients by indirect heat then add all the oils in it and stir well.
Phase 2: Dissolve the borax in water with the help of heat.
While still hot add the phase 1 into the phase 2 gradually with constant stirring to the wax and
oil mixture. Continue this process for 5 minutes, stir all the time then remove from the heat and
stir until it gets cold.
MATERIALS USED FOR THE PREPARATION OF CALAMINE LOTIONS
Ingredients Quantity
Calamine powder 33.75 gm
Zinc oxide 10 gm
Bentonite 10 gm
Sodium citrate 1.25 gm
Liquid phenol 1.25 gm
Glycerin 1.25 gm
Rosewater 1ml
Water Q.S. to 250
ml
METHOD OF PREPARATION
I. All glassware was washed and dried.
II. Required quantity of chemicals were taken and weighed.
III. Weigh and mix the calamine, zinc oxide, and bentonite in a mortar so that the bentonite
is well distributed.
IV. Dissolve sodium citrate in 700 ml rosewater, and gradually add to the mixture in the
mortar so that a smooth paste is produced.
V. Add the liquefied phenol and glycerin and mix well.
VI. Add sufficient rosewater to produce the required volume.

Evaluation: The evaluation of creams and lotions includes assessing their moisturizing

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potential, skin feel, and user acceptance. Consumer panels and clinical studies can be used
to evaluate a cream or lotion’s performance and user acceptance.
Shampoo
Design: The design of a shampoo involves selecting the active and inactive ingredients,
determining their concentrations, and creating a formula that effectively cleanses the scalp
and hair while also being gentle and non-irritating. Common active ingredients include
surfactants, such as sodium lauryl sulfate, and conditioners, such as silicones and proteins,
while inactive ingredients include thickeners and preservatives.
MATERIALS USED FOR THE PREPARATION OF LIQUID SHAMPOO
Ingredients Quantity
SLS 40%
NaCl 2‐4%
Water Up to 100%
Rose oil q.s
Amranth solution q.s
Parabens q.s
METHOD OF PREPARATION
Evaluation: The evaluation of a shampoo includes assessing its cleansing efficacy,
conditioning potential, and user acceptance. Consumer panels and clinical studies can be
used to evaluate ashampoo’s performance and user acceptance.
Toothpaste
Design: The design of a toothpaste involves selecting the active and inactive ingredients,
determining their concentrations, and creating a formula that effectively cleans and protects
teeth and gums. Common active ingredients include fluoride, which helps prevent tooth decay,
and baking soda, which helps remove stains, while inactive ingredients include abrasives,
thickeners, and flavorings.
MATERIALS USED FOR THE PREPARATION OF TOOTHPASTE
Ingredients (gm) Quantity Role
(W/W) %
Guar gum 0.5 Laxetive
Clove oil 0.2 Antibacterial
Sodium chloride 2 Anti Cavities
Calcium carbonate 50 Abrasive
Methyl Paraben 0.2 Preservative
Menthol 0.1 Cooling agent
Titanium dioxide 0.5 Whitening agent
Sodium Lauryl sulphate 2.5 Detergent
Glycerine 30 Humectant
Amranth solution 0.1 Colouring agent
Water q.s 100 Vehicle
METHOD OF PREPARATION

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Trituration method:
The binder is premixed with solid abrasive and triturate, which is then mixed with the liquid phase
containing humectants, and oils, Then add preservatives and sweetener into a mixer. After the
formation of a homogeneous paste, the flavor and the detergent added last under slow speed agitation
to minimize foaming, mixed, milled deairated and tubed.

Evaluation: The evaluation of a toothpaste includes assessing its cleaning and protective
efficacy, taste, and user acceptance. Consumer panels and clinical studies can be used to
evaluate a toothpaste’s performance and user acceptance.
Lipstick
Design: The design of a lipstick involves selecting the active and inactive ingredients,
determining their concentrations, and creating a formula that provides the desired color, texture,
and wear time. Common active ingredients include pigments and waxes, while inactive
ingredients include emollients and thickeners.
MATERIALS USED FOR THE PREPARATION OF LIPSTICK
Ingredients Quantity
Lanolin alcohol 9.2%
Castor Oil 25-30%
Dyes 0.5-2%
Silica 1-5%
Wax 20-25%
Glycerine 5-10%
Antioxidant 0.5-2%
Rose oil 0.1-3%

METHOD OF PREPARATION
1. All ingredients were weighted properly according to formula.
2. We used “White Bee’s wax” as wax.
3. White Bee’s wax and glycerin were weighted first and heated at 80° - 85° C.
4. Castor oil is then added to the heated mixture.
5. Mixture was stirred well at 100 rpm. It was done manually.
6. Then rest of the ingredients were added to the homogenous mixture.
7. Colorant was added at 65° - 75° C.
8. All ingredients were mixed in a mortar and pestle. This process lasts for 45-50 min.
9. After the mixture is prepared, it is cooled and fragrance is added in cooled mixture.
10. We prepared mold by passing the mixture in a test tube.
11. After cooling the semisolids mass solidifies in a mould and lipstic is prepared.
12. The sticks may be ‘flamed’ to produce a glossy finish to the surface.

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Observation:

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Result:

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PRACTICAL-17 Date: / /202_

Aim: Demonstration of Gel electrophoresis.

Reference:
Tan TT, Tan ZY, Tan WL, Lee PF. Gel electrophoresis: DNA science without the DNA!.
Biochemistry and Molecular Biology Education. 2007;35(5):342-9.
Gel Electrophoresis

Theory:
⚫ Gel electrophoresis is simple, rapid and sensitive analytical technique for the separation of
charged particle.
⚫ Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules,
such as RNA and proteins) based on their size and charge.
⚫ Electrophoresis involves running a current through a gel containing the molecules of interest.
Based on their size and charge, the molecules will travel through the gel in different directions or at
different speeds, allowing them to be separated from one another.
⚫ Gel electrophoresis, as a tool to separate DNA frag-ments generated by various analytical
methods in molecular biology, was developed in the 1960s and 1970s.
⚫ Starch, agar, or polyacrylamide were originally used asthe gel matrix. In 1973, Joseph Sambrook
and col-leagues at the Cold Spring Harbor Laboratory pioneeredthe use of agarose, a highly purified
polysaccharideextracted from seaweed.
⚫ The gels, however, are porous and the size of the pores relative to that of the molecule determines
whether the molecule will enter the pore and be retarded or will bypass it.
⚫ The separation thus not only depends on the charge on the molecule but also on its size. Needless
to say, that resolution of a sample is sharper and better in a gel than in any other type of medium.
⚫ Agarose gel is used as a supporting media for the separation of DNA, RNA or protein under the
influence of electric charge.
⚫ Most of the biomolecules has a net charge at any pH other than at their isoelectric point.
⚫ There is difference in the electrophoretic mobility of these charged molecules due to their
difference in size, shape, and charge.
⚫ The gels, however, are porous and the size of the pores relative to that of the molecule determines
whether the molecule will enter the pore and be retarded or will bypass it.
⚫ The separation thus not only depends on the charge on the molecule but also on its size. Needless
to say, that resolution of a sample is sharper and better in a gel than in any other type of medium.
⚫ Agrose gel is used as a supporting media for the separation of DNA, RNA or protein under the
influence of electric charge.
⚫ Most of the biomolecules has a net charge at any pH other than at their isoelectric point.
⚫ Gel electrophoresis involves an electrical field; in particular, this field is applied such that one
end of the gel has a positive charge and the other end has a negative charge.
⚫ Because DNA and RNA are negatively charged molecules, they will be pulled toward the
positively charged end of the gel. Proteins, however, are not negatively charged; thus, when
researchers want to separate proteins using gel electrophoresis, they must first mix the proteins with a
detergent called sodium dodecyl sulfate.
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This treatment makes the proteins unfold into a linear shape and coats them with a negative
⚫
charge, which allows them to migrate toward the positive end of the gel and be separated.
⚫ Finally, after the DNA, RNA, or protein molecules have been separated using gel electrophoresis,
bands representing molecules of different sizes can be detected.

Materials are used

1. Agarose
2. Polyacrylamide
3. Electrolytes
4. Gel
5. Starch
6. Capillary electrophoresis
7. Agarose Gel.
AGAROSE
❖ Agarose is a natural colloid which is isolated from the seaweed.
❖ It is linear polysaccharide.
❖ It is made up of repeating units of agarobiose, comprises alternating units of 3,6-anhydrolactose
and galactose.
⚫ This gel has generally larger pore size, which makes them suitable to separate larger molecules
having molecular mass more than 200 kDa.
⚫ It is most commonly used for the electrophoresis of both protein and nucleic acids.
⚫ Agarose is used in concentration between 1% and 3%.

⚫ POLYACRYLAMIDE GEL
❖ Polyacrylamide gel is consisting of chains of acrylamide monomers cross-linked with N, N’-
methylenebisacrylamide units, which is commonly termed as bisacrylamide.
❖ In this gel, pore size and resolving power is totally depends upon the concentration of acrylamide
and bisacrylamide.
❖ The concentration of the gel normally varies from 5% to 25%.
❖ This gel is used in electrophoresis for the separation of proteins ranging from molecular weight
<5000 to >200,000, and polynucleotides ranges from <5 to ~ 3000 base pairs in size.
Apparatus of Gel Electrophoresis
I.Horizontal gel apparatus:
It is used for immune electrophoresis, iso-electric focusing and electrophoresis of DNA and RNA in
the agarose gel.
II.Vertical gel apparatus:
It is commonly used IN SDS PAGE for the separation of proteins.
Types of Gel Electrophoresis
a. Agarose gel electrophoresis
b.SDS-PAGE
c. Pulse field gel electrophoresis (PFGE)
d.2D gel electrophoresis

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Advantages
I.The separation is more efficient than paper type as the rate of running of molecules is slow and area
of separation is larger by thickness.
II.The sample is applied and subjected to electric field which can ncaa lead to to separation of
molecules. These molecules form bands and can be be rece recognized by staining and comparing
with standard sample bands
III.The method is more effective than paper and for instance from serum sample, proteins bands can be
isolated.
Disadvantages
a. The technique requires more separation time.
b. It is costly than paper electrophoresis.
c. Use of high voltage may be dangerous so proper precautions should be taken.
Methods
A. Casting of the gel
❖ The acrylamide solution (a mixture of monomeric acrylamide and a bifunctional cross linker bis-
acrylamide) is mixed with the TEMED and APS and poured in between the glass plate fitted into
the gel caster.
❖ What is the mechanism of acrylamide polymerization? Ammonium persulfate in the presence of
TEMED forms oxygen free radicals and induces the polymerization of acrylamide monomer to
form a linear polymer.
❖ These linear monomers are interconnected by the cross linking with bis-acrylamide monomer to
form a 3-D mesh with pores.
❖ The size of pore is controlled by the concentration of acrylamide and amount of bis-acrylamide in
the gel. In a vertical gel electrophoresis system, we cast two types of gels, stacking gel and resolving
gel.
❖ First the resolving gel solution is prepared and poured into the gel cassette for polymerization. A
thin layer of organic solvent (such as butanol or isopropanol) is layered to stop the entry of oxygen
(oxygen neutralizes the free radical and slow down the polymerization) and make the top layer
smooth. After polymerization of the resolving gel, a stacking gel is poured and comb is fitted into
the gel for construction of different lanes for the samples. Different steps involves the vertical gel
electrophoresis is shown in the below Fig.

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Running of the gel

❖ The sample is prepared in the loading dye containing SDS, β-mercaptoethanol in glycerol to
denature the sample and presence of glycerol facilitates the loading of sample in the well.
❖ As the samples are filled vertically there is a distance drift between the molecules at the top 31 Vs
at the bottom in a lane.
❖ This problem is taken care once the samples run through the stacking gel.
❖ The pH of the stacking gel is 6.8 and at this pH, glycine is moving slowly in the front whereas Tris-
HCl is moving fast.
❖ As a result, the sample gets sandwiched between glycine-Tris and get stacked in the form of thin
band.
❖ As the sample enters into the resolving gel with a pH 8.8, the glycine is now charged, it moves fast
and now sample runs as per their molecular weight (due to SDS they have equal negative charge).
❖ After tracking dye reaches to the bottom of the gel, gel is taken out from the glass plate with the
help of a spatula.
❖ Gel is stained with Coomassie brilliant blue R250 dye.
Buffer and reagent for Gel electrophoresis
• The different buffer and reagents with their purpose for gel electrophoresis is as follows
1. N, N, N', N'-tetramethylethylenediamine (TEMED)-it catalyzes the acrylamide polymerization.
2. Ammonium Persulfate (APS)-it is an initiator for the acrylamide polymerization.
3. Tris-HCl- it is the component of running and gel casting buffer.
4. Glycine-it is the component of running buffer
5. Bromophenol blue- it is the tracking dye to monitor the progress of gel electrophoresis.
6. Coomassie brilliant blue R250-it is used to stain the polyacrylamide gel.
7. Sodium dodecyl sulphate-it is used to denature and provide negative charge to the protein.
8. Acrylamide- monomeric unit used to prepare the gel.
9. Bis-acrylamide- cross linker for polymerization of acrylamide monomer to form gel.
Application:
❖ Estimation of molecular weight of proteins and nucleic acids.
❖ Determination of subunit structure of proteins.
❖ Purification of isolated proteins.
❖ Monitoring changes of protein content in body fluids
RESULT:

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PRACTICAL-18 Date: / /202_

AIM: - Preparation & evaluation of liposome delivery system.

Reference: - Novel methods for liposome preparation, Chemistry and Physics of

Lipids,Volume 177,2014,Pages 8-18,ISSN 0009-3084,
Requirements:-
Chemical:-Paracetamol, Tween 80, Ethanol, water.
Apparatus:- Beaker, Pipette, Glass rod, Spatula, Butter Paper, Injection, magnetic stirrer,
magnetic bead.

Theory:- Liposomes are spherical vesicles made of phospholipids that are commonly used in
drug delivery systems. They can encapsulate a variety of compounds, such as drugs, peptides,
andnucleic acids, and protect them from degradation and clearance by the immune system. Here
are some steps to prepare and evaluate a liposome delivery system:
Preparation: Choose the appropriate lipids: The selection of lipids is crucial as it affects
the stability, size, and charge of the liposomes. Commonly used lipids include
phosphatidylcholine, cholesterol, and phosphatidylglycerol.
Determine the desired size and charge: The size and charge of liposomes can be adjusted
by varying the lipid composition, sonication conditions, and extrusion methods.
Encapsulate the cargo: The cargo can be encapsulated within the liposomes by several
methods, such as passive loading, active loading, or remote loading.
Purify the liposomes: The liposomes can be purified by several methods, such as
ultracentrifugation, gel filtration, or dialysis.
Evaluation: Size and distribution: The size and size distribution of the liposomes can be
measured by several methods, such as dynamic light scattering or transmission electron
microscopy.
Stability: The stability of the liposomes can be evaluated by monitoring changes in size,
aggregation, and leakage of the encapsulated cargo over time.
Cargo release: The release of the encapsulated cargo from the liposomes can be assessed
by various methods, such as dialysis, gel filtration, or centrifugation.
In vitro and in vivo efficacy: The efficacy of the liposome delivery system can be
evaluated invitro using cell-based assays or in vivo using animal models. The amount of cargo
delivered to the target site, as well as the safety and toxicity of the liposomes, should also
be evaluated.
Overall, preparation and evaluation of a liposome delivery system require careful selection
of lipids and encapsulation methods, as well as rigorous characterization of the physical
and biological properties of the liposomes.

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Procedure:
1. In 10 ml ethanol 400mg of lecithin soya was dissolved and mixed using a magnetic
stirrer.
2. In the above solution 50mg of PCM powder was added to the above solution and loaded
into an injection.
3. The ethanolic solution was then injected in 10ml of water in which 2% tween 80
surfactant was added and heated up to 70°C.
4. The injection rate was fixed and the liposomes were formed spontaneously with
continuous stirring at 500 rpm.

Observation Table:-

RESULT:

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