Bio Pharmaceutics

LAB MANUAL
Pharmaceutics VIII
(BIOPHARMACEUTICS)
(PHARM-472P)
B. Pharm. 4th Year

(VII Semester)
KUNWAR HARIBANSH SINGH COLLEGE OF PHARMACY

JAUNPUR (U.P.)
(College Code-453)
(Affiliated to GBTU, Lucknow)
LIST OF EXPERIMENTS
Pharmaceutics VIII (Biopharmaceutics) (PHARM 472P)
S.NO. EXPERIMENT
1. To establish a standard curve of salicylic acid in distilled water.
To study the effect of dilution and wetting agent on in-vitro release water soluble drug
2.
(Sodium Salicylate) from capsule dosages form.
To study the effect of diluents and wetting agents on is vitro release of water insoluble
3.
drug from a capsule dosage form.
4. To study the dissolution time profiles of Paracetamol tablet I.P.
5. To perform in-vitro bioequivalence of two marketed Paracetamol tablets.
To study the extent of protein binding with aspirin by changing the concentration of egg
6.
albumin.
7. To formulated and perform in-vitro dissolution of coated granules of ciprofloxacin.
8. To prepare & evaluate Aspirin matrix tablet.
9. To study in-vitro dissolution study of the various marketed sustained release product.
10. To study the effect of surfactant in enhancement of bioavailability using dissolution test.
To study the Effect of solubility enhancing agent (surfactant /co. solvent) on solubility/
11.
dissolution rate/ absorption of lipophilic drug (salicylic acid)
12.
13.
14.
15.
16.
17.
EXPERIMENT NO. 01
Object- To establish a standard curve of salicylic acid in distilled water.
Reference:
K.H.B.S College of Pharmacy, Jaunpur Page 2

1. Dr. Ali Javed, Dr. Ahuja, Dr. Baboo in Sanjula, Dr. Khar K. Roop, Bio-Pharmaceuticals and
pharmacokinetics, Theoretical concept & illustrate practical exercise 2nd edition 2008-09. Birla
Pharmaceuticals Publication Pvt. Ltd., Page No. 290.
2. Indian pharmacopoeia, Government of India, ministry of health & family welfare, 1996, volume II.
Requirement: - Salicylic acid, Distill water, Ethanol, Beaker, Volumetric flask.
Principle: Bears law can calculate the magnitude of the absorption of light at a fixed Wavelength. This
equation relates the amount of light absorbed (A) to the concentration of absorbing substance (in g/l)
& the length of the path of the sample B in (M) at.
A = abc
In which a = constant of absorptivity for a particular absorbing species (in units of
liter g-1 cm-1) if the unit of c moles-1 liter-1 then the constant is terminal as, is the molar absorptivity
(liter mole1 cm-1)
The absorptivity depends not only on the molecules where absorbance is being determined, but
also on the type of solvent being used as well as on the temperature & wavelength of the light used for
the analysis.
The A is termed as absorbance and is related to the transmittance of light (T) by
A = log Io Log I
In which Io is intensity of light after it emerges from the sample.
Salicylic acid forms a blue violet colored complex with ferric chloride. This is due to the interaction
between the ferric ion and phenolic group of salicylic acid.
Procedure:
(a) Preparation of colouring agent :
Dissolve ferric chloride 5 gm in 100 ml of distill water & prepare a 5% solution of ferric chloride.
(b) Preparation of solution of salicylic acid :
Weigh 100 gm of salicylic acid dissolve in 100 ml of distill water using 5 ml of ethanol. Take 1 ml
of this solution and dilute to 10 ml of water. This is required stock solution, from this solution of

concentration 1000 g/ml. Prepare dilution of concentration 10, 20, 30, 40, 50, g/ml. Now record
absorbance of all samples at max of 540 nm & plot calibration curve of absorbance against
concentration.
.
Observation Table:
Capsule No. Ingredients Quantity
1 Sodium Salicylate 100 mg
Lactose 50 mg
Dibasic Calcium Phosphate. 50 mg
Lactose 50 mg
Sodium Lauryl Sulphate 2%
Dibasic Calcium Phosphate 50 mg
Sodium Lauryl Sulphate 2%
Result: Standard curve of salicylic acid has been plotted.
EXPERIMENT NO. 02
Object: To study the effect of diluents and wetting agent on in-vitro release of water soluble drug
(Sodium Salicylate) from capsule dosages form.
References:
1. Lachman leon liberman Herbert- A kanig L. Joseph, The theory on practice of Industrial pharmacy. 4 th
1991 largehex publishing house.
2. Dr. Ali Javed, Dr. Ahuja Alka, Dr. Baboota Sanjula Dr. Khar K. Roo. Biopharmacuticals & Pharmacy
kineticsIInd ed. Birla publication Pvt. Ltd. P. No. 297.

Requirements: Capsules, Sodium salicylate, lactose, and dibasic calcium phosphate is used for
dissolution studies, calcium phosphate, Sodium Lauryl Sulphate.
Theory:
The tablets & capsules contain water soluble or insoluble excipients. The pharmaceutical excipients
which are used in different formulation are following:
1. Diluents:
They are added if the dose is inadequate to produce to necessary bulk hydrophilic diluents like
lactose; starch, micro crystalline cellulose (MCC), etc. are useful in promoting the dissolution of
poorly water soluble drugs like steroid. Hydrophobic diluents like dibasic calcium phosphate are also
used in the formation of capsules.
2. Binders:
Binders are used to bind powder to form granules or promote cohesive compacts for directly
compressible material and to ensure that the tablet remain intact after compression. Binders include
polymeric materials like starch, cellular derivatives, acacia, and PVP, gelatin & sugar solution.
3. Disintegrants:
These agents overcome the cohesive strength of tablet & break them up on contact with water.
Generally, all disintegrants are hydrophilic in nature. A decrease in the of distingrants can significantly
lower bioavailability, disintegrating agents such as starch tends to swell with wetting.
4. Lubricants:
Lubricants are often added to capsule or tablet dosages form so that the powder mass or
finished dosages form will not stick to the processing machinery. When the hydrophilic lubricating
agent SLS was added, 325mg salicylic acid tablet dissolved in 0.1 N HCl more rapidly than salicylic
acid tablets containing no lubricants. However, the hydrophobic lubricant magnesium stearate was
added, dissolution rate was decreased. Thus they must be properly formulated to avoid reducing rate
and bioavaibility.
5. Surfactants (Surface Active Agents):
Surface active agents are used in formulation as wetting agent solubilizers. The interaction of a
drug with a surfactant varies considerably, depending on where the concentration of surface active
agents is above or below CMC.

Procedure: Prepare 6 capsules as per the pattern given in the formula. The sixth capsule in an empty
one. In dissolution apparatus place 900 ml of phosphate buffer in each beaker. One capsule is placed in
each beaker & rotation speed is set at rate of 50 rpm. Samples 5 ml are withdrawn after 5, 10, 15, 20,
25 minutes from beakers (1-5) & volume of 5ml is replaced from beakers number 6. The sample is
withdrawn at the end of 25 minutes. There will be 5 samples for each capsule except for the sixth one.
To the sample in the text tube. Add 4 ml of coloring agent & make up the volume up to 10 ml with
phosphate buffer & record the absorbance at 540 nm. Calculate the concentration of salicylic acid by
interpolation using curve.
Observation table:
Formula:
Capsule Time (min) Absorbance at 540 nm Concentration (g/ml)
5
10
1 15
20
25
Capsule Time (min) Absorbance at 540 nm Concentration (g/ml)

5
10
2 15
20
25
Capsule Time (min) Absorbance at 540 mm Concentration (g/ml)
5
3 10
15
20
25
Capsule No. Ingredient Quantity

1 Salicylic Acid 100 mg
2 Salicylic Acid 100 mg
Lactose 50 mg

3 Salicylic acid 100 mg

Dibasic calcium phosphate 50 mg
Lactose 50 mg
SLS 2%
Dibasic phosphate 50 mg
SLS 2%
6 Blank
Results: In-vitro release of water soluble drug increases on addition of diluents & wetting agents.
EXPERIMENT NO. 03
Object: To study the effect of diluents and wetting agents on is vitro release of water insoluble drug
from a capsule dosage form.
References:
1. Lachman leon liberman Herbert- A kanig L. Joseph, The theory on practice of Industrial pharmacy. 4 th
1991 largehex publishing house.
2. Dr. Ali Javed, Dr. Ahuja Alka, Dr. Baboota Sanjula Dr. Khar K. Roo. Biopharmacuticals & Pharmacy
kineticsIInd ed. Birla publication Pvt. Ltd. P. No. 297.
Requirements: Capsules, salicylic acid, lactose, dibasic calcium phosphate, calcium phosphate,
sodium lauryl sulphate.
1. Diluents:

They are added if the dose is inadequate to produce to necessary bulk hydrophilic diluents like
lactose; starch, micro crystalline cellulose (MCC), etc. are useful in promoting the dissolution of
poorly water soluble drugs like steroid. Hydrophobic diluents like dibasic calcium phosphate are also
used in the formation of capsules.
2. Binders:
Binders are used to bind powders to form granules or promote cohesive compacts for directly
compressible material & to ensure that the tablet remain intact after compression. Binders includes
polymer material like starch cellulose derivatives, acacia, PVP, gelatin and sugar solution.
3. Disintregrants:
These agents overcome cohesive strength of tablet & break them up on contact with water.
Generally all disintegrants are hydrophilic in nature. A decrease in the disintegrants can significantly
lowers the bioavailability. Disintegrants agent such as starch tends to swell wetting. In swelling the
starch can break apart the dosages form. Absorbing disintegrants like bentonite & veegum should be
avoided with low dose drug like digitoxin, alkaloids & steroids.
4. Lubricants:
Lubricants are often added to capsule or tablet dosages form so that the powder mass or
finished dosages form will not stick to the processing machinery. When the hydrophilic lubricating
agent SLS was added, 325mg salicylic acid tablet dissolved in 0.1 N HCl more rapidly than salicylic
acid tablets containing no lubricants. However, the hydrophobic lubricant magnesium stearate was
added, dissolution rate was decreased. Thus they must be properly formulated to avoid reducing rate
and bioavaibility.
5. Surfactants (Surface Active Agents):
Surface active agents are used in formulation as wetting agent as solubilizers. The interaction
of a drug with a surfactant varies considerably, depending on where the concentration of surface active
agents is above or below CMC.
Procedure: Prepare 6 capsules as per the pattern given in the formula. The sixth capsule in an empty
one. In dissolution apparatus place 900 ml of phosphate buffer in each beaker. One capsule is placed in
each beaker & rotation speed in set at rate of 50 rpm. Samples 5 ml are withdrawn after 5, 10, 15, 20,
25 minutes from beakers (1-5) & volume of 5ml is replaced from beakers number 6. The sample is
withdrawn at the end of 25 minutes. There will be 5 samples for each capsule except for the sixth one.
To the sample in the text tube. Add 4 ml of coloring agent & make up the volume up to 10 ml with

phosphate buffer & record the absorbance at 540 nm. Calculate the concentration of salicylic acid by
interpolation using curve.
Observation Table:
Capsule No. Time (min) Absorbance at 540 nm Concentration (g/ml)
5
10
1 15
20
25

5
10
2 15
20
25

5
10
3 15
20
25
Result: Release of water insoluble drug increases on addition of diluents & wetting agent.

EXPERIMENT NO. 04
Object: To study the dissolution time profile of Paracetamol tablet as per I.P.
References: Indian pharmacopoeia, government of ministry of health & family welfare, 1996, volume
II, Page no. 554.
Requirements: Paracetamol tablet, Phosphate buffer (pH-7.4), volumetric flask, dissolution apparatus,
pipette etc.
Theory:
Absorption of drug is possible when it is in solution form. Where the molecules are
independent & assume molecular dispersion of each molecule is absorbed independently through the
biological membrane. Thus dissolution is prerequisite for drug absorption of drug is expected to be
released from solid dosages form & immediately goes into molecular dispersion this process is called
as dissolution.
Dissolution is expressed in the term of rate process .The dissolution may be defined as the
amount of drug that goes into solution. Per unit time under standard condition of liquid- liquid or
solid liquid interface, temperature & solvent composition.

Dissolution of Noyes Whitney theory depends on the first Ficks law to describe dissolution
process of the dry the rate of mass transfer of solute molecule or ions through a static diffusion layer is
directly proportional to-
(a) Area available for molecular or ionic strength
(b) Concentration diffusion (cg-c) across the boundary layer.

dC
Ks(CsC) K
dt
Where,
K= Dissolution rate constant
S = Surface area of Particles
Cs = Equilibrium solubility of drug
C = Concentration of drug in bulk of solution
Procedure:
Preparation of standard curve:
100mg of Paracetamol was weighed accurately & dissolved in 100 ml of phosphate buffer (pH
7.4). From this stock solution withdraw 10 ml & dilute up to 100 ml to get concentration (100 g/ml).
Now, dilution are made to get 2, 4, 6, 8, 10 g/ ml respectively. Absorption was measured at 257 nm
plotted the graph of concentration verses absorbance.
Dissolution study of Paracetamol tablet:
The Paracetamol tablet was placed in the basket of dissolution apparatus. The study was
performed by using 900 ml phosphate buffer pH 7.4 as the dissolution media & basket was rotated at
54 rpm and temperature was maintained at 37.5 5 . Sample was withdrawn at specified
period of time interval and replaced by same quantity of fresh buffer solution.
Then withdrawn 1ml sample was diluted to 20 ml with phosphate buffer & measure the
absorbance at 257 nm the amount of drug release was calculated & then % was determined.
Observation Table:
Standard Curve of Paracetamol:
S.No. Conc.g/m Absorbanc (x-x) (y-y) (x-x)2 (y-y)2 (x-x) (y-y)
l e

1. 2
2. 4
3. 6
4. 8
5. 10
Dissolution study of Paracetamol:

S.No. Time Absorbance Conc.g/ml Amount of Cumulative Cumulative %
(min) at 257 nm drug amount of drug release
dissolved in drug
mg/ml dissolved in
900ml
1. 5
2. 10
3. 15
4. 20
5. 25
6. 30
Result: Release of Paracetamol drug was found be.
EXPERIMENT NO. 05
Object: To perform in-vitro bioequivalence study of two marketed Paracetamol tablets.
References:
1. Remington The Science And Practice of Pharmacy, volume II, 21 st edition. Published by
Lipincott Williams & Wilkins, Indian Edition, New Delhi, Reprint 2006, Page No. 1040-1046.
2. Indian Pharmacopeia govt. of India ministry of health and family welfare, Volume II. Published
by controller of publication, Delhi, 1996, Page No. 550.
Requirements: Paracetamol tablets, phosphate buffer (7.4) & dissolution apparatus.
Principle:
Bioequivalence studies: The major reason for performing bioequivalence studies is that in the past
drug products which were considered pharmaceutical equivalents did not give comparative therapeutic
effect in patients.
The design and equation of well controlled bioequivalence studies require the co-operation of
pharmacokinetics, statistics clinical pharmacologist, bio-analyst, chemists & others.

In considering a bioequivalence study, one formulation of drug is chosen as a reference

standard against in which all other formulation of drug is compared.
The reference should contain active ingredient in the most bioavailable formulation & in some
quantity as in other formulation to which it is to be compared. The reference material should be
administered by same route as the comparison formulations unless the alternative route or additional
route is necessary to answer pharmaceutical question.
Procedure:
100mg of Paracetamol was weighed accurately & dissolved in 100 ml of phosphate buffer (pH
7.4). From this stock solution withdraw 10 ml & dilute up to 100 ml to get concentration (100 g/ml).
Now, dilution are made to get 2, 4, 6, 8, 10 g/ ml respectively. Absorption was measured at 257 nm
In-vitro bioequivalence study:
Two different Paracetamol tablets were placed in different basket in dissolution apparatus. The
study was performed by using 900 ml phosphate buffer solution as the dissolution media filled in both
baskets and both was rotated at 54 rpm, temperature was maintained at 37.5 5 . Sample was
withdrawn at specified period of time interval and replaced by same quantity of fresh buffer solution.
Absorbance was measured at 257nm. And of drug release was calculated & percentage drug release
was also calculated.
Observation Table:
Dissolution Study of first marketed Paracetamol tablet
Cumulative
Amount of
amount of
Time Absorbance drug Cumulative %
S.No. Conc.g/ml drug
(min) at 257 nm dissolved in drug release
dissolved in
mg/ml
900ml
1. 5
2. 10
3. 15
4. 20
5. 25
6. 30
Dissolution Study of Second marketed Paracetamol tablet

Cumulative
Amount of
amount of
Time Absorbance drug Cumulative %
S.No. Conc.g/ml drug
(min) at 257 nm dissolved in drug release
dissolved in
mg/ml
900ml
1. 5
2. 10
3. 15
4. 20
5. 25
6. 30
Result: Bioequivalence study of two marketed Paracetamol tablets were performed and percentage
drug release was found to be.and.
EXPERIMENT NO. 06
Object: To study the extent of protein binding with aspirin by changing the concentration of egg
albumin.
Reference: Dr. Ali javed, Dr. Ahuju, Dr. Baboota, Sujata, Dr. Khat K. Roop. Biopharmacutical &
Pharmacuitics, 2nd edition, Birla Publication.
Principle:
When the drug reaches the systemic circulation it can be bind to many component including
blood cells & plasma proteins. A protein molecule is macromolecules composed of many amino acids
linked together. The functional groups that at side chain of the amino acids provide sites for binding
small molecules. Proteins have many & varied binding sites. Drug protein binding site is a type of
complexation. Protein binding decreases the distribution of drug. The protein binding drug is big in
size which can not easily pass through cell membrane and only free drug pass through cell membrane
and contribute to tissues binding higher the protein binding lower the tissue binding.
Protein binding decrease the metabolism of drug and enhance the biological half life only that
fraction of drug which is free can get metabolized. Protein binding decrease the renal excretion of drug
and enhance biological half life.
Only free dry can get execrated through glomerulus filtration. In general protein bound drug is
not pharmacologically active. However, its activation may be reversed by addition of competitive
inhibitor to the binding system.
Procedure:


100 mg of drug was weighed accurately & dissolved in 100 ml of phosphate buffer (pH 7.8).
From this stock solution withdraw 10 ml & dilute up to 100 ml to get concentration (100 g/ml). Now,
dilution are made to get 2, 4, 6, 8, 10 g/ ml respectively. Absorption was measured at ..nm
Preparation of Sample Solution:
100 mg of drug was weighed accurately & dissolved in 100 ml of phosphate buffer (pH 7.8) and used
for further study as sample solution.
Preparation of dry protein complex:
Complex-I: 25 ml of sample solution is mixed with 25 mg of egg albumin.
Complex-II: 25 ml sample solution is mixed with 50 mg of egg albumin.
Complex-III: 25 mg of sample solution is mixed with 75 mg of egg albumin.
Diffusion study:
250 ml of phosphate butter pH 7.8 was placed in 500 ml of beaker. In open ended tube, closed
one side by dialysis membrane and 2.5 ml of complex solution is placed in it. Similarly another two
tube are prepared these tube are placed in contract surface of buffer solution with the help of burette
stand.
In the beaker magnet bead is placed beaker is put on magnetic stirrer and rotate at 75 rpm.
5 ml of sample was collected at different time interval & replace by same medium. Absorbance was
measured at .nm & percentage of drug release was calculated.
Observation Table:
Complex-I- 25ml. drug solution + 25 mg egg albumin
S.No. Time Absorbance Conc.g/ml Amount of Cumulative Cumulative % drug
(min) (nm) drug amount of release
dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90

Complex-II- 25ml. drug solution + 50 mg egg albumin

dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90
Complex-III- 25ml. drug solution + 75 mg egg albumin

dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90
Result: As the concentration of albumin increase protein binding of drug also increase & percent of
drug release decrease.

EXPERIMENT NO. 7
Object: To formulated and perform in vitro dissolution of coated granules of ciprofloxacin.
References: Lachman leou Uberman hearbert a konig L. Joseph the theory on practice of Industrial
pharmacy 4th ed. 1981, Varghese publishing house.
Principle: Coating is process in which the granules or particles or core are covered with thin film of
coating material. The coating process is mainly used to achieve to control the site of dissolution to
control absorption rate & pattern of action to protect dosage form from environmental interaction such
as oxidation to mask better taste of dry such as aspirin, to improve appearance, to convent liquid into
free flowing solid.
The process like pan coating, fluidized bed coating is used for this purpose. The coating
material such as sugar, waxes, shellac, cellulose derivatives gelatin are used for coating.
Procedure:
Dissolved l00 mg of pure drug in 100 ml of distilled water. Make dilution by using distilled
water to get different concentration of solution. Absorption was measured at 278 nm plotted the graph
of concentration verses absorbance.
Preparation of granules:
Weigh all ingredients & mix well. Then prepared starch paste (10%) & prepare the wet mass by
proper mixing. The wet mass is passed through mesh no. 12/14. The granules prepared were dried at
50 0C for 45 minutes. The granules then passed through mesh no. 16.
Coating of granules:
Prepare the coating solution by dissolving the HPMC propylene glycol in the iso-propyl
alcohol by using the solution; granules are coated by pan coating method. The coating granules are
dried by hot air oven. Dried granules used for the dissolution study.
Dissolution Study:

Dissolution study is carried out by using dissolution apparatus. Distilled water is used as
dissolution solvent. The study carried out at 54 rpm at temperature 37 0. 5 oC. 500 mg of granules
are feeded in empty capsules used for dissolution study. The 5ml sample is withdrawn at specific time
interval & replace by fresh media, absorbance was observed at 278 nm.
Observation Table:
Dissolution Study of Ciprofloxacin (uncoated)
(min) at 278 nm drug amount of release
dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90
6. 105
7. 120
Dissolution Study of coated granules Ciprofloxacin:

dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90
6. 105
7. 120

Result: Coated granules were prepared & evaluate for drug release study was done which is found to
be . after .. hours.
EXPERIMENT NO. 08
Object: To prepare & evaluate Aspirin matrix tablet.
Reference:
Indian journal pharmaceutical science sept oct 2004 P.No. 636.
Lachum team liberman herbent A. kanig Joseple The theory on prailice of Industrial
pharmacy 1991 verghese publishing house.
Requirements:
Principal:
The drug is dispersed in an insoluble matrix of rigid non-swellable hydrophobic material or
swellable hydrophobic materials. Materials used for matrix device are as polyvinyl chloride, fatty
material, hydrophilic gums.
The hydrophilic matrix lab is used to control drug release. Polyacetate, polyvinyl pyrroledone,
hydroxyl ethyl cellulose, methyl cellulose, natural gums can be used as a matrix material. The
hydrophilic material requires water to active the release mechanism. The immersion of water
hydrophilic matrix quickly forms a gel layer around the tablet. Drug release is controlled by a gel
diffusion barrier that is formed a tablet erosion.
Procedure:
Aspirin was passing through mesh no. 100. PVP & drug ratio 2:1 & 1:1 is used in preparation
of tablet. Other ingredient were mixed thoroughly to ensure complete mixing by using starch paste
(10%) wet mass is passed through mesh no. 16 after drying at 50 0C for 45 minutes talc and
magnesium stearate are mixed & compressed by using round flat punches.
100 mg of drug was weighed accurately & dissolved in 100 ml of distilled water and suitable
dilution are made to get 10, 20, 30, 40, g/ ml respectively. Absorption was measured at 540 nm using
ferric nitrate as colouring agent plotted the graph of concentration verses absorbance.

Observation Table:
Ingredients Quantity of drug 2:1 (gm) Quantity of drug 1:1 (gm)
Aspirin 3.00 3.00
P.V.P 6.00 3.00
MCC 5.55 5.55
Talc 2%(0.276) 2%(0.276)
Magnesium Sterate 1% (0.13) 1% (0.13)
Starch 10% (1.3) 10% (1.3)
HPMC 1.5 1.5
Standard curve of Aspirin

S.n CONCENTRATIO ABSORBANC x-x y-y (x x)2 (y y)2 (x - x)(y - y)
o N E
(g/ml) (nm)
1.
2.
3.
4.
5.
6.
7.
( x x )( y y)
Slope = ( x x )2
(x x )( y y )
Regression Coefficient = ( x x )2( y y)2

Dissolution Study of matrix tablet (2:1):

dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90
6. 105
7. 120
Dissolution Study of matrix tablet (1:1):

S.No. Time Absorbance Concentratio Amount of Cumulative Cumulative %
(min) at 540 nm n g/ml drug amount of drug release
dissolved in drug
mg/ml dissolved in
900ml
1. 15
2. 30
3. 45
4. 60
5. 90
6. 105
7. 120
Result: The matrix tablet of aspirin is prepared & evaluated for drug release and found to be
.. % after ..hours.
EXPERIMENT NO. 9
Object: To study in-vitro dissolution of the various marketed sustained release product.
Reference: 1. Lachman Leon, Liberman herbent. A kavig, L. Joseph The theory on practice of
Industrial pharmacy 4th edi 1991 varghese publishing house.

2. Remington The science & practice of pharmacy vol-II 2007, Published by

welfare klever health India pvt. Ltd. New Delhi P. No. 1040-1046.
Principle:
Dissolution is process in the drug particles are goes into solution form in particular time. To
prepare controlled release & sustain release by controlling the dissolution rate of drug that highly
water soluble. This can be done by preparing on appropriate salts or derivatives by coating the drug
with a poorly soluble material.
In order to maintain a constant level of drug is some desired target tissue. In zero order drug
release the above dose of drug is available to body. Hence, sustained action of drug in body, slow
down absorption, biotransformation & elimination rate.
Procedure:
100 mg of diclofenac was weighed accurately & dissolved in 100 ml of Phosphate buffer pH
7.4 and suitable dilution are made to get 10, 20, 30, 40, g/ ml respectively. Absorption was measured
at 540 nm using ferric nitrate as colouring agent plotted the graph of concentration verses absorbance.
Dissolution studies are carried out two different marketed tablets Voveron SR. & Diclonac 100 SR of
Diclofenac.
OBSERVATION TABLE:
Standard curve of Diclofenac
S.n CONCENTRATIO ABSORBANC x-x y-y (x x)2 (y y)2 (x - x)(y - y)
o N E
(g/ml) At 540 nm
1.
2.
3.
4.
5.
6.
7.
Calculation:
(x x )( y y)
Slope = (x x )2

( x x )( y y )
Regression Coefficient = (x x )2( y y)2
Dissolution Study of voveron:

dissolved in drug
mg/ml dissolved in
900ml
1. 10
2. 60
3. 120
4. 180
5. 240
6. 300
7. 360
8. 420
9. 480
Dissolution Study of Diclofenac:

dissolved in drug
mg/ml dissolved in

900ml
1. 10
2. 60
3. 120
4. 180
5. 240
6. 300
7. 360
8. 420
9. 480
Result: The percentage drug release from sustained release. Diclofenac marketed product was found
to be ..% & % for vaveron & Diclonac tablet respectively.
.
EXPERIMENT NO. 11
Object: To study the effect of surfactants in enhancement of bioavailability using dissolution study.
Reference:
1. Dr. Ali Javed, Dr. Ahuja, Dr. Baboo in Sanjula, Dr. Khar K. Roop, Bio-Pharmaceuticals and
pharmacokinetics, Theoretical concept & illustrate practical exercise 2nd edition 2008-09. Birla
Pharmaceuticals Publication Pvt. Ltd., Page No. 290.

2. Indian pharmacopoeia, Government of India, ministry of health & family welfare, 1996, volume II
3. Lachman Leon, Liberman Herbert A.,The Theory & Practice Of Industrial Pharmacy, CBS
Publication, New Delhi, Special Indian Edition, 2009, Page no..
Principle:
As per the definition of bioavailability, it concerns a drug with poor bioavailability is the one
with poor aqueous solubility & slow dissolution rate in the biological fluid on inadequate partition
coefficient. Thus poor permeability through the bio-membrane. To overcome the problem
bioavailability few approaches are done.
1. Pharmacokinetic approaches
2. Pharmaceutical approaches
3. Biological approaches
4. Surfactants
Surfactants are active agent which increases the dissolution rate, non ionic surfactant like polysorvate
are widely used.
Procedure:
Firstly, prepare standard curve of Paracetamol by 100mg of Paracetamol was weighed
accurately & dissolved in 100 ml of 0.1 N HCl to make stock solution from stock solution 1ml was
taken & diluted to 10 ml to get a series of 10, 20, 30, 40, 50, 60 g/ml and a graph was plotted as
concentration v/s absorbance.
Then dissolution study was done by using dissolution apparatus.
Observation Table:
Dissolution study: 0.1 N HCl- Dissolution media
dissolved in drug
mg/ml dissolved in
900ml
1. 5
2. 10
3. 15
4. 20
5. 25
6. 30

Dissolution study :( 0.25% tween 80 + 0.1 N HCl Dissolution media)

dissolved in drug
mg/ml dissolved in
900ml
1. 5
2. 10
3. 15
4. 20
5. 25
6. 30
Dissolution study:( 0.5% tween 80 + 0.1 N HCl Dissolution media)

dissolved in drug
mg/ml dissolved in
900ml
1. 5
2. 10
3. 15
4. 20
5. 25
6. 30
Result: As concentration of surfactant in dissolution medium increases. The percentage of drug

release also increases.

EXPERIMENT NO-12
Object: To study the Effect of solubility enhancing agent (surfactant /co. solvent) on solubility/
dissolution rate/ absorption of lipophilic drug (salicylic acid)
Reference:
1. Bramhanker D.M, Jaiswal B.S, A Treatise Biopharmaceutics and Pharmacokinetics Vallabh
Prakashan, Delhi; reprint edition; 2008; Page No. 45
2. Lachman Leon, Liberman Herbert A.,The Theory & Practice Of Industrial Pharmacy, CBS
Publication, New Delhi, Special Indian Edition, 2009, Page no..
Requirement: Beaker, Funnel, Conical flask, Filter Paper, weight box
Chemical Requirement: Salicylic acid, NaOH, Distilled water, oxalic acid, twin 80,
phenolphthalein
Theory:
Surfactant:
Surfactants are materials which have a tendency to preferentially get absorbed at the interface
between two phases. Their molecules consists of a polar and a non-polar part and when they are
placed between two phases of differing polarities the non-polar part gets oriented towards the phase of

low polarity while the polar towards the high polarity phase. As a result of their surface absorption the
tension between two phases gets lowered and the phases acquire a greater tendency to intermix with
each other. Hence, wherever there is need to lower the interfacial tension surfactants are used.
When a surfactant is placed in a solvent its molecules are present in a larger number at the
interface than in the bulk of the solution. Preferential absorption at the interface continues until all the
available space at the surface is occupied. Thereafter the surfactants molecules descend into the bulk
of the solution in the form of molecular aggregates or micelles of various shapes .The molecules are so
arranged in a micelle that their parts, corresponding to polarity of the solution, are oriented towards
the outside. The concentration of the surfactant at which it forms micelle is known as Critical Micelle
Concentration (C.M.C.). The following figure illustrates the surface adsorption of surfactants and
micelle formation.
Co solvent
The solubility of a weak electrolyte or non-polar compound in water can be improved by
changing the polarity of the solvent. This can be achieved by the addition of another solvent that is
both miscible with water &in which the compound is also soluble. This process is known as co-
solvency, & the solvents used in combination to increase the solubility of the solute are known as
co-solvents. Vehicles used in combination to increase the solubility of a drug are called as cosolvents,
and often the solubility in this mixed system is greater than can be predicted from the materials
solubility in each individual solvent.
Because it has been shown that the solubility of a given drug is maximal at a particular
dielectric constant of any solvent system, it is possible to eliminate those solvent blends possessing
other dielectric constants. The choice of suitable co solvents is somewhat limited for pharmaceutical
use because of possible toxicity and irritancy, particularly if required for oral or parentral use. Ideally,
suitable blends should possess values of dielectric constant between 25&80.The most widely used
system that will cover this range is a water/ethanol mixture. Other suitable solvents for use with water
include sorbitol, glycerol, propylene glycol& syrup. For example, a mixture of propylene glycol &
water is used to improve the solubility of co-trimoxazole, & Paracetamol is formulated as an elixir by
the use of alcohol, propylene glycol & syrup. For external application to the scalp, betamethasone
valerate is available dissolved in water/isopropyl alcohol mixture.
Procedure:

1. Take 500mg of salicylic acid and added 25ml of water to it and short in the help of glass rod
for 10 min. filter the mixture 10 ml of filtrate and titrate with 0.1N NaOH using
phenolphthalein as an indicator.
2. Again take 500mg SA+ 24.5 ml water & add 0.5ml tween 80 and stir for 10 min with the help
of glass rod & filter and take 10 ml of filtrate and titrate by & brave titration.
3. Again taken 500mg S A and 24 ml H2O water & 1ml tween 80 &stir for 10min take 10ml of
titrate and filtrate it.
4. Again tween 500 S A and add 23.5 ml water and 1.5ml tween 80 and stir for 10min take10ml
filtrate and titrate it.
5. Taken 500mg SA and add 23.0 ml water and 2ml tween 80 & stir for 10 min take 10ml filtrate
and titrate it.
6. Taken 500mg S A &22.5 ml water &2ml tween 80 &after stirring take 10 ml and titrate it.
RESULT:
EXPERIMENT NO. 13
KE t 1 /2
Object: To determine the pharmacokinetic parameters Cmax, Tmax, , , AUC0t*, AUMC and
MRT from the given data.
Reference: Dhachnamoorthi D., Venkateswaramurthy N., Biopharmaceutics and Pharmacokinetics

A Practical Manual, PharmaMed Press, Hyderabad, First edition, 2008, page no.78-81.
Data
A 150mg oral dose of a drug was administered to a healthy volunteer. Blood samples were
collected and plasma was separated from each blood sample and analyzed for drug concentration. The
collected data are shown in the table below.
S.No. Time in hrs Plasma drug concetraton in mg/L

1 0.25 1.726
2 0.5 2.837
3 0.75 3.43

4 1 3.885
5 1.25 4.147
6 1.5 4.17
7 2 4.009
8 3 3.396
9 4 2.834
10 6 1.887
11 9 1.029
12 12 0.549
13 15 0.296
14 18 0.161
15 24 0.046
Graphical Expression
Plot the graph in between the time in hours versus Plasma drug concentration in mg/L.
Data Analysis
1. Cmax
The point of maximum concentration of a drug in plasma is called as peak plasma concetration
and it is computed directly from the plasma level profiles.
2. Tmax
The time of the drug to achieve the peak plasma concentration is computed directly from the
plasma level profiles.
KE
3. Elimination Rate Constant ( )
Elimination rate constant is calculated from the terminal elimination phase of log plasma
concentration Vs time by least square regression analysis. From the slope of regression analysis,
KE
is calculated as
KE
= slope 2.303
t 1 /2
4. Biological Half Life
The time taken for the amount of drug in the body as well as plasma concentration to decline
by 50% of its initial volume. And it is calculated by the following formula.

0.693
t 1 /2 =
KE
AUC
5. t*
The extent of absorption is calculated from the area under the plasma concentration time curve
from O to t* hours by trapezoidal rule method.
C1 +C 2
AUC tt =2
( t 2 t1 )
1
2
6. AUMC 0t*
Plot of the product of concentration and time Vs time from 0 time to t* is often referred to as
the area under the first moment curve AUMC 0t*. The area under the curve 0 to t* is calculated by
means of trapezoidal rule method.
7. Mean Residence Time (MRT)

AUMC
MRT =
AUC
Result
The following pharmacokinetic parameters were calculated from the given data.
Cmax
1. = --------------- g /ml
T max
2. = --------------- hrs
Ke 1
3. Elimination rate constant ( ) = --------------- hrs
t 1 /2
4. Biological half life ( ) = --------------- hrs
5. AUC0t* = ---------------- g . hr /ml

2
6. AUMC0t* = --------------- g . hr /ml

7. MRT = --------------- hrs
EXPERIMENT NO. 14
Object: To determine the pharmacokinetic parameters of the given plasma time profile obtained by
intravenous bolus dose of a drug.

A Practical Manual, PharmaMed Press, Hyderabad, First edition, 2008, page no..
Principle
When a drug is given as an intravenous bolus and the drug distributes from the blood into the tissues
quickly, the serum concentrations often decline in a straight line when plotted on semi logarithmic
axes. In this case, a one-compartment model intravenous bolus equation can be used.
K E
C=C0
t

Where t is the time after the intravenous bolus was given (t=0 at the time the dose was
administered), C is the concentration at time =t, Vd is the volume of distribution, C0 is the initial
concentration and KE is the elimination rate constant.
If two or more serum concentrations are obtained after an intravenous bolus dose, the
elimination rate constant, half-life and volume of distribution can be calculated. By plotting the serum
concentration/time data on semi logarithmic axes, the time it takes for serum concentrations to
decrease by one-half can be determined and is equal to half life of the drug.
The elimination rate constant can be computed using the following relationship.
K E =0.693 /t 1/ 2
The concentration/time line can be extrapolated to the y-axis where time = 0. The extrapolated
concentration at time = 0 is called initial concentration (C 0), which can be used to calculate the volume
of distribution.
V d =Dose/C 0
The serum concentration at time = zero (C 0) can be computed using a variation of the intravenous
bolus equation:
t
C=CK
0
E
C0 KE
Area under curve (AUC) = /
Data
1. Following table contains plasma drug concentrations (C) obtained following an intravenous
bolus administration of a 400 mg dose of a drug that exhibited the characteristics of a one-
t 1 /2
compartment model and was eliminated exclusively by urinary excretion. Find out ,
KE C0 Vd
, and .

Plasma drug concentration (g/ml) Time (h)

88.0 0.5
74.0 1.0
50.0 2.0
38.5 3.0
26.0 5.0
21.8 7.0
Plot semi logarithmic graph by taking plasma drug concentration in x axis and time in hours in
y axis.
Data Analysis
t 1 /2 C0
Find out elimination half life ( ) and initial plasma concentration ( ) from the above
semi logarithmic plot.

t 1 /2
i. The elimination half life ( ) = .hours
C0
ii. Initial plasma concentration ( ) =..g/ml
KE t 1 /2
iii. Elimination rate constant = 0.693/ = ..h-1
Vd
iv. Apparent volume of distribution ( ) = Dose/C0 = .ml
EXPERIMENT NO. 15
Object: To determine the absorption rate constant from the given plasma drug concentration-time
profile.

Principle
Two methods are used to calculate absorption on rate constant from plasma concentration time data
following extra vascular dose. The first method is known as feathering, method of residuals or
curve stripping. The method allows the separation of the mono-exponential constituents of a bi-

exponential plot of plasma concentration against time. The second method is called as Wagner
Nelson method. The first method applies when absorption is first-order kinetics. The second method
makes no assumption about the nature of the absorption process. Both methods require that one
compartment linear disposition kinetics.
Method of Residuals
1. Plot plasma drug concentration Vs time in semi logarithmic paper (fig. No.1)
2. Back extrapolate the log linear portion of the curve in decline phase.
3. Plasma concentration values obtained from the extrapolated portion of the plasma drug
concentration Vs time.
4. Subtract the observed plasma concentration form the corresponding extrapolated value at each
time point the resulting value called as residual value.
5. Plot residual value versus time on the same graph paper.
6. Resulting residual straight line plot determine the absorption half life and absorption rate
constant.
Ka = 0.693/ t1/2 (obtained from residual straight line)
Fig. 2 model plot of plasma drug concentration Vs time gin semilagrathemic paper.
Data:

Following table contain the plasma drug concentration obtained following an oral administration.
Find out the absorption rate constant Ka by using curve stripping method (Method of residuals).
Time (hrs) Plasma drug concentration (a)

0.3 3.10
0.5 4.70
1 5.80
2 4.90
3 3.30
4 2.10
5 1.90
6 0.85
i. Plot semi logarithmic graph by taking plasma drug concentration in x axis and time in
hours in y axis.
ii. Back extrapolate the log linear portion of the curve in decline phase
iii. Plasma concentration values obtained from the extrapolated portion of the plasma drug
concentration Vs time.
iv. Subtract the observed plasma concentration from the corresponding extrapolated value at
each time point the resulting value called as residual value.
v. Plot residual value versus time on the same graph paper.
Time (hrs) Plasma Extrapolated plasma Difference between

concentration (a) concentration (b) b and a
0.3 3.10
0.5 4.70
1 5.80
2 4.90
3 3.30
4 2.10
5 1.90
6 0.85
Data Analysis:
From the residual straight line plot the absorption half life and absorption rate constant can be
calculated

Absorption half life (t1/2) will be obtained from residual straight line.
Absorption rate constant (Ka) = 0.693 / t1/2
= ____________h-1
EXPERIMENT NO. 16
Object: To determine the serum drug concentration at a specific time point after administration of oral
dose.

Data
Consider 600mg of oral antibiotic was administered to a patient. It is known from prior clinic
visits that the patient has a half life equal to 5 hours, an elimination rate constant of 0.138hr -1 and
volume of distribution of 170L. The capsule administered to a patient has an absorption rate constant
equal to 2hr-1 and an oral bioavailability fraction of 0.73. Find out serum concentration after 6 hours.
Data Analysis
K

V ( aK E) eK t e K t
E a
C=
FKaD

Where
D= Dose

C = Serum drug concentration

F = Oral bioavailability fraction
Ka = Absorption rate constant
KE = Elimination rate constant
V = Volume of distribution
t = time point
Serum drug Concentration after 6 hrs C = .mg/L
EXPERIMENT NO. 16
Object: To calculate the adjusted dose for renally impaired patient using the pharmacokinetic
parameter of a drug.

Principle
In patients with renal disease, there is a functional loss of nephrons. Depending on the etiology of the
renal disease; the patient with acute kidney failure may recoup their baseline renal function after a
period of supportive care and dialysis long enough for their kidney to recover. For drugs that are
excreted primarily by the kidneys, extreme care is needed with respect to the dose of drug and its
dosing interval in the case of kidney damage.
The renal impairment cases increased drug levels due to reduced excretion. Drug excretion that is
highly dependent upon the renal function dose adjustment necessary. Dose and dosing frequency
calculated for renal impaired patient from following formula.

dose of normal patient

dosing freq uency of Estimation rate constant of
normal patient normal patient
dose of renal impaired patient

dosing frequency of Estimation rate constant of
renal impaired patient renal impaired patient
Problem 1
a. The elimination rate constant of an anti-malarial drug in normal and renally impaired are found
to be 0.25 and 0.075 respectively. It is usually administrated once daily in a dose of 500mg.
How can be dose adjusted is renally impaired patient to provide similar plasma level in normal
patient by maintaining the one dosing interval of 24 hrs.
b. If it is decided to keep the dose of 500mg constant how would you change the dosing interval
of drug in renal impatient to provide plasma concentration as that of normal individual.
c. If it is decided to give the dose of 200mg, how would you change the dosing interval of drug in
the renal impaired patient to provide the concentration as that of normal individual?
Given Data
Dose of normal patient (DNP) = 500 mg
Dosing frequency of normal patient (Df NP) = 24 hrs
Dosing frequency of renal impaired patient (Df RIP) =24 hrs
Elimination rate constant of renal impaired patient (KE RIP) =0.075 hrs
Elimination rate constant normal patient (KE NP) = 00.25 hrs
Calculations:
a. Dose of renal impaired patient (DRIP) =?
D NP Df RIP K E RIP
DRIP = Df NP K E NP
=______________ mg.
b. Dosing frequency of renal impaired patient if the dose of 500mg.

Df NP K E NP DRIP
Df RIP = DNP K E RIP
=______________mg.
c. Dosing frequency of renal impaired patient if the dose of 200mg for renal impaired patient.
Df NP K E NP DRIP
Df RIP = DNP K E RIP
=______________mg.
EXPERIMENT NO. 17
Object: To determine the effect of partition coefficient of salicylic acid

Principle
Partitioning is the ability of a drug to distribute in two immiscible systems. The ability of drug to
permeate across biological membranes has traditionally been evaluated using its partitioning in octanol
(representing lipid membrane) and water system. Occasionally, other organic solvents like chloroform,
ether, and hexane have been used as lipid solvents instead of octanol to evaluate drug partitioning
behavior. When a drug is placed is an immiscible system comprising of octanol and water, the drug

distributes in each solvent and eventually reaches equilibrium. The ratio of drug concentration in each
phase is termed its distribution coefficient or partition coefficient.
The pKa of the drug determines the degree of ionization at a particular pH and only unionized drug
with sufficient lipid solubility is absorbed into the systemic circulation. For optimum absorption, the
drug should have sufficient aqueous solubility to dissolve in the GIT at the absorption site and lipid
solubility should be high enough to facilitate drug partitioning of the drug into the lipoidal membrane
and into systemic circulation. The partition coefficient (log P) value in the range of 1 to 3 has good
passive absorption across lipid membranes, and log P is greater or less than 3 have often poor transport
characteristics. Highly non-polar molecules form a depot in the lipophillic regions of the membrane,
and very polar molecules have poor bioavailability because of their inability to penetrate lipodal
membrane. The drug with high affinity for lipids may have poor release and dissolution from the
dosage form. Partition coefficient is generally determined as the distribution of a molecule between a
mixture of octanol (representing lipid membrane) and water.
Materials Required
Salicylic acid, n-octanol, hydrochloric acid, sodium hydroxide, potassium chlorde, potassium
hydrogen phthalate, potassium dihydrogen phosphate, ferric nitrate, digital electronic balance, pipette,
separating funnels and UV/Visible spectrophototmeter, etc.
Procedure
Construction of Calibration Curve for Salicylic Acid by Visible Spectroscopic Method
Preparation of Salicylic Acid Primary Stock Solution (1000g/ml).
Weigh accurately 100mg of salicylic acid and transferred in to 100ml volumetric flask. Add small
quantity of distilled water and shake the solution thoroughly to dissovle salicylic acid and make up the
volume.
Preparation of Salicylic Acid Secondary Stock Solution (400g/ml)
Transfer 40ml of the primary stock solution of 100ml volumetric flask and make up the volume with
distilled water.
Preparation of Working Standard Solution

Prepare a series of dilute solutions of 20, 40, 60, 80 and 100g/ml from the above secondary stock
solution by taking 0.5, 1, 1.5, 2, 2.5ml & add 5ml of 4% ferric nitrate solution and finally the volume
is make up to 10ml with distilled water. Keep the solution aside for 10 minutes to develop a colour.
Measure the absorbance at 547nm in UV/Visible spectrometer and note the values in Table1. Plot a
graph by taking concentration (g/ml) on x axis and absorbance on y exis. This plot gives a straight
line and the linearity can be determined using y=mx +c formula. Calculate the coefficient of
determination R2 value, slope m and intercept c using the following formula.

R 2=
( x+ X )( y + y )
( x+ X ) ( y + y )
2 2

( x + X )( y + y )
m
(x +X ) 2

c= y m x
Determination of Partition Coefficient

Preparation of Buffer Solutions
a. Acid Phathalate Buffer (pH 2.2)
50ml of 0.2M potassium hydrogen phthalate (dissove 40.846gm in 1000ml distilled water ) and
add 49.5ml of 0.2N hydrochloric acid (17ml diluted to 1000ml with distilled water) finally
make up the volume to 200ml.
b. Acid Phthalate Buffer (pH 3)
add 22.3ml of 0.2N hydrochloric acid (17ml diluted to 1000ml with distilled water) finally
c. Acid Phathalate Buffer (pH 4)
add 0.1ml of 0.2N hydrochloric acid (17ml diluted to 1000ml with distilled water) finally make
up the volume to 200ml.
d. Phosphate Buffer (pH 7.2)

50ml of 0.2M potassium dihydrogen phosphate (dissolve 2.72gm in 1000ml distilled water )
and add 34.7ml of 0.2M sodium hydroxide (dissolve 8gm in 1000ml of distilled water) finally
e. Phosphate Buffer (pH 8)
50ml of 0.2M potassium dihydrogen phosphate (dissove 2.72gm in 1000ml distilled water )
and add 46.1ml of 0.2M sodium hydroxide (dissolve 8gm in 1000ml of distilled water) finally
f. Alkaline Borate Buffer (pH 10)
50ml of 0.2M boric acid and potassium chloride solution g(dissolve 1.23gm of boric acid and
1.49gm of potassium chloride in 1000ml of distilled water) and add 43.7ml of 0.2M sodium
hydroxide (dissolve 8gm in 1000ml of distilled water) finally make up the volume to 200ml.
1. Take thoroughly cleaned and dried 6 numbers of separating funnels and labeled as S1, S2, S3,
S4, S5, S6.
2. Take 25ml of n-octanol and 25ml of Acid Phthalate buffer pH2.2 in S1 separating funnel.
100mg. of salicylic acid is added and shake for one hour.
3. From this mixture remove one ml of aqueous layer and transfer to 100 ml volumetric flask.
Finally make up to 1000 ml with distilled water.
4. Take 5 ml of the above solution then add 5 ml of 4 % ferric nitrate solution.
5. Keep the solution aside for 10 minutes to develop colour.
6. Measure the absorbance at 547 nm using 5 ml of water with 5 ml of 4 % ferric nitrate solution
as blank.
7. To the separating funnel S2, S3, S4, S5 and S6 add the respective buffer with varying pH and
repeat the same procedure.
8. From the absorbance at different pH the concentration of salicylic acid at different pH are
calculated in partition coefficient table.
Observation Table:
Calibration curve data for salicylic acid
Volume
Drug Secondary
of 4% Volume of Absorban
concentratio stock
S.No Ferric distilled ce of the (x- (y (x- (y (x-x)
n is solution of
. nitrate water in sample at x) -y) x)2 -y)2 (y-y)
salicylic acid
g/ml(x) solution ml 547nm(y)
in ml
in ml
1 0 0 5 5
2 20 0.5 5 4.5
3 40 1 5 4
4 60 1.5 5 3.5
5 80 2 5 3
6 100 2.5 5 2.5

Partition Coefficient determination
Concentration in Amount of salicylic acid Partition

S.No. Buffer pH Absorbance
g/ml on 25 ml of n octanol (y) coefficient (y)/(x)
1 2.2
2 3
3 4
4 7.2
5 8
6 10
Calculation:
Concentration g /ml 25 100
Amount of salicylic acid in 25 ml of buffer (x) = 1000
Amount of salicylic acid in 25 ml of n-octanol (y) 100( x)
y
Partition coefficient (P) = x
Result: The maximum partition coefficient of _______at ______ pH leads to maximum absorption.
EXPERIMENT NO. 17
Object: To determine the pH partition profile of diclofenac sodium and theoretical estimation of its
apparent volume of distribution.
Reference: Dhachnamoorthi D., Venkateswaramurthy N., Biopharmaceutics and Pharmacokinetics A

Practical Manual, PharmaMed Press, Hyderabad, First edition, 2008, page no.39-42.
Material Required: n-octanol, diclofenac sodium, potassium dihydragen phosphate, sodium

hydroxide, volumetric flask, separating funnel, digital electronic balance, UV/Visible
spectrophotometer, etc.
Principle

Biological membranes are lipophilic and drugs penetrate these barriers mainly in their molecular,
undissociated form. According to pH partition hypothesis drugs are adsorbed from the GI tract by
passive diffusion relative to the fraction of undissociated drug at the pH of the intestine. It is reasoned
that the partition coefficient between membranes and gastrointestinal fluids is large for the
undissociated drug species and favours transport of the molecular form the intestine through the
mucosal wall and into the systemic circulation. There is relationship between the volume of
distribution (expressed as distribution coefficient) and the fraction of protein binding (P), and the
apparent lipid/water partition coefficient (APC). The various volume s of distribution can be estimated
from the in vitro data of APC (n-octanol/pH7.4 Phosphate buffer) and from the extend of protein
binding. Apparent partition coefficient can be calculated by using the following formula.
( C1C 2 ) a
APC =
C2 b
Where, C1- drug concentration in aqueous phase before equilibrium

C2- drug concentration in aqueous phase after equilibrium
a- Volume of aqueous phase
b- Volume of organic phase
For One Compartment Open Model
Vd = (0.0955.APC+1.2232) (1-P) Body weight
For Two Compartment Open Model
Vc = (0.0397.APC+0.0273) (1-P) Body weight
Vdss = (0.1141.APC+0.6611) (1-P) Body weight
Vd = (0.1302.APC+0.9314) (1-P) Body weight
Procedure
Construction of Calibration Curve for Diclofenac Sodium
Preparation of Diclofenac sodium Primary stock solution (1000 g/ml)
Weigh accurately 100mg of diclofenac sodium and transfer in to a 1000 ml volumetric flask. Add
small quantity of methanol and shake the solution throughout to dissolve diclofenac sodium . finally
the volume to 100 ml with the help of distilled water.
Preparation of diclofenac sodium secondary stock solution (100 g/ml)
Transfer 10 ml of the primary stock solution to 100 ml volumetric flask and wit h distilled water.

Preparation of working standard solution.

Prepare a series of solute solution from the above secondary stock solution according to table 2 in 10
ml volumetric flask. Measure the absorbance of solution at 276 nm using distilled water as a blank.
Plot a graph by taking concentration (g/ml) on X axis and absorbance at on y axis. This plot gives a
straight line and linearity can be determined using y = mx + c formula. Model calibrated curve shown
in figure. Calculate the coefficient of determination R 2 value, slope m and intercept c using the
following formula.
R 2=
( x+ X )( y + y )
2 2
( x+ X ) ( y + y )

( x + X )( y + y )
m
(x +X ) 2

c= y m x
Preparation of Buffer Solutions

Preparation of 0.2M Potassium Dihydragen Phosphate Solution
Dissolve 2.72gm of potassium dihydragen phosphate in 1000ml distilled water.
Preparation of 0.2M Sodium Hydroxide Solution
Dissolve 8gm of sodium hydroxide in 1000ml distilled water
Prepare different buffer solutions of the following pH 5.8, 6.0, 6.6, 7.0, 7.4, 7.8 & 8.0 mixing 0.2M
Potassium dihydragen phosphate solution and 0.2M sodium hydroxide solution given in Table 1
Determination of Partition Coefficient
1. Take thoroughly cleaned and dried 7 numbers of separating funnels and labeled as S1, S2, S3,
S4, S5, S6, & S7
2. Take 20ml of n-octanol and 20ml of corresponding buffer in separating funnel. 50mg of
diclofenac sodium is added in each flask.

3. After 3 hours (before equilibrium) and after 24 hours (after equilibrium) from this mixture
remove one ml of aqueous layer and transfer to 100ml volumetric flask. Finally make up to
100ml with distilled water.
4. Measure the absorbance at 276nm and calculate the diclofenac sodium concentration.
5. Calculate the apparent partition coefficient (APC) for different pH (table 2) and plot a graph
between APC in y axis and pH in x axis (Fig. 1).
6. Calculate the Theoretical volume of distribution for diclofenac sodium in pH 7.4 by using the
corresponding APC values.
Observation Table:
S.No. pH Volume of 0.2M potassium Volume of 0.2M
dihydragen phosphate sodium hydroxide
solution in ml solution in ml
1 5.8 50 3.6
2 6.0 50 5.6
3 6.6 50 16.4 Volume make up
4 7.0 50 29.1 to 200ml with
5 7.4 50 39.1
6 7.8 50 44.5 distilled water
7 8.0 50 46.1
S.No. pH Concentration of Concentration of Apparent

Diclofenac sodium Diclofenac sodium partition
before equilibrium after equilibrium coefficient
1 5.8
2 6.0
3 6.6
4 7.0
5 7.4
6 7.8
7 8.0
Fig. 1 Model Plot: APC Vs pH

Model Calculation
For Example one compartmental open model (at pH 7.4)
1. Apparent partition coefficient =
2. Protein binding P = 0.997
3. Body weight (adult average weight ) = 70kg
Apply these values in the following formula
Vd = (0.0955 x APC + 1.2232) (1- P) B.W.
Result: Theoretical volume of distribution is =

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LAB MANUAL

B. Pharm. 4th Year

KUNWAR HARIBANSH SINGH COLLEGE OF PHARMACY

1. To establish a standard curve of salicylic acid in distilled water.

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Requirement: - Salicylic acid, Distill water, Ethanol, Beaker, Volumetric flask.

(b) Preparation of solution of salicylic acid :

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Result: Standard curve of salicylic acid has been plotted.

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Capsule Time (min) Absorbance at 540 nm Concentration (g/ml)

Capsule No. Ingredient Quantity

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3 Salicylic acid 100 mg

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Capsule No. Time (min) Absorbance at 540 nm Concentration (g/ml)

Capsule No. Time (min) Absorbance at 540 nm Concentration (g/ml)

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(b) Concentration diffusion (cg-c) across the boundary layer.

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Dissolution study of Paracetamol:

Requirements: Paracetamol tablets, phosphate buffer (7.4) & dissolution apparatus.

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In considering a bioequivalence study, one formulation of drug is chosen as a reference

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Preparation of standard curve:

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Complex-II- 25ml. drug solution + 50 mg egg albumin

Complex-III- 25ml. drug solution + 75 mg egg albumin

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Dissolution Study of coated granules Ciprofloxacin:

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Standard curve of Aspirin

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Dissolution Study of matrix tablet (2:1):

Dissolution Study of matrix tablet (1:1):

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2. Remington The science & practice of pharmacy vol-II 2007, Published by

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Dissolution Study of voveron:

S.No. Time Absorbance Conc.g/ml Amount of Cumulative Cumulative % drug

Dissolution Study of Diclofenac:

S.No. Time Absorbance Conc.g/ml Amount of Cumulative Cumulative % drug

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Dissolution study :( 0.25% tween 80 + 0.1 N HCl Dissolution media)

S.No. Time Absorbance Conc.g/ml Amount of Cumulative Cumulative % drug

Dissolution study:( 0.5% tween 80 + 0.1 N HCl Dissolution media)

S.No. Time Absorbance Conc.g/ml Amount of Cumulative Cumulative % drug

Result: As concentration of surfactant in dissolution medium increases. The percentage of drug

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MRT from the given data.

Reference: Dhachnamoorthi D., Venkateswaramurthy N., Biopharmaceutics and Pharmacokinetics

S.No. Time in hrs Plasma drug concetraton in mg/L

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