Practical Pharmacology4 25

Experiments

Experiment No. 6
cTUDY OF DIFFERENT ROUTES OF
DRUG ADMINISTRATION IN MICE/RATS

sleeTo study different

routes of drug administrationand blood withdrawal
from
experimentalanimals,
Drug Administration:
0 Intraperitoneal (IP)Injection:
1P injectionis the route of drug administrationinto the peritoneum.
Upon injection,
drug
Affuses into surrounding tissues, from there to capillary blood vesselsand lymph. Large
amount of fluids can be administered, whileitis not possiblewith intravenous routedue to
high blood pressure.

1 Restrainthe mouse by scuffing.

2. Prepare the area with an alcohol swab to disinfectthe skin at the site of
administration.

3. To preventthe injuryto internalorgans,handle the animal such that itshead is lower

than itsbody.

4. Insert the needle into the lower left/right

quadrant of the abdomen at a 30 angle,
administerdrug steadily.
5. Aspiratethe syringe to ensure proper placement. Any sign of blood in the syringe
indicatesimproper placement,and the needle needs to be repositioned.
such as a yellow or clearcolourindicatespunctureof the urinary
If upon aspiration,
6.
bladderor green/brown colour indicatespunctureofthe intestines.

injection

) SC injection
Fig.6.1:
Subcutaneous (Sc)Injection:
Intra-peritoneal

into the layer of

is the route of administration
skin below dermis and
(cutis)
to attain high
is advantageous to
raisethe concentrationin blood
epidermis.This route
efficacy.
to be administered.
thesyringewith thesolution
hll
anaesthesia.
or use an appropriate
Restrainthe mouse by scuffing
 Experiments
26
PracticalPharmacology-!

to disinfectthe skin.
3. Swab the area with an alcohol forefinger
fold between your thumb and
Insert the needle at the base of the skin
4. may pierce
an angle to the needle,it
the needle straight,because, if thereis
keeping
through the muscle ofyour finger.
in the syringe indicates
the syringe to ensure proper placement. Blood
S. Aspirate
needs to be repositioned.
placement, and in thiscase the needle
Improper
in a steady, fluid motion.
As you inject,you can feel the
6. Administer the solution
your fingers.
injectionarticlecreating a bulbous under the skin between
and the frontleg.This is
The location is to injectinto the flank,between the hind leg
7.
also thepreferredlocation forinjectingtumor cells.

(b) (c)
(a

Fig.6.2:Subcutaneousinjection
(u)Intradermal (D)Injection:
1 not typically carried out in mice, apart from the
Intradermal injection is

administration of certaincompounds via the footpador ear pinna.

2. must be performed under anaesthesia.

Intradermalinjection

3 When injecting on
the back of the mouse, take the scalpelholder and scalpel
carefullyin one hand and extend the skin between the fingersof the other hand.
Then with thescalpelremove thehair.
Visualize
the skin afterinjection.

4 the skin area with an alcoholswab and insertthe needle carefully

Disinfect through
the dermis at a 30° angle.Aspiratethe syringeto ensure proper placement.Any sign
of blood in the syringeindicatesimproper placement, so the needle needs to be
repositioned.
E Administerslowly,with a maximum volume of 50 ulforfootpadand ear pinna.

(TV) Injection:
(IV)Intravenous restraintdeviceor anaesthetize
it.
mouse into plastic
Placethe
a

1.
with an alcoholswab. Ensure that you can visualize
thelateral
the tailskin
2. Disinfect the use
This
can be assistedwith of a heatedlamp or by placingtheanimal
tailveins. or on the top of warming platefor
warmer
a
a few minutes prior to
in a cage
injection.
 27
Practical Pharmacology Experiments

,Withthe tailunder tension,inserttheneedle approximatelyparallel

tothe vein.
A Ensure proper needle placement by insertingtheneedle at least3 mm intothe lumen
ofthe vein.Once the lumen, the needle shouldfeelsmooth and thereshoulod be
in

no resistanceupon injection.
Intravenousinjectionutilizes
lateral tailveins.

5.Administerin a slow fluidmotion to avoid ruptureof thevessel.,

There is clearingof
thelumen as the injectionarticlereplacesthe blood in the vein.

Fig.6.3:
Intravenousinjection
needle
6. Ifthe solutionleaksinto the surrounding tissuesor forms a bleb,remove the
higher on the vein.
and insertagain slightly
or not.
7. Before returningthe mouse to its cage,ensure bleeding has stopped
(V) Oral Route (P.O.):
1 Selectthe correct-sizedgavage needle on animal intended to use,ensure that
based
sharp.Never use a
there is a metal ball on the end to prevent the tipfrom being
hypodermic needle fororal gavage.
should be no
2. Measure the needle length against the mouse's body; the needle
take care to only
is longer,
longer than from the nose to the last rib.If the needle
Shorter gavage
insertthe appropriatelength to prevent the damage of the stomach.
compounds, ensure thatthe needle fits
needles can be used; but if injectingacidic
to the esophagus.
adequately intothe stomach to prevent damage
amount of drug to be dosed.
the appropriate
3. Fillthesyringewith

4. Restrain the mouse byscuffing.

of the mouse.
5. Placethe tipofthe needle in the mouth
the frontin one fluid
6. Slidethe tip down the back of the mouth, moving it towards
motion.
placement.In such a case,
1. Take your time: any resistance felt indicatesimproper
enter
Do not apply force on the needle, as itmay
remove the needle and start again.
into the
The needle should slide down
the trachea and damage the epiglottis.
esophagus easily.
 28
PracticalPharmacology-!

placed,administerthe drug.
8. Once the needle is properly
so as not to damage the esophagus.
9. Remove the needle carefully

Fig.6.4:Oral route
(VI)Intramuscular(IM)Route:
which allows for
Intramuscularinjectionsdepositmedications intovascularmuscle tissue,
through muscle
rapidabsorptiondue to rich blood supply,allowing fasterrateof absorption
than through the subcutaneousroute.The IM siteis used fordrugs thatrequirea quick
fibers
absorptionrate(fasteronset of action),but alsoa reasonablyprolongeddurationof action.
vastuslateralis
A needle sizeof 21G is mostly used. Sitesfor IM include the ventro-gluteal,
and the deltoidmuscle. Complicationswith IM injectioninclude pain,muscle atrophy,injury
to bone,cellulitis,
abscesses.

Report:

QUESTIONS

1 Enlistthedifferentroutesof administrationin experimental animals.

2. What isthe maximum volume of drug solutionto be administeredin rodentsby oral

route?
3. Explainthe techniqueofadministeringdrug in IP,
LV.,IM,LD,P.O.
and S.C.routes.
4, forselectionofroutesofdrug administration.
Explain the criteria
 Experiments

Practical Pharmacology

ExperimentNo. 2
STUDY OF COMMON LABORATORY ANIMALS

Aim: To study about laboratoryanimals and theirhandling.

Discussion:
and find a cure for a
In pharmacological research,animal models are used to study
organs and body
efficacyof a new treatment. As animals'
diseaseand prove the safety and
animals are susceptibleto the same diseases,that affect
systems are similar to humans and
The
animals usage in experimental pharmacology.
humans, theirshort lifespan allows
speciesincluding dogs,
commonly used laboratory animals are mice, ratsand other animal

cats.rabbits,farm animals,fish,frogs,birds,non-human primates,etc.

1. Mice:
name Mus musculus
Scientific
Balb/Cand C-57
Other strain
37.4°C
Body temperature
90-160/minute
Respiratoryrate

Pulserate 300-750/minute

Blood pressure 120/75mm Hg Fig.2.1

Bloodvolume 70-80ml/kg

RBC COunt 4.9-12.5M/mm

Life span 1-2years

Humidity
30-70%

Room temperature 20-27°C

Mating age 6-8 weeks

Body weight 25-30 gm

Uses studies
Acute toxicity

Assay ofinsulin
Evaluation ofanalgesics
Musculardystrophy
and cancer
Screening of chemotherapeutic agents,teratogens
and geneticstudies.
Isolated
preparations Vas deferens,ileum.
bitten.
taken while handling to avoid being
anaing:Mice will bite,so care should be
Pickthe mouse thumb and forefingerand place it.
grasping at base of the tail
by with
 Experiments

PracticalPharmacology-l

2. Rat:
name
Scientific Rattusnorvegius
Dawley rat
Otherstrain Wistar rat,Spraque
37.5°C
Body temperature

Respiratory rate 80-150/minute

Pulse rate 260-450/minute

129/91mm Hg
Blood pressure
Blood volume 50-65 ml/kg
Fig.2.2
2-3 years
Lifespan
Humidity 30-70%
Room temperature 18.5-27°C

Mating age 70-84 days

|Body weight 120-300gm

• Evaluation of psychopharmacological agents, analgesics, anti
Uses
convulsants.

• Toxicity carcinogenicity,
studies-teratogenicity, gastric secretion
studies(anti-ulcer
activity) physiologicalstudies.
and liver
Isolated Uterus, colon,stomach.
fundus strip,
preparations

Handling:Rats will bite.The best method is pick up rats gently by firmlygraspingtherat

around thechestwith the thumb and forefinger of one hand without compressing the chest
wall.Picking up a rat by grasping the tipof the tailwill usuallycause the skin to separate
from the bone.
3. Guinea Pig:
Scientific
name Caviaporcellus
Body temperature 37.6-38.9°C

Respiratoryrate 60-110/minute
Pulserate 150/minute
Blood volume 65-90ml/kg
Lifespan 2 years
Humidity 40-70%
Room temperature 18.5-27°C Fig.2.3

Mating age 12-20weeks

Gestationperiod 59-72days
size
Litter 4 per year

Body weight 200-1000gm

 PracticalPharmacology Experiments

Uses
• Evaluation of bronchodilatorsagainst experimentally induced
asthma. Wo
• Sensitizationstudies(antigenslike egg albumin, horseserums).
• Study of localanaesthetics.
Bioassay-Digitalis,
histamine and acetylcholine. Ted
• Screening ofspasmodic and antispasmodic compounds.
• Study of vitamin C metabolism (since it requiresexogenous S
vitamin C).
• Study of anti T.B,drugs. Y
Ileum,trachealchain and anococcygealmuscle prevention.
Isolated
preparations
Guinea pigshave four digitson the forelegand threedigitson the hind leg and have a
tail.
vestigial
Handling:The best method is,pick animal up gently by firmlygraspingaround the chest
with theone hand and support the rear legs with the otherhand.
4. Rabbits:
name
Scientific Oryctolagus
Cuniculus

Body temperature
38.7-39.1°C
Respiratoryrate 55/minute

Pulse rate 135/minute

Bloodvolume 45-70 ml/kg

Lifespan 4-5 years Fig.2.4
Humidity 50%
Room temperature 15.5-18.5°C

Mating age 6-9 months

Gestational
period 28-31days
Littersize 4 per year
Body weight 2 to3 kg
Uses • Pyrogen testingin intravenousfluids.
• Antidiabeticstudies.
• Bioassay of insulinand d-tubocurarine.
•Pharmacokinetic studies.
• Capillarypermeabilitystudies.
• Screening of embryo toxicagents and teratogens.
Isolated colon,stomach.
Uterus,fundus strip,
preparations

Handling:Improper handling of rabbitscan cause serious injuries.Pick up a rabbitby

placingone hand under the chest and the otherhand under the animal'srearpart.
 Experiments
10

PracticalPharmacology

5. Hamster: Cricetulusgriseus
auratus or winter white dwart
name
Scientific Mesocricetuz campbelli),
hamster (Phodopus hamster (Phodopus
Other strain Campbell'sdwarf Roborovski
sungorus),
hamster (Phodopus
roborovski)

36.2-37.5C
Body temperature
74/minute
Respiratoryrate

Pulse rate 280-412/minute

Lifespan 2-3years

Humidity
40-60%

Room temperature 37°C Fig.2.5

Mating age 6-8weeks

Body weight 85-140 gm

Feed Soya bean meal and corn

starch

Uses • Cytologicalinvestigation,
immunological and diabetic
studies.
Assay of prostaglandinsE and F,genetics,tissuecultureand
radiationresearch.

Host in investigations, virology.

parasitological toxicology.
nutrition
research,reproductive
physiology.
Isolated Ileum,colon,stomach,trachea,spinalcord,heart.
preparations

Report:

QUESTIONS
1 Listoutthe commonly used experimental
2 animals.
Give the scientific
names and uses forthe
following:
) Mice, (i)Rat,(i) Rabbit,(iv)
Guinea pia.
 Esperiment No. 3

DETERMINATION OF VEIN ISLET

NUMBER, VEIN
TERMINATION AND PALISIDE RATIO ISLETd
Vein-Islet
Number:
The vein-islet
is the small area of
green tissue surrounded by the veinlets(lateral
veins).

Island-
thepieceof land surroundedby water.
Definition:
The vein-islet
number is the averagenumber of vein-isletsper square millimeter
ofa
leafsurfacemidway between the midrib
and margin.
Itis determined by countingthe number of vein-islets
in the area of 1 sq.mm. ofthe
centralpart ofthe fullymatured leafbetween the midrib and
the margin. At leastfour
determinationshould carry out.

Reagent:
Chloralhydrate -15gm of Chloralhydratein 20 ml of water.
Procedure:
Take the fullygrown leafof the plantunder studyand make
suitablesizepiecesfrom
the centralpartsbetween midriband the margin.

Dechlorophyllizedthe pieces of the leaf by boilingin choral hydrate

solutionfor
about thirtyminutes.
Arrange camera lucidaand drawing board for making drawings to scale.
Place a stage micrometer on the microscope and using
10x objectives and 5x
1
eyepiece,draw a lineequivalentto mm as seen throughthe microscope
and draw a
squareon thisline.
Adjust the black paper on board so that the square is seen
in the eyepiece, in the
centerof thefield.Place the slidewith the cleared leaf(epidermis on the
stage).
Trace allthe veins which seen as superimposed on the 1
sq. mm square.Draw the
vein islets
which are more than 60% presentwithin the
square.
Count thenumber of vein isletsin the sauare millimeter.Find the average
number of
vein islets
from thefourdeterminations,
to getthe averagevalues forone Sq. mm.

-3.1
 Experiment-3

Pharmacognosy & Phytochemistry-l (Practical)

Vein islets
TerminationNumber: islet.
of the vein
A veintermination is the free end (termination)

Definition: per sq.

termination
of vein islet
Vein islet the number
as
terminationnumber isdefined
margin.
mm ofthe leaf midrib of the
leaf and its
surface,midway between

Fig.3.1:
Veins and Vein Islet
Enlarged
Procedure:

Take the fullygrown leafof the plant

under study and make suitablesize pieces from
thecentralpartsbetween midrib
and the margin.
Dechlorophyllizedthe piecesof the leaf
by boiling in choral
about thirtyminutes. hydrate solutionfor

Arrange camera lucidaand

drawing board formaking
Place a stage drawings to scale.
micrometer on the
microscope and using 10x
eyepiece,draw a line objectivesand 5x
equivalentto 1 mm as seen
Squareon thisline. through the microscope and
draw a
Adiust the black
paper on board so that
the square is seen
centerofthefield. in the
Place the slidewith eyepiece,in the
the clearedleaf
Trace all the veins (epidermis on the stage).
terminationwhich seen as
Count the number of superimposed on the sq. 1
vein isletsin the mm square.
square millimeter.
of vein isletsterminationfrom the four Find the average number
one sq.mm.
determinations, gettheto
average valuesfor

-3.2
 Pharmacognosy & Phytochemistry-i(Practical) Experiment - 3

Palisaderatio

:
It is average number of palisadecells beneath one
epidermal cell of a leaf.It is
determinedby countingthe palisadecellsbeneath fourcontinuous
epidermalcells.
Table:Palisaderatiosof variousleaf-drugs
Sr. Name of Plant
Range
No.

1 Adhatoda vasica 5.5– 6.5

2. Andrographispaniculata 03 – 4.5
3 Atropabelladonna 05 -7.0
4. Azaderachta indica 03-3.5
5. Bacopa monneira 1.5-2.25
6. Cassiaangustifollia
5.5–10.0(upper) 4.0-7.4
(lower)
7. Centellaasiatica 3.5-5.75
8. Daturametel 05 -6.5
9 Daturastramonium 4.2-6.5
10. Digitalis
lanata 2.5–6.5
11. Digitalis
purpurea 3.7–4.2
12. Eucalyptus globules -
5.5 6.5(upper) 3.5–5(lower)
13. Hyoscyamus niger 03-06
14. Lawsonia inermis 07-8.5(upper) 4.5–5.7(lower)
15. Mentha arvenis
3.5 -0.5
16. Mentha piperita 6.1-8.5
17. Momordica charantia 2.5-3.8
18.. Nicotianatabacum
3.5-0.4
19. Ocimum sanctunm 0.2-3.5
20. Thymus vulgaris 1.7-3.2

-3.3
 Eperiment No. 5

DETERMINATION OF FIBRE LENGTH AND NIDTH

Requirements:
Stage micrometer,eyepiecemicrometer, compound microscope,powdered crude
drug sample (Ceylon cinnamon,Cassiabark or Cinchona bark).

Procedure:

• Calibratethe eyepice micrometer using stage micrometer, calibrateand calculate

the factor(average distance between two lines of one division of eyepiece
micrometer in microns).
quantityof powder drug in a testtube,decolorized by boilingwith clearing
Take little
solution(chloralhydrate).
Wash the decolorizedpowder drug with water in a watch glass.

Stain the lignifiedfibreswith the staining reagent(phloroglucinoland concentrated

hydrochloric
acid).
Mount this treated powder thinly,uniformly scattered,without overlapping of

particlesin glycerinewater and observe the slide under low power and focus
a

stained intactfibre.
the
By rotatingthe scale of eyepiecemicrometer, note the numbers of divisionsof
eyepiece micrometer covered by the length of the fibreagain rotatethe eyepiece
micrometer without disturbingthe slideand find the numbers of the divisionof the

eyepiece micrometer covered by the width ofthe same fibre.

calculatethe length and width of about 25 fibres,
Similarly, and write the readings in

two separatecolumns.
Calculatethe mean length and width of fibre
in micron.

-5.1
-
 18
(Practical) Experiments
PhysicalPharmaceutics-II
Experiment
No.5
by determining
determine the flow properties following
flow
Object To
givenpowder:
parameters of
(a) Angle ofrepose
Index
(b)Carr'sCompressibility
(c) Hausner'sRatio
measuring di
Requirements : Powder sample, funnel, bulk density apparatus,

stand,balance,simple graph paper.

Theory:
Flow Properties
A bulk powder is somewhat analogous to a which
non-Newtonian liquid, exhibitsnlsek

flow and sometimes dilatancy,the particlesbeing influencedby attractiveforces to varyjing

or cohesive("sticky").
degrees.Accordingly,powders may be free-flowing Neumann discussed

the factorsthataffectthe flow propertiesofpowders. Of specialsignificanceare particlesize

shape,porosityand density,
and surfacetexture.
With relativelysmall particles(less than 10 um), particleflow through an orifice is
restrictedbecause the cohesive forces between particlesare of the
same magnitude as
gravitationalforces.Because these latterforces are a function of the
diameter raised the to
third power, they become more significant
as the particlesize increases and flowis
A maximum
facilitated. flow rateis reached,afterwhich the flow
decreasesas the size of the
particlesapproaches thatofthe orifice 50.Ifa powder containsa reasonablenumber of small
particles,the powder's flow propertiesmay
be improved by removing the
adsorbing them onto the larger "fines"

or
particles.Occasionally,poor flow may resultfrom
presence of moisture, in which the
case drying the particleswill
Elongated or flatparticlestend
reduce the cohesiveness.
to pack, albeitloosely,
to give powders with a high
Particles with a high density porosity.
and a low internal
porosity tend to possess
properties. This can be offset
by surfaceroughness, free-flowing
which leads to poor
due to friction and flow characteristics
cohesiveness.
Angle of Repose
The frictional
forces a loosepowder can be
in
defined as,"maximum measured by the angle of repose
angle possiblebetween the which is
surface of a pileof
horizontal
plane".The flowofpowder the powder and the
and theangle of
repose is given by:
tan o =

where, h- Heightofpilein cm
r-Radius ofthe base ofthepilein
cm
-Angle of repose
Lower the angle of repose
betteristhe fiowproperty.
Rough and irreqular
surfaceofthe
particles
give higherangle ofrepose.
 Dhvsical Pharmaceutics-I(Practical)
19
Sr.No.
1
Angle of
repose
<25
) Flow property
Experiments

2 Excellent
25-30
3 Good
30-40
4 Passable
> 40
Very poor
Angle of repose
helps inpredictingflowproperties
halns in predicting the during compression.
conditions to be adopted in Compressibility
the
imnrove the flow properties of the material,glidants compression of dosage forms.To
magnesium stearate, are added into the
starch and talc. granules such as
Generally,optimum
concentration is desirableto concentration of 1% or less
improve the flow properties of
concentration, decreasein flowrate granules. Above this
was observed
Carr'sConsolidationIndex

CCI = Tapped density- Fluffdensityx

100
Tapped density
Thispropertyis known as compressibility.It is
somewhat relatedto the flow rate,
cohesiveness,shape and sizeofthe particle
and moisture content.
Tapped Density
Itis "the ratioofmass of powder to the tapped volume". Tapped volume is "the volume
occupiedby thesame mass of powder aftera standard tapping
measurement".
FluffDensity
Itis"theratioof mass of powder to the fluffvolume".Fluff volume is "thevolume
occupied by a certain mass when poured intoa measuring cylinder"
Sr.No. CCI Flow property
1. 5-12 Excellentflow

2 12-16 Good

3 18-21 Fair

4 >23 Poor

Hausner Ratio

Itmay bedefined as, "theratioof volume (Vo)of powder bed at initialstage (m) to the
volume(V)ofpowder bed aftertapping (ml)".Hausner ratiois relatedto themorphology of
thepowder.

Hausner ratio

If

Hausner ratiovalue is egual to or less than 1.2-g0od flow property is obseved

because of lessinter-particle
friction.

hausner'sratiovalue ismore than 16-poor flowpropertyis observed.

 20
(Practical) Experiments
Pharmaceutics-II
Physical

Procedure: of 6.4 cm from the surface of working shelf with

the help
at a height
1. Place funnel of
stand.
bottom and closethe end of funnel with cotton.
2. Place a graph at the
in the funnel.
3. Keep 20 g ofpowder
the graph paper by removing the cotton.
4, Allow the flow ofpowder on
and heightof itand calculateangle of ren.
5. Afterformationofheap,find the radius
by given formula.
6. Repeat itthriceand findthe mean angle of repose.

7. index, place a known

For Carr'scompressibility weight of sample in a measurinn

and note the bulk volume.

cylinder
8. Allow tapping of cylinder in bulk density apparatus 50 times and
again note the

tapped volume.
9. CalculateCCI and HR using given formula.
ObservationTable:
Sr Height(cm) Diameter (cm) Radius (cm) Angle of repose
No. tan (h/r)
1.
2.

3.

Angle ofrepose = tan

Bulk density
CCI = 1 x 100
(Tapped density

HR = Tapped density
Bulk density
Report :The calculatedflow parameters were found
to be which indicates
excellent/good/bad
flow ofgivenpowder.
 Experiment No. 6
Object:
To determine effect
oflubricanton angleofrepose.
Requirements:Powder sample, funnel,
stand,balance,talc, simplegraph paper.
Theory:
Flow Properties
A bulk powder is
somewhat analogoustoa non-Newtonian liquid,
flow and sometimes dilatancy, which exhibitsplastic
the particlesbeing
influencedby attractiveforcesto varying
dearees.Accordingly, powders may be free-flowing or cohesive("sticky).
Neumann discussed
the factorsthat affecttheflow properties
of powders. Of specialsignificanceare particle
size,
shape,porosityand densityand surfacetexture.
 physical Pharmaceutics-I(Practical)
21
with relatively Experiments
small particles(less
because the than 10 um),
restricted cohesive forces particle flow
through an orifices
forces. Because between particles
are of the
gravitational theselatterforces same magnitude as
are a function
third power, they become more of the
diameter raisedtothe
Ifa powder containsa significantas the particlesize
ilitated.
reasonablenumber of small increases and flow is
roDertiesmay be improved particles,the powder'sflow
by removing the
rticles. Occasionally, poor flow may resultfrom
"fines" or adsorbing them onto the
the presence of larger
ning theparticles moisture,in which case
will reduce the
albeitloosely, togive powders with cohesiveness. Elongated or flatparticlestend
to pack,
a high porosity. Particleswith a high
internalporositytend to densityand a low
possess free-flowingproperties.
rOughness, which leadstopoor This can be offsetby
flowcharacteristics surface
due to frictionand
Angle ofRepose : cohesiveness.
The frictionalforcesin a loosepowder can be
measured by the angle of repose
defined as,"maximum which Is
angle possiblebetween the surfaceofa pile
of the powder and the
The flowof powder and the
horizontal plane".
angle ofrepose is given by:
h
tan =

where, h- Heightofpilein cm
r - Radius of the base of the pilein cm
- Angle ofrepose
Lowertheangle of repose,better is the flow property.Rough and irregularsurfaceof the
particlesgivehigherangle ofrepose.
Sr.No Angle of repose () Flow property
1 < 25 Excellent

2 25-30 Good

3 30-40 Passable

4 > 40 Very poor

Angle ofrepose helps in predictingflow properties during compression. Compressibility

of dosage forms. To
helpsin predictingthe conditionsto be adopted in the compression
are added into the granules such as
improve the flow propertiesof the material,glidants
of 1 % or less
magnesium stearate,starch and talc.Generally,optimum concentration
Above this
improve the flow properties of granules.
Concentration is desirable to
observed.
Concentration decrease in flowratewas

.
Procedure :
shelf with the help of
at a height of 6.4 cm from the surfaceof working
1. Placefunnel
stand.
with cotton.
and closethe end offunnel
<. Place a graph at the bottom
3. Keep 20 g of powder in the funnel. by removing the cotton.
on thegraph paper
Allow the flow of powder ofitand calculateangle of
repose
the radiusand height
3. formationof heap find
After
by givenformula.
 22
(Practical) Experiments
PhysicalPharmaceutics-II

thrice and find the mean angle of repose,

6. Repeatsame
and 0.75%)of talc in the powder
ea

c
different oncentrations (0.25%, 0.5%
7. Add of repose.
forcalculation of angle
and repeatthe same procedure
ObservationTable

:
Radius (cm) Angleofrepose
Sample Sr. Height Diameter
No. (cm) (cm)
tan

Powder sample 1
2
3

Sample + 0.25% talc 1

2.

3.

Sample + 0.5%talc 1
2.

3.

Sample + 0.75% talc 1

2.

3.

Angle of repose = tan

Report:
The angle ofreposewas decreased/increasedby additionof lubricant.
 AtropineSulphate
12:
Experiment

Aim: To perform assayofatropine

sulphate.

Requirements: acetic
acid, anhydrous glacial acetic acid,
Chemicals: Atropine sulphate,perchloric
crystalviolet.
anhydride,potassium hydrogen phthalate, burette
measuring
burette, cylinder,
flask,pipette,pipette filler,
Apparatus:Beaker, conical
bench mat.
glassfunnel,white tile,
stand and holder,

Principle:
be determined by titration
in non-aqueous
Atropine sulphate can quantitatively
solvents.

The total alkaloids of the atropine group are determined by titration

against

acidin anhydrous aceticacid.

perchloric

The method can suppositories

be applied to tablets, and eye drops containing
atropine.

or with crystalvioletas indicator.

The endpointis determined potentiometrically
Theory:

(A)Atropinesulphate(Analyte):
1. Molecularstructure:
CH,

OH
H,so,

2
2. Molecularformula:
(CH23NO;)2H,SO4.H0
3. Molecularweight:676.8
 Medicinal Chemistry - I
Experimental 45
Experiments
Chemicalnames:
(RS)-(1R,3r,5S)-3-tropoyloxytropanium
5. Melting point:190-194°C sulphatemonohydrate

Itiscolorlesscrystals
Description:
or a white,crystalline
, Itis very solublein ethanol;
Solubility:
powder;odourless.
slightlysolublein chloroform;
ethylether. insolublein

8. Pharmacologicalactivity:
()AtropineSulfateis the sulfatesalt of
atropine,a naturally-occurringalkaloid
isolatedfrom the plantAtropa
belladonna.
(ii)Atropine functions as a
sympathetic,competitive antagonist of
cholinergic receptors,thereby abolishing the muscarinic
effectsof parasympathetic
stimulation.

B) acid (Titrant):
Perchloric ReferAssay of ChlorpromazineHydrochloride.
(g Crystalviolet (ndicator):
ReferAssay ofChlorpromazineHydroch loride.

Preparationsof Reagents/Solutions:
(a) 0.1
M Perchloricacid:
ReferAssayofChlorpromazineHydrochloride.
(b) Crystalviolet
solution:
ReferAssay ofChlorpromazine Hydrochloride.ab
STANDARDIZATION:
ofPerchloricacid:ReferAssay ofChlorpromazineHydrochloride.
Standardization
Factor:1 mL of 0.1M HCIO, 0.02042g of CgH;04K

ASSAY OF ATROPINE SULPHATE:

Procedure (Volumetric):
30 mL of
Weigh accurately about 0.5 g of atropinesulphate powder and dissolvein
anhydrous glacialaceticacid.
Titratewith 0.1 M perchloric acid, determining the end-pointpotentiometrically.
Carry out a blank titration.
Procedure(Potentiometric):
differenceversusthe volume of the titrant
Plota graph of the variationsof potential
(0.1M perchloricacid)added.
the presued equivalence point.
Continuing the additionof the titrantbeyond
sharp variationof potentialdifference.
The end- point correspondsto a omitting
by
Perform a blank titration repeatingthe procedure in the same manner

the substance under examination.

under examination is the
Ihe actualvolume of titrant equivalentto the substance
and that consumed
between the volume consumed in the blank titration
difference
with the substance under examination.
In the titration
 Experiments
46

Experimental Medicinal Chemistry-I

ObservationTable: (mL ofHCIO,)

BuretteReading Mean
Sr.
Final
No. Initial A+ B+C
A mL
0.0mL 3
1.
B mL
2. A mL =X mL
C mL
3. B mL

Reaction: HQO
HO
OH
OCIO,
OH

Atropinesulphate Perchloric acid

oncituiczlol
FactorCalculation:
acid
1 mole of Perchloric l mole of Atropinesulphate

1000 mL of 1 M Perchloric
acid 676.8gm of (CiyHz3NO)zH,SO4

1 mL of 1 M Perchloric
acid = 0.6768gm of (C7HzsNO,)z H,SO4
1 mL of 0.1M Perchloric
acid = 0.06768gm of(C1yHz3NO)2-H,SO4
Factor:

1 mL of0.1M Perchloric
acid = 0.06768gm ofAtropinesulphate
Calculations:

StepI:
(NormalityofHCIO)
1 mL of0.1M HCIO4 = 0.02042g of potassium hydrogen phthalate
XmlBurete reoding of Of y
standardzotion) M HCIO4 = 0.35g of CeHsKO.
0.35x 1 x 0.1
=
X mL) x (0.02042)
y = ......
M
Step I:
(Amount of AtropineSulphate)
1 mL of 0.1M Perchloricacid 0.06768 gm of Atropinesulphate
X mLBurettereading ofassay)of y M HCIO4 = Agof Atropinesulphate

X mLxy x 0.06768g
Ag = 1x0.1
 |
Bxperimental
Medicinal Chemistry- I 47
Experiments
(%Purity)
StepII:
For 0.5g ofAtropine
sulphate 100%
Agof Atropine
sulphate P%
Hence,

P= L00)
0.5
x (A)

henit Atropinesulphatecontainsnot less

than 99.0%and not more than 101.0%
ofatropine
ailphate,
(CiHzaNO)2.HzSO4, calculatedon the anhydrous basis.
Result:
1. The molarityof Perchloric
acidsolution(prepared,0.1M) was found to be
2. The percentpurityof givensample ofAtropinesulphatewas ...M.
found tobe....%w/v
 Experimnent 15:
Furosemide (Frusemide)

Aim: To performassay of Furosemide(Frusemide)

Requirements:
Chemicals: Furosemide, dimethylformamide, sodium
hydroxide, bromothymol blue

indicator.

burette,measuring cylinder,burette
Apparatus:Beaker,conicalflask,pipette,pipettefiller,
stand and holder,
glassfunnel,whitetile,
bench mat.
Principle:

1 The assay offurosemideis carriedout by aqueous acid-basetitration

which is based
on simpleneutralization
reaction.
2. Itisa directtype of titration.

3. Furosemide dissolvedin dimethylformamide and directlytitratedwith

is
sodium
hydroxideusing bromothymol blue as an indicator.
Theory:
(A)Furosemide(Frusemide)
(Analyte):
1.Molecularstructure:
OOH
H

H,N
C
2. Molecularformula:
CyHuCIN,0;S
3. Molecularweight:330.7.
 Medicinal
Experimental Chemistry-I 6
Chemicalnames: Experiments
Frusemideis
6 Meltingpoint:203- 206°c. 4-chloro-N-furfuryl-5-sulphamoylanthranilic
acid.
Itis a white or
pescription:
almostwhite,
, Solubility:Itis slightlysoluble crystalline
powder.
in water.Slightly
Solublein acetone, solublein chloroformand
methanol, DMF, aqueous solutions ether.
ethanol. above pH 8.0.Less soluble
in

8.Pharmacologicalactivity:
Furosemide is a sulfamoylanthranilic
acidderivative,
also known as furosemide,
and potentloop diuretic.
Itiswidely used totreathypertensionand edema.
This agent is highlybound to albumin and is largelyexcretedunchanged in the
urine.

B) Bromothymol blue solution(Indicator):

1.Molecular structure:
HO Br

H,C H5
-CHs
CH,
H,C
-OH

CHa B

2. Molecular formula:CzpHBr;0;S
3. Molecular weight:624.38
Bromothymol blue,Bromothymol
phthalein,
4. Chemical names: Bromothymol sulfone
7.1).
blue (BTB) is a pH indicator(pka
202°C
5. Melting point:
over a pH range
blue is 6.0 to 7.6.Itchanges color
6. The pH range of bromothymol
from 6.0(yellow)to 7.6 (blue).
less solublein non-polar
soluble in water, ether,
7. Solubility:Bromothymol blue is

tolueneand xylene.
solventssuch as benzene, be in
a weak acid in solution.Itcan thus
as
8. Mechanism:
Bromothymol blue acts The
appearing yellow or blue, respectively.
form, thus
protonated or deprotonated blue has its peak absorption
at 692 nm,

protonated form
of bromothymol has itspeak
And the deprotonatedform
lightin acidicsolutions.
transmittingyellow blue in more basic solutions.
light
at 602 nm thus transmitting
absorption
 Experiment

Chemistry-I
Experimental Medicinal

2.5g (85% w/w).

yield:
Theoretical
171-172°C.
Melting point:
Physicalconstant:
Table: Actual Molar
Calculation
ActualQuantity Moles taken
Reactant Molecular Ratio
Sr
(g) taken (Unit)
No. Weight 0.025 1
2.79
108
OPD
13 mL 0.34 13.6
2. 46
90% Formic acid (density= 1.2)

3. NaOH 40

PracticalYield x
100
The percentyield= Theoretical
Calculation: Yield

Result:

1. The percentageyield of Benzimidazolewas

found to be .....%
w/w.

2. Physicalconstant:

3. Appearance: