Experiment No.

1
Aim: To Perform Preliminary Phytochemical Screening of Crude Drugs.

Theory
The process of detection of various constituents in a plant extract is known as phytochemical
screening. Plant contains numerous chemical constituents that are responsible for eliciting
various physiological and therapeutic responses. Therefore, plants are generally tested for the
presence of biologically active and medicinally useful phytochemical constituents responsible for
a particular biological activity. Some of the examples of phytoconstituents include alkaloids,
glycosides, carbohydrates, Flavonoids, phytosterols, saponins, tannins etc.

The following aspects should be taken into consideration while choosing a method for
phytochemical screening.

1. It should be simple and rapid.

2. It should be designed such that it uses minimum equipments.
3. It should be suitable for studying all the types of compounds.
4. It should be quantitative in approach.
5. It should have an advantage of detecting the presence or absence of specific compounds of a
group being evaluated thereby furnishing additional information.
Importance
l. Phytochemical screening helps in expanding the classification of plants based on their
chemistry.
2. It helps in the discovery of new therapeutic agents and in the production of semi synthetic
derivatives.
3. It helps in isolating and characterizing the chemical constituents present in plant extracts.
4. It helps in understanding the herbal drugs and their preparations.
5. According to Ayurveda, plants are known to possess chemical constituents capable of curing
various diseases. Phytochemical screening helps to prove the claims of Ayurveda and other
folkloric remedies.
Qualitative Preliminary Phytochemical Analysis
The purified extracts either organic, alcoholic or water extracts are then subjected to the various
qualitative tests for the detection of plant constituents like alkaloids, glycosides, fixed oils,
tannins, resins, proteins, amino acids, volatile oils, gums and mucilages etc.

Questions
Q.1. Give different types of phytochemiocal screening.
Q.2. Which different compounds present in plant.
Q.3. Define secondary metabolites.
Q.4.Which is the aspects should be taken into consideration while choosing a method for
phytochemical screening.
Q.5. What is the significance of phytochemical screening?
I. Detection of Carbohydrates.

Requirements
Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Distilled water, alcoholic solution of α– napthol, Fehling’s solution A and B,
Barford’s reagent, Benedict’s reagent.

Tests
Stock solution: About 100mg of the extract is dissolved in 5 mL of Distilled water and filtered.
The filtrate is subjected to the following tests.

1. Molisch’s Test
Principle: Molisch's test: The test is named after Austrian botanist Hans Molischis a
sensitive chemical test for the presence of carbohydrates, based on the dehydration of the
carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which condenses with
two molecules of phenol (usually α-naphthol, though other phenols (e.g. resorcinol, thymol) also
give colored products), resulting in a red- or purple-colored compound.

Procedure: To 2 mL of filterate, two drops of alcoholic solution of α– napthol is added. The

mixture is shaken well and 1 mL of concentrated sulphuric acid is added slowly along the sides
of the test tube, the test tube is cooled in ice water and allowed to stand. A violet ring at the
junction of two liquids indicates the presence of carbohydrates.

2. Fehling’s Test

Principle: Fehling's solution is a chemical reagent used to differentiate between water-

soluble carbohydrate and ketone functional groups, and as a test for reducing sugars and non-
reducing sugars, supplementary to the Fehling test. The test was developed by German
chemist Hermann von Fehling in 1849.
Procedure: 1 mL of filtrate is boiled on a water bath with 1 mL each of Fehling’s solution A and
B. Formation of red precipitate indicates the presence of sugar.

3. Barfoed’s Test

Principle: Barfoed's test is a chemical test used for detecting the presence of monosaccharide’s.
It is based on the reduction of copper (II) acetateto copper(I) oxide (Cu2O), which forms a brick-
red precipitate.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
(Disaccharides may also react, but the reaction is much slower.) The aldehyde group of the
monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate. A
number of other substances, including sodium chloride, may interfere.
It was invented by Danish chemist Christen Thomsen Barfoed and is primarily used in botany.
The test is similar to the reaction of Fehling's solution to aldehydes.
Procedure: To 1 mL of the filtrate, 1 mL of Barfoed’s reagent is added and heated on aboiling
water bath for 2 minutes. Red precipitate indicates the presence of sugar.

4. Benedict’s Test
Benedict's reagent (often called Benedict's qualitative solution or Benedict's solution) is a
chemical reagent named after American chemist Stanley Rossiter Benedict.
It is a complex mixture of sodium carbonate, sodium citrate and copper (II)
sulfate pentahydrate. It is often used in place of Fehling's solution to detect the presence
of reducing sugars. The presence of other reducing substances also gives a positive reaction.
Such tests that use this reagent are called the Benedict's tests. A positive test with Benedict's
reagent is shown by a color change from clear blue to a brick-red precipitate.
Generally, Benedict's test detects the presence of aldehydes and alpha-hydroxy-ketones, also
by hemiacetal, including those that occur in certain ketoses. Thus, although the ketose fructose is
not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive test because it is
converted to the aldoses glucose and mannose by the base in the reagent.
The principle of Benedict's test is that when reducing sugars are heated in the presence of an
alkali they are converted to powerful reducing species known as enediols. Enediols reduce the
cupric compounds (Cu2+) present in the Benedict's reagent to cuprous compounds (Cu+) which
are precipitated as insoluble red copper(I) oxide(Cu2O).The color of the obtained precipitate
gives an idea about the quantity of sugar present in the solution, hence the test is semi-
quantitative. A greenish precipitate indicates about 0.5 g% concentration; yellow precipitate
indicates 1 g% concentration; orange indicates 1.5 g% and red indicates 2 g% or higher
concentration.

Procedure: To 0.5 mL of filtrate 0.5 mL of Benedict’s reagent is added. The mixture is heated
on a boiling water bath for 2 minutes. A characteristic colored precipitate indicates the presence
of sugar.

Questions
Q. 1. Define carbohydrates.
Q. 2. Classify the carbohydrates.
Q. 3. Who invented the Molish test?
Q. 4. What is the basic unit of carbohydrates?
Q. 5. Which colour given by Benedicts test.
II. Detection of Alkaloids.

Requirements
Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Mayer’s reagent, Wagner’s reagent, Hager’s reagent, Dragendroff’s reagent

Tests
Stock solution: About 50 mg of solvent free extract is stirred with little quantity of dilute
hydrochloric acid and filtered. The filtrate is tested with various alkaloidal reagents as follows:

Mayer’s Test
Principle: Mayer's reagent is an alkaloidal precipitating reagent used for the detection
of alkaloids in natural products. Mayer’s reagent is freshly prepared by dissolving a mixture
of mercuric chloride (1.36 g) and of potassium iodide (5.00 g) in water (100.0 mL).Most
alkaloids are precipitated from neutral or slightly acidic solution by Mayer’s reagent (potassium
mercuric iodide solution) to give a cream coloured precipitate. This test was invented by the
German Chemist, Julius Robert Von Mayer (1814–1878).

Procedure: To a few mL of filtrate, two drops of Mayer’s reagent is added along with the sides
of the test tube. If the test is positive, it gives white or creamy precipitate.

Wagner’s Test
Procedure: To a few mL of the filtrate, few drops of Wagner’s reagent were added along with
the sides of the test tube. Formation of reddish brown precipitate indicates test as positive.

Hager’s Test
Procedure: To a few mL of filtrate 1 or 2 mL of Hager’s reagent is added. A prominent yellow
precipitate indicates positive test.
Dragendroff’s Test
Principle: Dragendorff’s reagent is a color to detect alkaloids in a test sample. Alkaloids, if
present in the solution of sample, will react with Dragendorff’s reagent and produce an orange or
orange red precipitate. This reagent was invented by the German pharmacologist, Johann Georg
Dragendorff (1836–1898) at the University of Dorpat.

Procedure: To a few mL of filtrate, 1 or 2 mL of Dragendroff’s reagent is added. Aprominent

reddish brown precipitate indicates positive test.

Questions
Q. 1. Define alkaloids.
Q. 2. In plant the alkaloids are found in which form.
Q. 3. Give the solubility parameter of alkaloids.
Q.4. Give the composition of Hager’s Reagent.
Q. 5. Classify the alkaloids according to presence of N atom.
III. Detection of Glycosides.

Requirements
Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Concentrated hydrochloric acid, ethylacetate, ammonia solution, pyridine, Sodium
nitroprusside solution, sodium hydroxide solution

Procedure: For detection of glycosides, about 50 mg of extract is hydrolyzed with concentrated

hydrochloric acid for 2 hrs on a water bath, filtered and the hydrolysate is subjected to the
following test.

Borntrager’s Test
Principle: Borntrager's test is a chemical test for the identification of anthraquinone glycosides.
Here anthraquinone glycosides are four type which O, N, S, C, and borntrager's test is use for O
glycosides and modify borntrager's test use for C glycosides

Procedure: To 2 mL of filtrate hydrolysate, 3mL of ethylacetate is added and shaken,

ethylacetate layer is separated and 10% ammonia solution is added to it. Formation of pink color
indicates the presence of anthroquinone glycosides.
Legal’s Test
Procedure: About 20 mg of the extract is dissolved in pyridine. Sodium nitroprussidesolution is
added and made alkaline using 10% sodium hydroxide solution.Presence of glycoside is
indicated by a characteristic pink color.
Questions
Q. 1. Define glycosides.
Q.2. Give the different types of glycoside on the basis of linkage between glycon and aglycon
Part.
Q. 3. The solubility of glycosides is depend on.
Q. 4. Pharmacologically active part in glycoside is known as.
IV. Detection of Saponins.

Requirements
Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Distilled water, acetic anhydride, concentrated sulphuric acid.

Foam or Froth Test

Procedure: A small quantity of the extract is diluted with Distilled water to 20 mL. The
suspension is shaken in a graduated cylinder for 15 minutes. A two centimeter layer of foam or
froth which is stable for 10 minutes indicates the presence of saponins.

V. Detection of Phytosterols and Triterpenoids

Libermann – Burchard’s test

Procedure: The extract is dissolved in acetic anhydride, heated to boiling, cooled and then 1 mL
of concentrated sulphuric acid is added along the side of the test tube. Red, pink or violet color at
the junction of the liquids indicates the presence of steroids / triterpenoids and their glycosides.

Salkowoski test
Procedure: Few drops of concentrated sulphuric acid is added to the extract, shaken on standing,
red colour in the lower layer indicates the presence of steroids and golden yellow color indicates
the presence of triterpenoids.

Questions
Q. 1. Give any two crude drugs which gives positive chemical test for foam or Froth test.
Q. 2. What is the basic unit molecule of triterpenoids.
VI. Detection of Phenolic Compounds and Tannins.
Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Distilled water, ferric chloride solution, sodium chloride, lead acetate solution,
ammonium hydroxide solution, magnesium turnings, con. hydrochloric acid.

Ferric chloride test

Procedure: About 50 mg of extract is dissolved in Distilled water and to this few drops of
neutral 5% ferric chloride solution is added. Formation of blue, green and violet color indicates
the presence of phenolic compounds.

Gelatin test
Procedure: A little quantity of extract is dissolved in Distilled water and 2 mL of 1%solution of
gelatin containing 10% sodium chloride is added to it. Development of white precipitate
indicates the presence of phenolic compounds.

Lead acetate test

A small quantity of extract is dissolved in Distilled water and to this; 3 mL of 10% lead acetate
solution is added. A bulky white precipitate indicates the presence of phenolic compounds.

Alkaline reagents
An aqueous solution of extract is treated with 10% ammonium hydroxide solution – yellow
fluorescence indicates the presence of flavonoids.

Shinoda test or Magnesium – Hydrochloric acid reduction

A little quantity of extract is dissolved in alcohol and few fragments of magnesium turnings and
con.hydrochloric acid (drop wise) were added. If any pink orcrimson – red color develops,
presence of flavonol glycoside is inferred.
Questions
Q. 1. Give any two drugs having phenol as active compound.
Q. 2. Give any two drugs having tannin as active compound.
Q. 3. What is the taste drugs having tannin as active compound.
VII. Detection of Flavonoid.

Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Distilled water, furfuraldehyde, sodium hydroxide, ethanol, zinc dust lead acetate
solution, ammonium hydroxide solution, magnesium turnings, con. hydrochloric acid.

Procedure: 0.5mL of aqueous solution of extract is added to 2mL of 2% (V/V) furfuraldehyde

in a test tube– Red color indicates the presence of flavonoids.

1) Alkaline reagent test: Extract is treated with 10 % NaOH solution; formation of intense
yellow color indicates presence of Flavonoid.
2) NH4OH test: 3 mL of extract is 10 % NH4OH solution development of yellow fluorescence
indicates a positive test.
3) Mg turning test: Extract were treated with Mg turning and add conc. HCl to this solution add
5mL of 95 % ethanol, formation of crimson red colour indicates Flavonoid.
4) Zn test: 2 mL extract were treated with Zn dust and conc. HCl development of red colour
indicates presence of Flavonoid.

Questions
Q. 1 .Give any two drugs having flavonoids.
Q. 2 .General uses of flavonoids are.
Q. 3. Effects of flavonoids on free radicals prepared by body.
VIII. Detection Amino Acids

Apparatus: Funnel, measuring cylinder, filter paper, pipet, test tube, test tube holder, test tube
stand, water bath
Chemicals: Distilled water, ferric chloride solution, Ninhydrin Reagent, concentrated nitric acid,
sodium hydroxide, Folin’s phenol reagent, sodium carbonate, Million’sreagent, sodium nitrate,
sulphanilic acid.

Procedure: The extract (500 mg) is dissolved in 5 mL of Distilled water. To this, few drops of
neutral 5% ferric chloride solution were added. A dark green color indicated the presence of
phenolic compounds.
Ninhydrin test : To 1mL of sample, add 5drops of Ninhydrin Reagent. Heated in a boiling water
bath for2 min. A purple color indicates the presence of amino acids.
Xanthoproteic test : To 3mL of the sample, add 1mL of concentrated nitric acid and heated for
3min. Then cooled and added 0.5 mL of NaOH. Reddish orange color indicates the presence of
aromatic amino acids.
Folin’s test :To 1mL of sample, add 1mL of Folin’s phenol reagent followed by the addition of
1N sodium carbonate. The blue color indicates presence of tyrosine and tryptophan.
Million’s test :To 1mL of sample, add 1mL of Million’sreagent and heated for 3 minutes. Then
1% sodium nitrate is added. Red color formed indicates the presence of Tyrosine.
Pauly’s test To 1mL of sample, add 1mL of 1% sulphanilic acid and cooled in ice. Then 1mL of
sodium nitrite added. After 5 min, 2mL sodium carbonate added. Presence of cherry red color
indicates the presence of Histidine.

Questions
Q. 1 .Give the ninhydrine solution.
Q. 2. Give the uses of amino acid.
 Experiment No. 2
Aim: Determine The Alcohol Content of Asava And Aristha.

Standardization of Asava and Aristha

Procedure: Various methods are there for standardization of asava and aristha
1. Organoleptic evaluation
Organoleptic evaluation is carried out for following parameters colour, odour, taste, nature and
texture of given sample of asava and aristha.
2. Determination of alcohol content in asava and aristha.
Requirements:
Apparatus: Digital weighing balance, beaker, measuring cylinder, distillation flask, separating
Funnel, heating mental, water bath, specific gravity bottle.
Chemicals: Water.
Procedure: Measure 25 mL of formulation by measuring cylinder and transfer to distillation
flask having capacity of 500 mL. Wash the measuring cylinder with150 mL of water and add it
to the flask. Add some porcelain pieces to the flask and Distilled it. Collect about 90 mL of
distillate from this take 25 mL of distillate and dilute it to 100 mL with water and with the help
of specific gravity bottle determine the specific gravity of liquid at 25 ºc. Follow the alcohol
content table with specific gravity and determine the V/V alcohol content in sample.
Calculate the % alcohol content by using multiplication factor.
3. Determine the specific gravity

Procedure: A tube of known weight (W) was filled first with essential oil and then with water
and the respective weight w1 and w2 was determined. Then, the specific gravity was calculated
using the following formula: d

Relative density Per cent ethanol content Per cent ethanol content
At 25ºC w/w at 15.56ºC v/v at 15.56ºC
0.8158 90 93.3
0.8146 90.5 93.6
0.8131 91 94
0.8118 91.5 94.3
0.8104 92 94.7
0.8090 92.5 95
0.8076 93 95.4
0.8062 93.5 95.8
0.8048 94 96.1
0.8034 94.5 96.5
0.8020 95 96.8
0.8006 95.5 97.1
0.7992 96 97.5
0.7977 96.5 97.8
0.7962 97 98.1
0.7957 97.5 98.4
0.7932 98 98.8
0.7917 98.5 99.1
0.7902 99 99.4
0.7886 99.5 99.7
0.7871 100 100
Determine the pH
Procedure: Use pH meter to determine the pH of the formulation

Determine the loss on drying

Requirements:
Apparatus: Digital weighing balance, petridish, hot air oven
Chemicals: Physical parameter so no need of chemicals
Procedure: Pipette out 10 mL of asava or aristha and transfer in to a pre weight china dish (W2)
and kept in hot air oven at 100-110 ºC till get the constant reading(W1). Loss on drying is the
weight difference between initial and final weight.
Determination of viscocity
Principle: The time of flow of liquid for given capillary is directly proportional to the viscosity
and inversely proportional to the driving force.
That is t1 α -----------------1

Where h – mean difference in the level of the liquidin the two

limbs
– density of liquid
– acceleration due to gravity
– coefficient of viscosity
If t2 is the time of flow of meniscus from the bulb mark ie
A-B when the same volume of another liquid is placed in the
viscometer then we have
t2 α ------------------2

From 1 and 2

The absolute viscosity can thus determine measuring t1, t2, and knowing the viscosity of
standard liquid.
Requirements:
Apparatus: Ostawald’s viscometer, stop clock, rubber tube, pipette, specific gravity bottle,
Distilled water, liquid (sample).
Chemicals: Physical parameter so no need of chemicals.
Procedure:
1. Clean the viscometer thoroughly with mixture of warm chromic acid and then rinse it
with Distilled water. Clamp it vertically onto stand.
2. The liquid whose viscosity is to be determine is deliver from a pipette into the limb with
bulb E. the quantity of liquid should be such that, when it is sucked through the tubein to
the next limb, upper level stand above A mark and the lower level stand in the other limb
at the bottom of bulb E.
3. First such Distilled water until its upper meniscus is above A mark and then allow to flow
down. Start the stop clock when is reaches to mark A and stop it when the level reaches
to mark B. Note down the flow time in seconds.
4. Repeat the procedures till get agreement values.
5. Clean the viscometer again and take equal volume of sample liquid and determione the
flow time in seconds as above.
6. Determine the density of the liquid with specific gravity bottle and calculate the viscosity.

Observations:
Liquid Flow time in seconds T Mean
/Parameters 1 2 3 seconds
Distilled water
Sample

Density g/cc =0.997

Calculations
1) To determine the density of the given liquid:
Weight of empty bottle W1 = gm
Weight of bottle + Distilled water W2 = gm
Weight of bottle + sample W3 = gm
So weight of liquid = W 3 – W1
Weight of Distilled water = W2 – W1

Density of given liquid = × density of water

Weight of water at 25ºC = 0.997g/cc

Viscosity of given liquid η= × ηw

Where
Density sample
Density of water at 25º
t1 T mean of sample
t2 T mean of Distilled water in seconds
ηw Viscosity of water at 25ºC = 0.8904
 Experiment No. 3
Aim: Evaluation of Excipients of Natural Origins.

I. Chemical Test for Agar

Synonym : Agar-agar, Japanese isinglass.
Biological source: Agar is the dried gelatinous substance, obtained from Gelidium amansii
Lamouroux, belongs to Family Gelidiaceae, Rhodophyceae (red Algae).
Macroscopical characters:
Form: Thin, membranous strips or flattened bands.
Colour: Colourless, translucent, greyish yellow.
Size: 30 to 50 cm length and 4 mm wide.
Surface: Micaceous coiled.
Odour: None.
Taste: Mucilaginous
Chemical constituents:
Carbohydrate- Polysaccharides.
Heterogeneous polysaccharides consisting of two components.
(a) Agarose (70%):
A neutral galactose polymer. It is free from sulphate. The gel strength of agar is due to this
component. Agarose also called as Agarobiose is a disaccharide consisting alternate residues of
1, 4-α-linked 1, 3-β-D galactose and 3, 6-anhydro-L-galactose. While the disaccharide unit is
called agarobiose or neoagarobiose, the linear chain is called agarose.
(b) Agaropectin:
An acidic sulphonated component where in 1, 3 linked D-galactose and the galactouronic acid
(an uronic acid) are partly esterified with sulphuric acid. Agaropectin comprises 90% and more
of sulphur. In addition, the sulphate group may also get linked to calcium, magnesium, potassium
or sodium.
Uses: Agar has been used as an ingredient in desserts , and also as a solid substrate to
contain culture mediafor microbiological work. Agar can be used as a laxative, an appetite
suppressant, a vegetarian substitute for gelatin, a thickener for soups, in fruit preserves, ice
cream, and other desserts, as a clarifying agent in brewing, and for sizingpaper and fabrics.
Requirements:
Chemicals: Cold water, ruthenium red, iodine solution, HCl, Caustic soda solution, Fehling’s
solution A & B, Barium chloride solution, KOH solution, tannic acid, millons reagent.
Apparatus: Water bath, test tube, test tube holder, test tube stand.

Sr.
Test Observation Inference
No.

Solubility:
May be presence of
a) Agar powder is treated with
Insoluble (It swells) agar
1. cold water.
Dissolves in hot Presence of Agar
b) 1gm of agar boiled with 10ml
water and forms jelly (distinguished from
of water.
on cooling Acacia & Gelatin)
Test for mucilage:
Add 1 or 2 drops of ruthenium red Particles are stained Presence of
2.
to powdered drug on glass slide pink colour Mucilage in agar
and observed under microscope.
Add N/50 iodine solution to Deep crimson to Presence of agar and
3.
0.5gms of agar powder. golden brown colour absence of starch
Test for reducing sugars:
A mixture of equal volume of dil.
HCl and aqueous solution of agar
is heated on a water bath for 30 A red precipitate of
Presence of reducing
4. mins. cuprous oxide is
sugars in agar.
In a portion of the resulting formed.
mixture add 3ml of 10% Caustic
soda solution. Add Fehling’s
solution A & B and heat on a
 water bath.
Sulphate test:
A white precipitate of Presence of agar
To the other portion of
5. barium sulphate is (distinguished from
hydrolysed solution add Barium
formed Acacia)
chloride solution.
A small quantity of agar is A canary yellow
6. Presence of agar
warmed with KOH solution. precipitate
Agar solution is added to aqueous AbsencBSe of
7. No precipitate
solution of tannic acid. tannins in Agar
Agar powdered drug is added to Absence of proteins
8. No red colour
million’s reagent. in Agar

Questions
Q. 1 .What is Pharmaceutical aids.
Q. 2. What is the use of agar as excipients.
Q. 3. What is the biological source of agar?
II. Chemical Test for Acacia
Synonym
Indian gum, gum Arabica.
Biological sources
Acacia is obtained from dried gummy exudation from a plant Acacia
Senegal or Acacia Arabica, family leguminosae.
Macroscopical characteristics:
Colour : Cream brown to red colour (gum) light brown (powder).
Shape :Iirregular brown tears with varying size.
Odour : Odourless.
Chemical constituents
The chief chemical constituent is arabin. On hydrolysis it will yield L-rhaminose, t –arabinose,
D- galactose and D- galactouronic acid.
Uses
Demulcent, binding agent, suspending agent, for microencapsulation.
Requirements:
Chemicals: Cold water, alcohol, dilute lead acetate, ruthenium red, iodine solution, HCl, Caustic
soda solution, Fehling’s solution A & B, ferric chloride solution,alcoholic benzidine..
Apparatus: water bath, test tube, test tube holder, test tube stand.

Sr.
Test Observation Inference
No.
Solubility: Soluble
a) A small amount of drug is mixed May be presence of
with 5ml of water. Insoluble acacia
b) Small amount of drug is taken May be presence of
1. with 3ml of alcohol in a test tube. The solution becomes acacia
c) Small amount of drug add 3ml of acidic in nature. May be presence of
water is taken in a test tube and boil. acacia
Test for acidic /alkaline nature.
d) To a small quantity of drug in White precipitate
 water add dilute lead acetate. presence of acacia

Test for mucilage:

Add 1 or 2 drops of ruthenium red to No Pink colour Absence of Mucilage
2.
powdered drug on glass slide and particles in acacia
observed under microscope.
On adding Iodine solution to the No blue or brown Absence of starch and
3.
solution of gum. colouration dextrin
Test for reducing sugars:
Boil a solution of gum in dil. Hcl
Red precipitate of Presence of reducing
and make alkaline by adding few ml
4. cuprous oxide is sugars in acacia
of caustic soda. Then Fehling’s
formed
solution A & B are added and keep
it on a water bath.
Absence of sulphur in
To a boiled solution of gum in dil.
5. No white precipitate acacia (distinct from
HCl add Barium Chloride solution.
agar).
6 To aqueous solution of gum add
No bluish black colour Absence of tannins
ferric chloride solution.
Aqueous solution of gum is added to
7 0.5 ml of H2O2. Add 0.5 ml of Presence of Oxidase
Blue colour is formed
alcoholic benzidine and shake enzyme in acacia
vigorously (Carcinogenic) .

Questions
Q. 1 .What is Pharmaceutical aids.
Q. 2. What is the use of acacia as excipients?
Q. 3. What is the biological source of acacia gum?
Q. 4. What is the solubility test about acacia?
III. Chemical Test for Tragacanth
Synonym
Gum tragacanth
Biological source
It is obtained from dried gummy exudation of stem and branches of
Astragalus gummifer belonging to the family leguminoseae.
Macroscopical characteristics:
Appearance : Occurs as flakes
Shape : Thin, flattened ribbon like flakes, curved.
Colour : Odourless
Taste : Mucilaginous
Solubility : It swells in water, it form a gelatinous mass only a little portion dissolves. It is
insoluble in alcohol.
Size : 3cm length, 1.2 cm width
Surface : Transluscent
Fracture : Short
Chemical constituents
It contains two fractions, Soluble one - tragacanthin ( 8 to 10 %), Insoluble one – bassorin (60 –
70%), Tragacanthin contains mainly tragacanthic acid. On hydrolysis of acid yield galactouronic
acid, galactose and fructose.Starch and protein also found in little quantiy.
Uses
Used as a thickening, suspending, emulsifying agent, dimulscent, emollient in various agents

Requirements:
Chemicals: cold water, ruthenium red, iodine solution, HCl, Caustic soda solution, Fehling’s
solution A & B, Ammonium hydroxide solution, ferric chloride solution, sodium hydroxide
solution.
Apparatus: water bath, glass slide, test tube, test tube holder, test tube stand.
Sr.
Test Observation Inference
No.
Solubility:
a) The powder drug is dissolved in Partially Soluble (but May be presence of
cold water. Swells) tragacanth
1. b) The powder drug is boiled with Soluble May be presence of
water tragacanth
c) The drug is treated with alcohol Insoluble May be presence of
tragacanth
Test for mucilage:
Add 1 or 2 drops of ruthenium red to
2. Pink colour particles Presence of Mucilage
powdered drug on glass slide and
in tragacanth
observed under microscope.
On adding Iodine solution to the Absence of starch in
3. Green colour
solution of gum. tragacanth
Test for reducing sugars:
Boil a solution of gum in dil. Hcl
Red precipitate of
and make alkaline by adding few ml Presence of reducing
4. cuprous oxide is
of caustic soda. Then Fehlings sugars in tragacanth
formed
solution A & B are added and keep it
on a water bath.
Sample is dissolved in Ammonium
5. A stingy precipitate Presence of tragacanth
hydroxide solution
Tragacanth solution is boiled with Deep yellow Absence of tannins in
6.
10%ferric chloride solution. precipitate tragacanth
The sample is warmed with NaOH Canary Yellow
7. Presence of tragacanth
solution colour
Questions
Q. 1. Give the uses of Tragacanth as pharmaceutical aids.
Q. 2. Give the biological source of tragacanth.
Q. 3. Which colour precipitate will form after boiled with ferric chloride.
Q. 4. What happens when tragacanth warmed with NaOH solution.
IV. Chemical Test for Starch
Aim : To carry out the chemical test for starch.
Synonyms:
Amylum.
Biological source: Starch consists of polysaccharide granules
obtained from the grains of Maize zea mays belongs to family
Graminae or from the tubers of potato Solarium tuberosum L.
belongs to family Solanaceae.
Macroscopical characters:
Shape: Irregular, angular masses or in form of white powder
Solubility: it is insoluble in cold water and forms a colloidal
solution on boiling. After cooling, the starch solution becomes
translucent jelly.
Chemical constituents:
(i) Starch contains generally a mixture of two polysaccharides, amylopectin (α- amylose) and
amylose (β-amylose).
(ii) Amylopectin it is the main constituent of most of the starches (more than 80%) and is present
in outer parts of granules. It contains both strait chained and branched glucose unit. It is insoluble
in water and is responsible for gelatinizing property. It gives bluish black colour with iodine
solution.
(iii) Amylose most starches contain 20% amylose. It contains straight chained glucose units and
is present in inner parts of granules. It is soluble in water and produces blue colour with iodine
solution
Uses: It is mainly used as a dusting powder, as a Pharmaceutical aid, Used as an antidote for
iodine poisoning, Source of Food-nutrition.
Protective and demulcent, It is also used as a tablet disintegrating agent and diluents.
Requirements:
Chemicals: Cold water, alcohol, α-napthol, Conc.H2SO4, Fehling’s solution A & B, Benedict’s
reagent, Iodine solution.
Apparatus: Water bath, test tube, test tube holder, test tube stand.
 Sr. No. Experiment Observation Inference

Solubility test: The translucent May be Presence of

1. Boil 1g of starch with 15ml of viscous jelly is starch. (Presence of
water and cool produced Amylopectin)
Molisch’s test: .
Drug was treated with Violet ring is formed Presence of
2. α-napthol in alcohol. To this add between the junctions Carbohydrate
equal volume of Conc.H2SO4 of two liquid.
along the sides of the test tube.

Reducing sugar test:

Absence of
The sample is added with volume No brick red
3. reducing sugar &
of Fehling’s A & B solution and precipitate is formed
presence of non-
warmed in a water bath.
reducing sugar.

Benedict’s test:
Absence of
The sample solution is mixed
4. No reddish brown reducing sugar &
with Benedict’s reagent and
precipitate presence of non-
heated in a water bath.
reducing sugar.
Blue Colour which
The given drug is treated with On heating blue
5. Presence of starch
Iodine solution and heated. colour disappears. On
cooling it reappears.

Questions
Q. 1. What is the biological source of starch?
Q. 2. What is the use of starch as excipients?
Q. 3. What is the solubility test for starch?
V. Chemical Test for Honey
Aim: Tocarry out the chemical test for honey.
Synonym: Honey
Biological source:
Honey is the saccharine liquid prepared from the nectar of the
flowers by the hive-bee Apis mellifica and bees of other
species of Apis.
Chemical constituents:
Honey contains a number of enzymes, includinginvertase, which converts sucrose to glucose and
fructose; amylase, which breaks starch down into smaller units; glucose oxidase, which converts
glucose to gluconolactone, which in turn yieldsgluconic acid and hydrogen peroxide; catalase
Uses:
Manuka honey is a common natural healing agent that has been used for centuries as
a topical antibiotic on wounds and acne. It can also be used for sore throats, colds, and other
common ailments due to these properties. Other typesof honey with antibacterial properties are
eucalyptus and linden honey
Requirements:
Chemicals: Alcohol, Fehling’s solution A & B, α-napthol, conc. H2SO4, ether, solution of
resorcinol in HCl.
Apparatus: Water bath, test tube, test tube holder, test tube stand, china dish.

Sr. No. Experiment Observation Inference

Solubility test:
May be presence
1. 1ml of Honey is mixed with 3ml of
Slight turbidity. of honey
alcohol. Shake well and keep aside.
(Dextrin).
Reducing sugar test:
To the solution of honey, add equal
2. A brick red precipitate Presence of
quantity of fehlings solution A & B
is formed. reducing sugars.
and heat it on a water bath.
3. Molisch test:
 To the solution of honey add alpha Purple coloured ring is Presence of
napthol and Conc. H2SO4 along the formed at the junction soluble
sides of the test tube. of two liquids. carbohydrate.
Fiehe’s test:
(To detect adulterants)
5ml of honey is thoroughly mixed
Presence of
with 5ml of ether. Shake well and
artificial invert
4. keep aside and separate the ethereal Persistent deep cherry
sugar (commercial
layer. The ethereal layer is red colour.
glucose).
evaporated in a china dish. To the
Adulterant honey.
residue add 1 or 2 drops of 1%
solution of resorcinol in HCl.
Seliwanoff’s test:
To the solution of honey add a
5. crystal of resorcinol and add equal Rose colour is Due to presence of
volume of Conc. HCl. Warm in the obtained. Ketones in honey.
mixture on a water bath.

Questions
Q. 1. What is the biological source of honey?
Q. 2. Which type of sugar present in honey?
Q. 3. Why honey should not get heated while preparation of any formulation
Q. 4. What type of excipient honey is?
VI. Chemical Test for Benzoin
Aim: Tocarry out the chemical test for benzoin.
Biological source:
It is a balsamic resin obtained from Styrax benzoin.belongs
to family Styraceae.
Macroscopical characteristics:
Appearance - Crystalline
Colour - Greyish brown
Odour - Aromatic and characteristics, balsamic
Shape - Tear
Taste - Sweet and slightly acrid
Requirements:
Chemicals: Water, alcohol, ether, conc. H2SO4, ether, ferric chloride solution, KMNO4 solution.
Apparatus: Water bath, test tube, test tube holder, test tube stand, porcelain dish.

Sr. No. Experiment Observation Inference

Solubility:
a) 0.5gms of drug is mixed with Insoluble May be presence of
1. 5ml water. benzoin.
b) 0.5gms of drug is mixed with Partially soluble, Presence of benzoin.
2ml alcohol. gives milky white
solution.
Heat small quantity of benzoin in a
test tube with a glass slide. Cool Crystals of cinnamic
2. Presence of benzoin.
the contents of test tube. Examine acid shall observe.
glass slide under microscope.
To 2.5gms of benzoin, add 10ml of
ether, shake it well. Pour 2 to 3ml Presence of Sumatra
3. A deep brown colour
of this extract in a porcelain dish. benzoin.
Add 2-3 drops of H2SO4.
 To 1gm of drug and shake with
Presence of Sumatra
4. 3ml of alcohol. Filter adds to an No colour.
benzoin.
alcoholic solution of Fecl3.
Add 4ml of KMnO4 solution to Odour of Presence of Sumatra
5.
1gm benzoin and warm. benzaldehyde occurs. benzoin.

Questions
Q. 1. What is the biological source of benzoin and give its uses.
Q. 2. What is the solubility test for benzoin.
Q. 3. What happens if KMnO4solution warm with benzoin.
Q. 4. What istype of excipient benzoin.
 Experiment No. 4
Aim: Incorporation of Preparation and Standardized Extract in Cosmetics Formulation.
I. Creams
Introduction
A cream is a preparation usually for application to the skin. Creams for application to mucous
membranes such as those of the rectum or vagina are also used. Creams may be
considered pharmaceutical products as even cosmetic creams are based on techniques developed
by pharmacy these are medicated and non-medicated creams. Creams are semi-olid emulsions of
oil and water. They are divided into two types: oil-in-water (O/W) creams which are composed
of small droplets of oil dispersed in a continuous water phase, and water-in-oil (W/O) creams
which are composed of small droplets of water dispersed in a continuous oily phase. Oil-in-water
creams are more comfortable and cosmetically acceptable as they are less greasy and more easily
washed off using water. Water-in-oil creams are more difficult to handle but many drugs which
are incorporated into creams are hydrophobic and will be released more readily from a water-in-
oil cream than an oil-in-water cream. Water-in-oil creams are also more moisturising as they
provide an oily barrier which reduces water loss from the stratum corneum, the outermost layer
of the skin.
General uses of cream
vehicle for drug substances such as local anaesthetics, anti-inflammatories, hormones, antifungal
santibiotics or counter-irritants, Creams are also used to treat sun burns.
General composition of cream
1. Water
2. Oil
3. Emulsifier
4. Thickening agent.
5. Active pharmaceutical ingredient ( synthetic / herbal).

Preparation of herbal creams

Ingredients
250mL herb infused oil.
250mL warm water.
20 gm beeswax (not paraffin).
8 drops essential oil (optional).
Procedure
Combine infused oil and beeswax, and heat mixture just enough to melt the wax. In a separate
pot heat water just enough to be warm to the touch. Put the warm water into your blender/food
processor. Take the center ring from the lid. Then, with the blender running at high speed, slowly
add the oil-wax mixture in a steady stream. Half way through (or thereabouts) you’ll see the
mixture turn white and thicken. This is the time to add the essential oil, if you are using any.
Keep adding oil until all used. Don’t turn off the blender until all the water has been
amalgamated. The mixture will be too thick to pour, so use a spatula to fill your wide mouth jars
while the cream is still warm.
Stored at room temperature, this cream will keep for 6 months.
Precautions
Any product containing water is a breeding ground for bacteria. Make sure all your equipment is
very clean and dont dip your fingers into the finished product. Always use a small spatula or
other applicator to keep products free of contaminants. Beeswax, essential oils and vitamin E are
natural preservatives which should give your products a shelf life of 6 months. You can also use
grapefruit seed extract which is a strong antiviral agent.
Ingredients
Skin cream 2 TBS
Herbs – dried 1 TBS
Mesh strainer - fine

Procedure
First, melt the cream in a double boiler a bowl placed over a pot that is with boiling water. Next,
add the dried herbs. Stir the mixture until the cream takes on the herbs color. Take the mixture
off of the heat, then strain the cream using the mesh strainer. Place the strained cream in a glass
bowl to cool, then transfer the cream to dark bottles. Store the bottles in a dark, cool place, where
the cream will be preserved to use for up to a year.
Precautions
Any product containing water is a breeding ground for bacteria. Make sure all your equipment is
very clean and do not dip your fingers into the finished product. Always use a small spatula or
other applicator to keep products free of contaminants. Beeswax, essential oils and vitamin E are
natural preservatives which should give your products a shelf life of 6 months. You can also use
grapefruit seed extract which is a strong antiviral agent.

Evaluation of creams
Requirements
Apparatus pH meter,
Analysis of physical parameters
Determination of organoleptic properties
The appearance of the cream was judged by its color, pearlscence and roughness and graded.

pH : Accurately weighed 5 g of the sample was dispersed in 45 mL. of water. The pH of the
suspension was determined at 27°C using digital pH meter.

Viscosity:
Principle: The time of flow of liquid for given capillary is directly proportional to the viscosity
and inversely proportional to the driving force.
That is t1 α -----------------1

Where h – mean difference in the level of the liquidin the two limbs.
– density of liquid
– acceleration due to gravity
– coefficient of viscosity
If t2 is the time of flow of meniscus from the bulb mark ie A-B when the same volume of
another liquid is placed in the viscometer then we have:
t2 α ------------------2

from 1 and 2
 =

The absolute viscosity can thus determine measuring t1,t2, and knowing the viscosity of
standard liquid.
Requirements: Ostawald’s viscometer, stop clock, rubber tube, pipette, specific gravity bottle,
Distilled water, liquid (sample).
Procedure:
1. Clean the viscometer thoroughly with mixture of warm chromic acid and then rinse it
with Distilled water. Clamp it vertically onto stand.
2. The liquid whose viscosity is to be determine is deliver from a pipette into the limb with
bulb E. the quantity of liquid should be such that, when it is sucked through the tubein to
the next limb, upper level stand above A mark and the lower level stand in the other limb
at the bottom of bulb E.
3. First such Distilled water until its upper meniscus is above A mark and then allow to flow
down. Start the stop clock when is reaches to mark A and stop it when the level reaches
to mark B. Note down the flow time in seconds.
4. Repeat the procedures till get agreement values.
5. Clean the viscometer again and take equal volume of sample liquid and determione the
flow time in seconds as above.
6. Determine the density of the liquid with specific gravity bottle and calculate the viscosity.
Observations
Liquid / Flow time in seconds T Mean
parameters 1 2 3 seconds
Distilled water
Sample

Density g/cc =0.997

Calculations
To determine the density of the given liquid:
Weight of empty bottle W1 = gm
Weight of bottle + Distilled water W2 = gm
Weight of bottle + sample W3 = gm
So weight of liquid = W 3 – W1
Weight of Distilled water = W2 – W1
Density of given liquid = × density of water

Weight of water at 25ºC = 0.997g/cc

Viscosity of given liquid η= × ηw

Where
Density sample
Density of water at 25º
t1 T mean of sample
t2 T mean of Distilled water in seconds
ηw viscosityof water at 25ºC = 0.8904
Determination of wetness:
It was determined by applying cream on skin surface of human volunteer.
Determination of type of smear:
It was determined by applying the cream on the skin surface of human volunteer. After
application of cream, the type of film or smear formed on the skin were checked.

Determination of emolliency:
Emolliency, slipperiness and amount of residue left after the application of fixed amounts of
cream was checked.
Stability studies
Stability testing of products begins as a part of drug discovery and ends with the demise of the
product. To assess the drug and formulation stability, stability studies were done according to
ICH guidelines.
The cream filled in bottle and kept in humidity chamber maintained at 30 ± 2 °C/ 65 ± 5
% RH and 40 ± 2 °C / 75 ± 5 % RH for two months. At the end of studies, samples were
analyzed for the physical properties and viscosity.
Spread ability studies
An important criteria for semisolids is that it possess good spreadability. Spreadability is a term
expressed to denote the extent of area to which the cream readily spreads on application to the
skin. The therapeutic efficacy of a formulation also depends on its spreading value.
An apparatus designed to study the spreadability of the formulations.
Spreadability is expressed in terms of time in seconds taken by two slides to slip off from the
formulation, placed between, under the application of a certain load. Lesser the time taken for the
separation of the two, better the spreadability. Two glass slides of standard dimensions were
selected. The formulation whose spreadability had to be determined was placed over one of the
slides. The other slide was placed on top of the formulations was sandwiched between the two
slides across the length of 5 cm along the slide. 100 g weight was placed up on the upper slide so
that the formulation between the two slides was pressed uniformly to form a thin layer. The
weight was removed and the excess of formulation adhering to the slides was scrapped off. One
of the slides was fixed on which the formulation was placed. The second movable slide was
placed over it, with one end tied to a string to which load could be applied by the help of a
simple pulley and a pan. A 30g weight was put on the pan and the time taken for the upper slide
to travel the distance of 5.0cm and separate away from the lower slide under the direction of the
weight was noted. The spreadability was then calculated from the following formula:

Spreadability=

m = weight tied to the upper slide (30g)

l =length of glass slide (5cm)
t =time taken in seconds.
Determination of type of emulsion Dilution test
In this test the emulsion is diluted either with oil or water. If the emulsion is o/w type and it is
diluted with water, it will remain stable as water is the dispersion medium" but if it is diluted
with oil, the emulsion will break as oil and water are not miscible with each other. Oil in water
emulsion can easily be diluted with an aqueous solvent, whereas water in oil emulsion can be
diluted with an oily liquid.

Dye solubility test

In this test an emulsion is mixed with a water soluble dye (amaranth) and observed under the
microscope. If the continuous phase appears red, it means that the emulsion is o/w type as the
water is in the external phase and the dye will dissolve in it to give color. If the scattered
globules appear red and continuous phase colorless, then it is w/o type. Similarly, if an oil
soluble dye (Scarlet red C or Sudan III) is added to an emulsion and the continuous phase
appears red, then it is w/o emulsion.

Questions
Q. 1. What is the ingredient in general composition of creams?
Q. 2. Which are the precautions should be taken while using of creams.
Q. 3. Give the uses of creams.
Q. 4. Which instrument is use to determine the viscosity.
II. Lotion
Introduction
Lotions are applied to external skin with bare hands, a brush, a clean cloth, cotton wool,
or gauze. While lotion may be used as a medicine delivery system, many lotions, especially hand
lotions and body lotions are meant instead to simply smooth, moisturize and soften the
skin. These may be used in anti-aging lotions, which can also be classified as a cosmetic in many
cases, and may contain fragrances.
Dermatologists can prescribe lotions to treat or prevent skin diseases. It is not unusual for the
same drug ingredient to be formulated into a lotion, cream and ointment. Creams are the most
convenient of the three but are inappropriate for application to regions of hairy skin such as the
scalp, while a lotion is less viscous and may be readily applied to these areas (many
medicated shampoos are in fact lotions). Historically, lotions also had an advantage in that they
may be spread thinly compared to a cream or ointment and may economically cover a large area
of skin, but product research has steadily eroded this distinction. Non-comedogenic lotions are
recommended for use on acne prone skin.
Lotions can be used for the delivery to the skin of medications such as:
Antibiotics
Antiseptics
Antifungals
Corticosteroids
Anti-acne agents
Soothing, smoothing, moisturizing or protective agents (such as calamine)
Most lotions are oil-in-water emulsions using a substance such as cetearyl alcohol to keep the
emulsion together, but water-in-oil lotions are also formulated. The key components of a skin
care lotion, cream or gel emulsion (that is mixtures of oil and water) are the aqueous and oily
phases, an emulgent to prevent separation of these two phases, and, if used, the drug substance or
substances. A wide variety of other ingredients such as fragrances, glycerol, petroleum
jelly, dyes, preservatives, proteins and stabilizing agents are commonly added to lotions.
Since thickness and consistency are key factors in lotions and creams, it is important to
understand the manufacturing process that determines viscosity.
Manufacturing lotions and creams can be completed in two cycles:
 1. Emollients and lubricants are dispersed in oil with blending and thickening agents.
2. Perfume, color and preservatives are dispersed in the water cycle. Active ingredients are
broken up in both cycles depending on the raw materials involved and the desired
properties of the lotion or cream.
A typical oil-in-water manufacturing process might go like this:
Step 1: Add flake/powder ingredients to the oil being used to prepare the oil phase.
Step 2: Disperse active ingredients.
Step 3: Prepare the water phase containing emulsifiers and stabilizers.
Step 4: Mix the oil and water to form an emulsion. (Note: This is aided by heating to between
110-185ºF (45-85º C) depending on the formulation and viscosity desired.).
Step 5: Continue mixing until the end product is completed.
Careful note should be taken in choosing the right mixing equipment for lotion manufacturing to
avoid agglomerates and long processing times. It can make all the difference in manufacturing
time and costs. Conventional agitators can present a number of problems including agglomerates
and longer processing times. On the other hand, high shear in-line mixers can produce quality
lotions and creams without many of the complications encountered with conventional mixers.
Preparation 1
Ingredients (general)
Oil
Wax
Water
Borax
Any additional ingredients and essential oils
The Process
1. Place your oils and wax in a heat proof container. Alternatively, can place a stainless steel
bowl over a pot of boiling water for a makeshift double boiler, melting your oils and waxes in
it. Either way, the mixture needs to get very hot, almost to boiling. If any wax remains
unmelted, stir it and it will melt. Add any butter, like shea butter, to the hot oil and continue to
melt.
2. In another container, heat the water to boiling. Add the borax and stir. This mixture needs to
be kept very hot as well. Use a towel or oven mitt when handling.
3. When both mixtures hot and melted, slowly pour a small amount of the water into the oil. Be
careful as this can bubble and come to the top. Keep stirring in the water until it’s all
incorporated. It should turn to a creamy consistency. If it doesn’t, or if it separates, don’t
panic. It just means that one or the other wasn’t hot enough. It’s the only way to get it creamy
if it separates or is too cool. Blend for about 5 minutes, possibly less. It begins to thicken up
and turn creamy.
4. When it’s blended and creamy, add a few drops of your essential oil. Stir and sniff. When the
desired consistency and scent is achieved, transfer to clean containers.
Preparation 2
Ingredients
Oil phase
3 mL sweet almond oil
3 mL grapeseeds oil
3 mL herbal oil
3 mL glycerin
Water phase
10 mL of herbal infusion
4 mL aloe vera gel
2 mL of vit. E
2mL of vit. A
3-5 drops of essential oil
Procedure:
1. Place bees wax and coconut oil on the top of double boiler let it melt completely add sweet
almond oil and grapeseeds oil this will make the bees wax semisolid.
2. Take out the mixture from boiler and let it cool.
3. Mix the water ingredient thoroughly.
4. Add the water ingredients to oil and add vit. E and vit. A.
5. Blend the mixture at high speed.
6. Take out the lotion and pack in wide mouth container and store it in dry place.
Standardizing parameters are same as that of creams.

Evaluation
The evolutionary parameters of lotion is as same of creams (refer evaluationary parameters of
cream).
Questions
Q. 1.What is the general composition of lotions.
Q. 2.Which types of excipients are use in lotions.
Q. 3.What is the general uses of lotions.
Q. 4.Which preservatives use in lotions.
III. Shampoo Preparation and Standardization.
Introduction
Definition: A shampoo is a preparation of a surfactant (i.e. surface active material) in a suitable
form – liquid, solid or powder–which when used under the specified conditions will remove
surface grease, dirt, and skin debris from the hair shaft and scalp without adversely affecting the
user.

Requirements of a Shampoo:
1. It should effectively and completely remove dust or soil, excessive sebum or other fatty
substances and loose corneal cells from the hair.
2. It should produce a good amount of foam to satisfy the psychological requirements of the
user.
3. It should be easily removed on rinsing with water.
4. It should leave the hair non‐ dry, soft, lustrous with good manageability and minimum fly
away.
5. It should impart a pleasant fragrance to the hair.
6. It should not cause any side‐ effects / irritation to skin or eye.
7. It should not make the hand rough

Types:
Powder Shampoo
Liquid Shampoo
Lotion Shampoo
Cream Shampoo
Jelly Shampoo
Aerosol Shampoo
Specialized Shampoo
Conditioning Shampoo
Anti‐dandruff Shampoo
Baby Shampoo
Two Layer Shampoo
Preparation 1
Material Quantity
Reetha extract 2.5 g
Amla extract 2.5 g
Sheekakai extract 2.5 g
Sidar extract 2g
Lemon juice 1 mL
Methyl paraben 1 mL of 0.05% solution
Gelatin solution q.s
Citric acid q.s
Essential oil 0.1 mL
Formulation
1) Mix the plant extracts in different proportions to obtain a shampoo.
2) Add herbal extracts to 10% gelatin solution and shake it for 20 min.
3) Add Lemon juice (1 mL) and Methyl paraben with stirring.
4) Finally adjust the pH of the solution by adding sufficient quantity of 1% citric acid solution.
5) Add few drops of rose essential oil to impart aroma to the prepared shampoo.
6) Make up the final volume to 100 mL with gelatin solution.
7) Store it in airtight container.

Evaluation
Physical appearance/visual inspection: The formulation prepared was evaluated for the
clarity, color, odor and fragrance.

Determination of pH:
Requirements:
Apparatus: pH meter, Physical parameter so no need of chemicals.
Procedure: Measure the pH of 10% v/v shampoo solution in Distilled water d by using pH
meter at room temperature.
Determination of % of solid contents:
Requirements:
Apparatus: Evaporating dish, hot plate weighing balance.
Physical parameter so no need of chemicals.

1) Take clean and dry evatporating dish and weigh it.

2) Place 4 gm of shampoo in evaporating dish. (W1).
3) Again weigh the dish to confirm to confirm the weight of shampoo.
4) Place the dish on hot plate to evaporate the liquid portion.
5) Take periodic weight of dish till get the constant reading. (W2).
6) Calculate the total solid content (W1- W2 ).
7) Calculate the % of solid contents.

Calculation:
% of solid contents

Dirt dispersion test:

Requirements:
Apparatus: Largetest tube
Physical parameter so no need of chemicals
Procedure:
1) Take a clean and dry large test tube.
2) Take 10 mL Distilled water in it.
3) Add two drops of shampoo in 10 mL of Distilled water.
4) Add one drop of Indian ink to tube.
5) Place stopper on the test tube and shake it for ten times.
6) The rubric nature of ink such as None, Light, Moderate or Heavy indicates the dirt dispersion
nature of shampoo.
Surface tension measurement
Requirements:
Apparatus:
A clean and dry capillary tube.
A tipped pointer.
A beaker containing water.
A travelling microscope.
Adjustable wooden stand.
Clamps and stand.
Procedure
To set up the apparatus:
1) Place the adjustable height stand on the table and make its base horizontal by leveling the
screws.
2) Fix the capillary tube and the pointer in a cork, and clamp it in a rigid stand so that the
capillary tubes and the pointer become vertical.
3) Adjust the height of the vertical stand, so that the capillary tubes dip in the water in an
open beaker.
4) Adjust the position of the pointer, such that its tip just touches the water surface.
To find the capillary rise:
1) Find the least count of the travelling microscope for the horizontal and the vertical scale.
2) Make the axis of the microscope horizontal.
3) Adjust the height of the microscope using the height adjusting screw.
4) Bring the microscope in front of the capillary tube and clamp it when the capillary rise
becomes visible.
5) Make the horizontal cross wire just touch the central part of the concave meniscus.
6) Note the reading of the position of the microscope on the vertical scale.
7) Now, carefully remove the beaker containing water
8) Move the microscope horizontally and bring it in front of the pointer.
9) Lower the microscope and make the horizontal cross wire touch the tip of the pointer.
10) Corresponding vertical scale readings are noted.
 11) The difference in the two readings (i.e., height of water meniscus and height of the tip of
pointer) will give the capillary rise of the given liquid.
12) We can repeat the experiment by changing the height of the wooden stand.
To find the internal diameter of the capillary tube:
1) Place the capillary tube horizontally on the adjustable stand.
2) Focus the microscope on the end dipped in water.
3) Make the horizontal cross- wire touch the inner circle at A. Note microscope reading on
the vertical scale.
4) Raise the microscope to make the horizontal cross wire touch the circle at B. Note the
vertical scale reading.
5) The difference between the two readings will give the vertical internal diameter (AB) of
the tube.
6) Move the microscope on the horizontal scale and make the vertical cross wire touch the
inner circle at C. Note microscope reading on the horizontal scale.
7) Move the microscope to the right to make the vertical cross wire touch the circle at D.
Note the horizontal scale reading.
The difference between the two readings will give the horizontal internal diameter (CD) of
the tube. We can calculate the diameter of the tube by calculating the mean of the vertical
and horizontal internal diameters. Half of the diameter will give the radius of the capillary
tube.
Observations
Least count of the travelling microscope = Value of one MSD / Number of divisions on the
Vernier
=

= 0.01 mm
= 0.001 cm
 To find the capillary rise:

Radius of the water meniscus Reading at tht tip of pointer

Height, h
Sr. Total = Total =
M.S.R. V.S.R. M.S.R. V.S.R. = h1-h2
No MSR+(VSR×LC) MSR+(VSR×LC)
(cm) (div.) (cm) (div.) (cm)
h1 (cm) h2 (cm)

5
Mean h = ……….......... cm
= ............. ×10-2 m
To find the internal diameter of the capillary tube:

Microscope readings
for cross wire in Internal diameter Internal
position,

Vertical, radius,
A B C D Horizontal, (cm)
Y= B-A
(cm) (cm) (cm) (cm) X=D-C (cm) Mean, (cm)
(cm)
Calculations
Density of water at observed temperature, ρ = ............ kgm-3
Angle of contact of water in glass, θ = 8o
Cos θ = 0.99027
1
Note the values of h in the first table and r in the second table for each capillary tube separately
and find the value of T in each case.

Surface tension,

= .................. N/m
Foam and foam stability

Requirements:
Apparatus: 250 mL graduated cylinder
Procedure:
1) Take clean and dry 250 mL of graduated cylinder.
2) Place 50 mL 1% of shampoo formulation in cylinder.
3) Cover the test tube with one hand and shake it for 10 times.
4) Record the total volume of foam content after 1 minute of shaking.
5) To evaluate the foam stability record the foam volume after 1 minute and 4 minute of shake
test.

Wetting time test

Requirements:
Apparatus: canvas paper, beaker, stop watch
Procedure:
1) Take the canvas paper.
2) Cut the paper in 1 inch of diameter.
3) In beaker take 1% v/v of shampoo solution and place the smooth surface of paper on
shampoo solution.
4) Start the stop watch.
5) Record the time required for the disc to start to sink.
6) The time require for the disc to start to sink is the wetting time

Evaluation of conditioning performance

Requirements:
Apparatus: hairs, conical flask
Procedure:
1) Obtain the hair tress of an Asian woman from local saloon.
2) Cut it in to four swatches with approximately length of 10 cm and weight about 5 gm.
3) A swatch without wash takes it as the control.
4) Wash the remaining three swatches with t10 gm of sample and 15 g of water in conical flask
for 2 minutes.
5) Rinse the swatch with 50 mL of water.
6) Allow the tress to air dry at room temperature.
7) Maximum conduct ten cycles.
8) Evaluate smoothness and softness, a blind touch test and rate the four tresses for conditioning
performance from score 1 to 4 (1 poor; 2 satisfactory; 3 good; 4 excellent).

Questions
Q. 1.Which is the ideal characteristics of shampoos.
Q. 2. What is the main excipient present in shampoo?
Q. 3. Which test gives idea about the cleaning property of shampoo?
Q. 4. What is the significance of wetting time test?
Q. 5. Why pH of shampoo is to determine.
 Experiment No. 5.
Aim: Incorporation of Preparation and Standardized Extract in Cosmetics Formulation.

I. Syrup
Syrups consist of sugar in water. These are highly concentrated, obviously viscous preparations.
And they may or may not contain therapeutic agent. In simple syrup sucrose at the concentration
of 85% is dissolved in water. These are sometimes used as a coating on to the surface of the
tablets. If some therapeutic agent is present, then it is called as medicated syrup.
Some syrups does not contain therapeutic agent, instead they consists of flavouring agents. These
are called flavoured syrups and are generally used as vehicles. For the preparation of
extemporaneous products, flavoured syrups are used as the vehicles.
These are of advantageous as they contain very less amount or no alcohol, it can be administered
to small children. But patients who have to take food having less calories should be aware of
amount of sugar present in liquid orals.
General method of preparation of the syrup:
There are four methods. Based on the physical and chemical properties on the ingredients, the
choice of the method is selected.
A. Solution with heat: The temperature of purified water is increased to 80 to 85º C and then
taken off from the heat source. Then add sucrose and shake it thoroughly. Those ingredients
which are resistant to high temperatures are added. Those substances that are heat sensitive and
volatile agents are added after the solution attain the room temperature.

B. Agitation without heat: This is a simple technique in which a vessel is taken generally made
up of stainless steel or glass. The vessel should be larger than the desired volume of syrup
required. Then the ingredients according to the formulation are added to watr and mixed. It is
better to dissolve solid ingredients in the water first and then to add them to syrup.

C. Addition of sucrose to liquid medicament: This method is generally used for fluid extracts.
But those substances which are soluble in alcohol will precipitate out as soon as the addition of
water. An alternation is to first dissolve all the ingredients in water. Now after sometime all the
precipitates formed are filtered out. Now add sucrose. But this method is of no use if the
precipitate formed has active ingredients.

D. Percolation method: As the name suggest, the principle of percolation is used. A sucrose bed
is prepared and then water or vehicle containing therapeutic agent is passed. Here the sucrose
bed should be coarse and shape of percolator must be cylindrical or cone shapped.
Sucrose syrups are replaced by agents like sorbital solution or mixture of sorbital and glycerin.
Sorbital is osmotic laxative and so it must be administered within the limits. Sorbital solution
contains about 64% of sorbital.
Herbal syrup preparation procedure
a) Formulation:- ( Each 500 g of powder contains)
Aswagandha - 139 g
Asparagus - 139 g
Bombax - 139 g
Yastika - 069 g
Ela - 007 g
Darusita - 007 g

b) Method of preparation of decoction:

1) Take above formulation and mix with 4000 mL of water.
2) Boil the mixture till the total volume becomes 1000 mL.
3) Cool the decoction and filter it.

c) Method of preparation of simple syrup (USP):

1) Weigh 666.7 gm of sucrose.
2) Take the sugar in stainless steel container.
3) Add sufficient purified water.
4) Heat the mixture so that sucrose is get dissolve in water.
5) Stir the solution occasionally.
6) When sucrose is get fully dissolve then add sufficient of boiling water to produce 1000 mL.
d) Method of preparation of final herbal syrup:
1) Mix the one part of decoction with five parts of simple syrup.
2) Add 0.2% of sodium benzoate to the resultant solution as preservative.
3) Pack the syrup and store it in dry place.

Evaluation of herbal syrup

1) Physical parameters:
Color examination:
Requirements:
Apparatus: Watch glass, white back ground.
Procedure:
1) Take 5 mL of syrup in watch glass.
2) Observe it against white back ground in white tube light.
3) Observe for its color by naked eye.

Odor examination: Smell two mL of final syrup. Keep 2 minute time interval among two
smelling to nullify the effect of previous smelling.

Taste examination: Take pinch of final syrup to taste.

Determination of pH:
Measure the pH of 10% solution of syrup with the help of digital pH meter.
Specific gravity at 25 :
The complete procedure is given in evaluation of creams.

Determination of Density:
The complete procedure is given in evaluation of creams.
Questions
Q. 1. What are the general methods of preparation of syrups?
Q. 2. Which is the main component of syrup?
Q. 3. What are the uses of syrup?
Q. 4. Why pH of syrup is to be determine.
II. Mixtures
Generalize definition of mixtures is the one product of a mechanical blending or mixing
chemical substances such as elements and compounds, without chemical bonding or other
chemical change, so that each ingredient substance retains its own chemical properties and
makeup. Despite that there are no chemical changes to its constituents, the physical properties of
a mixture, such as its melting point, may differ from those of the components. Some mixtures
can be separated into their components by using physical (mechanical or thermal) means.
Churna is the best known example of mixture. Churna is a powdered form of medications used in
the treatment of disorders in the Ayurvedic system of medicine. It is the most basic and simplest
form of Ayurvedic medicine containing a fine powder of herbs. Churna may contain a single
herb or a mixture of several herbs.
General method of preparation of Churna
All the ingredients required for the Churna should be cleaned thoroughly and dried well in the
shade or in the sun separately to ensure the complete absence of moisture in the same.
Each ingredient is powdered finely and then, sieved to remove any coarse particles. The
ingredients are weighed separately and then, mixed together. It should be noted that some herbs
contain a fibrous matter. Hence, the weight of such herbs may vary before and after drying.
Hence, it is important to powder and weigh them separately so that the correct quantity of each
herb can be present in the final product.
Formulation for 50 gm of churna
Zingiber officinale (powder) 16.7gm
Foeniculum vulgarae (powder) 16.7gm
Cinnamomum zeylanicum (powder) 8.7gm
Trachyspermum ammi (powder) 8.7gm
Procedure
1) Take all the ingredients in given proportion.
2) Mix the powders thoroughly.
3) Pass the mixture through sieve number 60.
4) Pack it in well close container and store it in dry place.
Evaluation
1) Physical parameters:
Color examination:
Requirements:
Apparatus: Watch glass, white back ground
Procedure:
1) Take 2 gm of syrup in watch glass.
2) Observe it against white back ground in white tube light.
3) Observe for its color by naked eye.

Odor examination: Smell two gm of final churna. Keep 2 minute time interval among two
smelling to nullify the effect of previous smelling.

Taste examination: Take pinch of final churna to taste.

pH determination:
Requirements:
Apparatus: small beaker, pH meter.
The pH of 1% solution of formulated churna was determined using pH meter.
Procedure:
1) Prepare 1% solution of churna with Distilled water.
2) With the help of pH meter determine the pH value of churna.
Determination of Moisture content:
Requirements:
Apparatus: Glass stopper weighing bottle, hot air oven, weighing balance
Physical parameter so no need of chemicals
1) Take clean and dry glass stopper weighing bottle and weigh it.
2) Place 2 gm of churna in glass stopper weighing bottle. (W1).
3) Again weigh the dish to confirm to confirm the weight of shampoo.
4) Open the glass stopper weighing bottle and place it in hot hot air oven to evaporate the
moisture portion.
5) After dying cool the bottle in desiccator.
6) Take periodic weight of weighing bottle till get the constant reading. (W2).
7) Calculate the moisture content (W1- W2).
8) Calculate the % of moisture content.

Calculation:

% of moisture content

1. Determination of Total ash

Principle: The ash value of any organic material is composed of their non - volatile inorganic
compounds controlled incineration of crude drugs result in an ash residue consisting of an
inorganic material (metallic salt and silica). This is an important parameter for the purpose of
evaluation of crude drugs.
Requirements:
Apparatus: furnace crucible, muffle furnace, weighing balance.
Procedure
1) Weigh accurately furnace crucible which was previously ignited and weighed (W1).
2) Weigh accurately about 2gm of sample transfer it to furnace crucible (W2).
2) The drug sample was spread as a thin layer.
3) The crucible was incinerated by gradually increasing of heat until all the carbon matter are
burned off to red hot whitish matter at a temperature not exceeding 150 ͦ c.
4) Crucible is removed and cooled in desiccator and dried and weighed (W3).
5) Calculate the percentage total ash with reference to the air dried drug.
Calculations
Total ash value W2 W1

% total ash
2. Acid- insoluble ash
Requirements:
Apparatus: Furnace crucible, ash less filter paper, muffle furnace, weighing balance.
Chemicals: Dilute hydrochloric acid, Distilled water.
1. Boil the total ash (W1) with for five minutes with 25 ml of dilute hydrochloric acid.
2. Collect the insoluble matter in a Gooch crucible or on an ash less filter paper.
3. Wash with hot water, ignite, and weigh W2.
4. Calculate the percentage of acid- insoluble ash with reference to the air dried drug.
Calculations:
Acid- insoluble ash W2

3. Water- soluble ash:

1) Boil the total ash for 5 minutes with 25 ml of water.
2) Collect the insoluble matter in a Gooch crucible or on an ash less filter paper.
3) Wash with hot water, and ignite to constant weight at a low temperature.
4) Subtract the weight of insoluble matter from the weight of the ash.
5) The difference in weight represents the water- soluble ash.
6) Calculate the percentage of water- soluble ash with reference to the air dried drug.
4. Sulphated ash:
1) A silica crucible was heated to redness for 10 minutes, allowed to cool in desiccators and
weighed.
2) 1 g of substance was accurately weighed and transferred to the crucible.
3) It was ignited gently at first, until the substance was thoroughly charred.
4) Then the residue was cooled and moistened with 1 ml concentrated sulfuric acid, heated
gently until white fumes are no longer evolved.
5) Ignited at 800° ± 25°C until all black particles have disappeared.
6) The ignition was conducted in a place protected from air currents.
7) The crucible was allowed to cool, and a few drops of concentrated sulfuric acid were added
and heated.
8) Ignited as before, allowed to cool, and weighed.
9) The operation was repeated until two successive weighing does not differ by more than 0.5
mg.
10) Calculate the percentage of Sulphated ash with reference to the air dried drug.
Water soluble extractive value
Requirements
Apparatus: Measuring cylinder, stoppered flask, hot air oven, dessicator, flat bottomed shallow
dish.
Chemicals: Distilled water.
Principle
Extractive values of crude drugs are used for evaluation especially when the constituents of a
drug cannot be readily estimated by any other means. Further these values indicate the nature of
the constituents present in a crude drug. These constituents may be soluble in different polar,
semi polar or non- polar solvents. Total soluble components of the drug in any particulate
solvent, mixture of solvent may be called as extractive value or percentage value.
Procedure
1) Macerate about 3g accurately weighed, coarsely powdered, air - dried sample with 100 ml of
Distilled water in a stoppered flask for24 hours.
2) Shaking frequently during 6 hours.
3) Filter rapidly through filter paper.
4) Evaporate 25 ml of a water extract to dryness in a flat bottomed shallow dish.
5) Dry at 105 ͦC and weigh accurate. Keep it in desiccator.
6) Calculate the percentage w/w of water soluble extractive with reference to the air- dried drug.
Alcohol Soluble Extractive Value
Requirements
Apparatus: Measuring cylinder, stoppered flask, hot air oven, desiccator, flat bottomed shallow
dish.
Chemicals: 90% alcohol.
Principle
Values of crude drugs are use for evaluation especially when the constituents of a drug cannot be
readily estimated by any other means. Further these values indicate the nature of the constituents
present in a crude drug. These constituents may be soluble in different polar, semi polar or non-
polar solvents. Total soluble components of the drug in any particulate solvent, mixture of
solvent may be called as extractive value or percentage value.
Procedure
1) Macerate about 2g accurately weighed.
2) Coarsely powdered, air – dried sample with 100 ml of 90% alcohol in a stoppered flask for 24
hours.
3) Shaking frequently during 6 hours.
4) Filter rapidly through filter paper taking precaution against excessive loss of alcohol.
5) Evaporate 25 ml of a water extract to dryness in a flat bottomed shallow dish.
6) Dry at 105 ͦ c and weigh accurate. Keep it in desiccator.
7) Calculate the percentage w/w of water soluble extractive with reference to the air- dried drug.

Questions
Q. 1. What is the significance of determination of extractive value?
Q. 2. What is the significance of determination of ash values?
Q. 3. What are the general characteristics of mixtures?
Q. 4. What will be the impact of moisture content?
Q. 5. What is the ideal particle size of churna.
III. Tablet
A tablet is a pharmaceutical dosage form. Tablets may be defined as the solid unit dosage form
of medicament or medicaments with or without suitable excipients and prepared either by
molding or by compression. It comprises a mixture of active substances and excipients, usually
in powder form, pressed or compacted from a powder into a solid dose. The compressed tablet is
the most popular dosage form in use today. About two-thirds of all prescriptions are dispensed as
solid dosage forms, and half of these are compressed tablets. A tablet can be formulated to
deliver an accurate dosage to a specific site; it is usually taken orally, but can be
administered sublingually, buccally, rectally or intravaginally.

Types
A pill
A pill was originally defined as a small, round, solid pharmaceutical oral dosage form of
medication.
Caplet
Variations on a common tablet design, which can be distinguished by both color and shape. A
caplet is a smooth, coated, oval-shaped medicinal tablet in the general shape of a capsule. Many
caplets have an indentation running down the middle so they may be split in half more easily
Orally disintegrating tablet (ODT)
An orally disintegrating tablet or or dispersible tablet (ODT), is a drug dosage form available for
a limited range of over-the-counter (OTC) and prescription medications.
The advantages of the Tablet dosage form are:
1. They are unit dosage form and offer the greatest capabilities of all oral dosage form for the
greatest dose precision and the least content variability.
2. Cost is lowest of all oral dosage form.
3. Lighter and compact.
4. Easiest and cheapest to package and strip.
5. Easy to swallowing with least tendency for hang‐up.
6. Sustained release product is possible by enteric coating.
7. Objectionable odour and bitter taste can be masked by coating technique.
8. Suitable for large scale production.
9. Greatest chemical and microbial stability over all oral dosage form.
10. Product identification is easy and rapid requiring no additional steps when employing an
embossed and/or monogrammed punch face.

Disadvantages of Tablet dosage form are:

1. Difficult to swallow in case of children and unconscious patients.
2. Some drugs resist compression into dense compacts, owing to amorphous nature, low density
character.
3. Drugs with poor wetting, slow dissolution properties, optimum absorption high in GIT may be
difficult to formulate or manufacture as a tablet that will still provide adequate or full drug
bioavailability.
4. Bitter testing drugs, drugs with an objectionable odor or drugs that are sensitive to oxygen
may require encapsulation or coating. In such cases, capsule may offer the best and lowest cost.
Formulation
Composition

Ingredients (mg) F1 F2 F3 F4
Clove 100 - 100 –
Cinnamon - 100 - 100
Lactose 290 290 - -
Mannitol - - 290 290
Sodium Sachrine 2 2 2 2
Talc 4 4 4 4
Magnesium Stearate 4 4 4 4
Formulation
1) Weigh all the excipients along with API as shown in Table.
2) Passed through sieve no. 20.
3) Mix all ingredients following geometric mixing excluding glidant and lubricant thoroughly for
15min.
4) Mix the powder blend was thoroughly with talc and magnesium stearate.
5) Compress 400mg tablet using single rotatory punching machine

Evaluation of Tablet

1. General Appearance: The general appearance of a tablet, its identity and general elegance is
essential for consumer acceptance, for control of lot‐to‐lot uniformity and tablet‐to‐tablet
uniformity. The control of general appearance involves the measurement of size, shape, color,
presence or absence of odor, taste etc.

2. Size & Shape: It can be dimensionally described & controlled. The thickness of a tablet is
only variables. Tablet thickness can be measured by micrometer or by other device. Tablet
thickness should be controlled within a ± 5% variation of standard value.

3. Unique identification marking: These marking utilize some form of embossing, engraving or
printing. These markings include company name or symbol, product code, product name etc.

4. Organoleptic properties: Color distribution must be uniform with no mottling. For visual
color comparison compare the color of sample against standard color.

5. Hardness: Tablet requires a certain amount of strength or

hardness and resistance to friability to withstand mechanical
shakes of handling in manufacture, packaging and shipping.
Hardness generally measures the tablet crushing strength.
6. Friability: Friability of a tablet can determine in laboratory
by Roche friabilator. This consist of a plastic chamber that
revolves at 25 rpm, dropping the tablets through a Distance of
six inches in the friabilator, which is then operate for 100
revolutions. The tablets are reweighed. Compress tablet that
lose less than 0.5 to 1.0 % of the Tablet weigh are consider
acceptable.

Drug Content and Release:

1) Weight Variation test (U.S.P.): Take 20 tablet and weighed individually. Calculate average
weight and compare the individual tablet weight to the average. The tablet pass the U.S.P. test if
no more that 2 tablets are outside the percentage limit and if no tablet differs by more than 2
times the percentage limit.
2) Content Uniformity Test: Randomly select 30 tablets. 10 of these assayed individually. The
Tablet pass the test if 9 of the 10 tablets must contain not less than 85% and not more than 115%
of the labeled drug content and the 10th tablet may not contain less than 75% and more than
125% of the labeled content. If these conditions are not met, remaining 20 tablet assayed
individually and none may fall outside of the 85 to 115% range.
3) Disintegration Test (U.S.P.): The U.S.P. device to test
disintegration uses 6 glass tubes that are 3” long; open at
the top and 10 mesh screen at the bottom end. To test for
disintegration time, one tablet is placed in each tube and
the basket rack is positioned in a 1‐L beaker of water,
simulated gastric fluid or simulated intestinal fluid at 37 ±
2 0 C such that the tablet remain 2.5 cm below the surface
of liquid on their upward movement and not closer than
2.5 cm from the bottom of the beaker in their downward
movement. Move the basket containing the tablets up and
down through a distance of 5‐6 cm at a frequency of 28 to
32 cycles per minute. Floating of the tablets can be prevented by placing perforated plastic discs
on each tablet. According to the test the tablet must disintegrate and all particles must pass
through the 10 mesh screen in the time specified. If any residue remains, it must have a soft
mass. Disintegration time: Uncoated tablet: 5‐30 minutes coated tablet: 1‐2 hours.
4. Dissolution Test (U.S.P.):
Two set of apparatus:
Apparatus‐1: A single tablet is placed in a small wire mesh basket
attached to the bottom of the shaft connected to a variable speed
motor. The basket is immersed in a dissolution medium (as specified
in monograph) contained in a 100 mL flask. The flask is cylindrical
with a hemispherical bottom. The flask is maintained at 37±0.50 by
a constant temperature bath. The motor is adjusted to turn at the
specified speed and sample of the fluid are withdrawn at intervals to
determine the amount of drug in solutions.

Apparatus‐2: It is same as apparatus‐1, except the basket is replaced by

a paddle. The dosage form is allowed to sink to the bottom of the flask
before stirring. For dissolution test U.S.P. specifies the dissolution test
medium and volume, type of apparatus to be used, rpm of the shaft and
time limit of the test and assay procedure for. The test tolerance is
expressed as a % of the labeled amount of drug dissolved in the time
limit.

Questions
Q. 1. Define tablets.
Q. 2. Types of tablet formulation
Q. 3. What are the advantages of tablets?
Q. 4. What is the significance of tablet friability?
Q. 5. What is the significance of weight variation test?
 Experiment No. 6
Aim: Monograph Analysis of Herbal Drug from Recent Pharmacopoeia.

I. Acacia
Acacia is the air-hardened, gummy exudation from the stem and
branches of Acacia nilotica (Linn.) Del. subsp. Indica (Benth.)
Brenan (syn. A. arabica Willd. var. indica Benth.)(Fam.
Leguminosae), or other species of Acacia.It is available as pieces
(tears) or in the form of a powder.

Description
Tears — Irregular and broken pieces of varying size, yellowish, white, yellow or amber in
colour, with numerous minute fissures; brittle fractured surface, glassy and occasionally
iridescent; odourless. Powder — A white or yellowish-white powder; odourless; on treatment
with water it dissolves to give a mucilaginous liquid which is colourless or yellowish, dense,
viscous, adhesive and translucent.

Identification
A. An aqueous solution is gelatinised by the addition of lead subacetate sol3either dextro-
rotatory or slightly laevo-rotatory.
B. To 5 mL of a 10 per cent w/v solution add gradually, while shaking, 10 mL of ethanol (95 per
cent). The cloudy liquid, on addition of 0.5 mL of acetic acid, gives a white precipitate. Filter
and add to the clear filtrate 50 mL of ammonium oxalate solution; the filtrate becomes cloudy.
C. A 10 per cent w/v solution is either dextro-rotatory or slightly laevo-rotatory.

Tests

Sterculia gum and agar. To 50 mg of the powdered substance under examination add 0.2 mL of
freshly prepared ruthenium red solutionand examine microscopically; the particles do not acquire
a red colour after irrigation with water.
Agar and tragacanth. To 10 mL of a 10 per cent w/v solution add 0.2 mL of lead acetate
solution; no precipitate is produced.

Starch and dextrin. Boil 10 mL of a 10 per cent w/v solution and cool, add 0.1 mL of 0.05 M
iodine; no blue or brown colour is produced.

Tannins. To 10 mL of a 10 per cent w/v solution add 0.1 mL of ferric chloride test solution; a
gelatinous precipitate is formed, but neither the precipitate nor the liquid shows a dark blue
colour.

Sucrose and fructose. To 1 mL of a 10 per cent w/v solution add 4 mL of water, 0.1 g of
resorcinol and 2 mL of hydrochloric acidand heat on a water-bath; no yellow or pink colour
develops.

Water-insoluble matter. Dissolve 5 g, in fine powder, in 100 mL of waterin a 250-mL flask,

add 10 mL of dilute hydrochloric acidand boil gently for 15 minutes. Filter by suction while hot
through a sintered-glass crucible, previously tared, wash thoroughly with hot water, dry at 105º
and weigh; the residue does not exceed 50 mg.

Microbial contamination 1.0 g is free fromEscherichia coli.

Sulphated ash Not more than 5.0 per cent.

Acid-insoluble ash Not more than 1.0 per cent.

Loss on drying Not more than 15.0 per cent, determined on 1.0 g by drying in an oven at 105º.

Storage. Store protected from heat, moisture and against attack by insects and rodents.
II. Arachis Oil
Groundnut Oil; Peanut Oil.

Arachis Oil is the refined fixed oil obtained from the seed
kernels of one or more of the cultivated varieties of Arachis
hypogaea Linn. (Fam. Leguminosae) and may contain suitable
antioxidants as stabilisers.

Description
A clear, colourless or pale yellow oily liquid;odour, faint and
nutlike.

Identification
Determine by the thin layer chromatography, coating the plate with kieselguhr G, Mobile phase.
Glacial acetic acid.

Test solution. Dissolve 20 mg (one drop) of the substance under examination in 4 mL of

chloroform.

Reference solution (a). Dissolve 20 mg (one drop) of arachis oil in 4 mL of chloroform.

Reference solution (b). Dissolve 20 mg (one drop) of cottonseed oil in 4 mL of chloroform.

Reference solution (c). Dissolve 20 mg (one drop) of sesame oil in 4 mL of chloroform.

Impregnate the plate by placing it to a depth of about 5 mm in a tank containing a shallow layer
of a mixture of 95 volumes of light petroleum (60º to 80º)and 5 volumes of liquid paraffin,
allowing the solvent to rise at least 14 cm, removing the plate and allowing it to dry in air for 5
minutes; use with the flow of the mobile phase in the direction in which impregnation was
carried out. Apply to the plate 2 μl of each solution. Allow the mobile phase to rise 12 cm. Dry
the plate at 110º for 10 minutes, allow to cool and expose it to iodine vapour in a saturated
chamber.
Remove the plate and allow it to stand for a few minutes until the brown background colour has
disappeared. Spray the plate with starch solution; blue spots appear which become brown on
drying and become blue again on spraying with water. The spots in the chromatogram obtained
with the test solution correspond to those in the chromatogram obtained with reference solution.

Tests
Weight per mL 0.908 g to 0.920 gm.

Refractive index 1.467 to 1.470.

Alkaline impurities. Mix 10 mL of recently Distilled acetone and 0.3 mL of water in a test-
tube, add 0.05 mL of a 0.04 per cent w/v solution of bromophenol blue in ethanol (95 per
cent)and neutralise the solution, if necessary, with 0.01 Mhydrochloric acid or 0.01 M sodium
hydroxide. Add 10 mL of the substance under examination, shake and allow to stand.
Not more than 0.1 mL of 0.01 M hydrochloric acid is required to change the colour of the upper
layer to yellow.

Semi-drying oils. Boil 1 g in a flask under a reflux condenser for 5 minutes with 5 mL of a
mixture of 3 volumes of 2 Methanolic potassium hydroxide and 1 volume of ethanol(95 per
cent), add 1.5 mL of 6 M acetic acid and 50 mL of ethanol (70 per cent), warm until the solution
is clear. Cool slowly with a thermometer in the liquid; the temperature at which the solution
becomes turbid is not lower than 36º.

Peroxide value Not more than 5.0.

Acid value Not more than 0.5.

Iodine value 85 to 105.

Saponification value 185 to 196.

Rancidity. Shake 1 mL of a 10 per cent v/v solution in etherwith 1 mL of hydrochloric acid, add
1 mL of a 0.1 per cent w/v solution of phloroglucinol in ether; no red or pink colour is produced.

Unsaponifiable matter (2.3.39). Not more than 1.0 per cent.

Cottonseed oil. In the Identification test, the chromatogram obtained with the test solution does
not correspond to that obtained with reference solution (b).

Sesame oil. In the Identification test, the chromatogram obtained with the test solution does not
correspond to that obtained with reference solution (c).

Heavy metals 2.0 g complies with the limit test for heavy metals, Method B (10 ppm).

Water Not more than 0.3 per cent, determined on 3.0 g.

Storage. Store protected from light and moisture, in a wellfilled container. Arachis Oil intended
for use in the manufacture of parenteral preparations should be stored in a glass container.

Labelling. The label states

(1) Whether the contents are suitable for use in the manufacture of parenteral preparations;
(2) When the addition of antioxidants is authorised, the name and quantity of the added
antioxidants.
III. Castor Oil
Castor Oil is the fixed oil obtained by cold expression from
the seeds of Ricinus communis Linn. (Fam. Euphorbiaceae).
It may contain suitable antioxidants.

Description
A pale yellowish or almost colourless, transparent, viscid
liquid; odour, slight and characteristic.

Tests
Light absorption Absorbance of a 1.0 per cent w/v solution in ethanol (95 per cent) at the
maximum at about 269 nm, not more than 1.0.

Weight per mL 0.945 g to 0.965 g.

Refractive index 1.4758 to 1.4798.

Optical rotation +3.5º to +6.0º.

Peroxide value Not more than 5.0.

Acid value Not more than 2.0.

Acetyl valueNot less than 143.

Hydroxyl value Not less than 150.

Saponification value176 to 187.

Iodine value 82 to 90.

Foreign fatty substances.

A mixture of 2 mL of the substance under examination and 8 mL of ethanol (95 per cent) is
clear.

B. Shake 10.0 mL with 20.0 mL of light petroleum (60º to 80º)and allow to separate; the volume
of the lower layer is not less than 16.0 mL.

Storage. Store protected from light and moisture at a temperature not exceeding 15º.

Labelling. The label states (1) the name and quantity of any added antioxidant; (2) whether the
contents are suitable for use in the manufacture of parenteral preparations.
IV. Mentha Oil
Mentha Oil is the volatile oil Distilled with steam from various
species of Mentha (Fam. Labiatae) and rectified if necessary.
Mentha Oil contains not less than 50.0 per cent w/w of total
menthol, C10H20O.

Description
A colourless or yellowish, clear liquid; odour, characteristic
and pleasant.

Tests

Acidity or alkalinity. A solution of 1 mL in 3.5 mL of ethanol(70 per cent) is neutral to litmus.

Weight per mL 0.892 g to 0.910 g.

Specific optical rotation –18.0º to –33.0º.

Assay. Place about 10.0 g in an acetylation flask, add 10 mL of acetic anhydride and 1 g of
anhydrous sodium acetate, attach a reflux condenser and boil for 2 hours. Cool, add 30 mL of
water and warm on a water-bath for 15 minutes with occasional shaking. Transfer the contents of
the flask to a separating Funnel, reject the water layer and wash the remaining oil with water
until the last washing no longer shows acid reaction. Dry the resulting oil by shaking with 2 g of
anhydrous sodiumsulphate, allow to stand for 30 minutes and filter through adry filter paper.
Weigh accurately about 1.5 g of the dry acetylated oil, add 3 mL of ethanol (95 per cent) and 0.1
mL of phenolphthalein solution and dropwise, 0.5 M ethanolicpotassium hydroxide until the
solution acquires a faint pink
colour. Add a further 20.0 mL of the alkali, attach a reflux condenser and boil for 1 hour on a
water-bath. Cool, add 1 mL.of phenolphthalein solution and titrate the excess of alkali with 0.5
M hydrochloric acid. Repeat the operation with the same quantities of the same reagents in the
same manner without the oil and calculate the amount of total menthol from the following
expression. where, S is the amount, in g, of the acetylated sample taken, a is the amount, in mL,
of 0.5 M hydrochloric acid consumed in the blank test, and b is the amount, in mL, of 0.5 M
hydrochloric acid consumed in saponification of the acetylated oil.

Storage. Store protected from light and moisture.

V. Starch
Starch consists of polysaccharide granules obtained from
the caryopsis of maize or corn, Zea mays Linn., or of rice,
Oryza sativa Linn., or of wheat, Triticum aestivum Linn.,
or from the tuber of potato, Solanum tuberosum Linn., or
from the rhizomes of tapioca, Manihot utilissima Pohl.

Description
A very fine, white or slightly yellowish powder or irregular white masses which are readily
reducible to powder, creaks when pressed between the fingers; odourless and tasteless. The
presence of granules showing cracks or edge irregularities is exceptional in starches other than
wheat starch; wheat starch may contain granules with cracks on the edges.

Identification
A. Corn or maize starch — Polyhedral granules, 2 to 23 μm in size, or rounded granules, 25 to
32 μm in diameter. The central hilum consists of a distinct cavity or two- to five-rayed cleft; no
concentric striations. Viewed between crossed nicol prisms, a distinct black cross is seen
intersecting at the hilum.

Potato starch — Single granules, either irregular, ovoid or pear-shaped, 30 to 100 μm in size, or
rounded, 10 to 35 μm in size; compound granules consisting of groups of two to four elements
are rare. Eccentric hilum; clearly visible concentric striations. Viewed between crossed nicol
prisms, a distinct black cross is seen intersecting at the hilum.
Rice starch — Polyhedral granules, 2 to 5 μm in size, either isolated or aggregated in ovoid
masses, 10 to 20 μm in size. Central hilum poorly visible; no concentric striations. Viewed
Calculate the content of p-methoxy cinnamic acid ethyl ester.

Storage. Store protected from heat, moisture and against attack by insects and rodents.
VI. Tragacanth
Tragacanth is the air-hardened gummy exudate,
flowing naturally or obtained by incision, from the
trunk and branches of Astragalus gummifer Labill.
and certain other species of Astragalus.

Description.
Colour: Pale yellow
Odour: Odourless and almost
Taste: Tasteless.
Solubility: on the addition of about 10 times its weight of water, it forms a mucilaginous gel.

Identification
A. Macroscopic — Occurs as thin, flattened pieces, 30 mm long, 10 mm wide and up to 1 mm in
thickness, more or less curved, marked on the surface by fine longitudinal striae and concentric
transverse ridges; white, translucent, horny; fracture, short. May also be in the form of thicker,
less brittle pieces, white to pale yellow and more opaque.

B. Microscopic — Reduce to powder. Examine under a microscope; the powder shows in the
gummy mass numerous stratified cellular membranes which turn violet on the addition of
iodinated zinc chloride solution. The mass includes starch granules, isolated or in small clusters,
rounded or occasionally deformed, diameter 4 to 10 μm, and up to 20 μm, with a central hilum,
visible in polarised light.

C. Moisten 0.5 g of the powdered material with 1 mL of ethanol (95 per cent) and add gradually,
while shaking, 50 mL of water until a homogeneous mucilage is obtained. To 5 mL of the
mucilage add 5 mL of water and 2 mL of barium hydroxide solution. A slightly flocculent
precipitate is formed which, when heated for 10 minutes on a water-bath, gives an intense yellow
colour.
D. Add 4 mL of 0.5 per cent w/v dispersion in water to 0.5 mL of hydrochloric acid and heat on
a water-bath for 30 minutes. To one half of the resulting liquid add 1.5 mL of sodium hydroxide
solution and 3 mL of alkaline cupric-tartrate solution and heat on a water-bath; a reddish brown
precipitate is formed. To the other half of the liquid, add a few drops of barium chloride
solution; no precipitate is formed (freedom from agar).

Tests
Acacia and other soluble gums. To 20 mL of a 2.5 per cent w/v suspension of the powdered
material prepared with freshly boiled water add 10 mL of lead acetate solution; a flocculent
precipitate is formed. Filter and add to the filtrate 10 mL of lead subacetate solution; a slight
cloudiness may appear, but there is no precipitate.

Karaya gum. Boil 1 g with 20 mL of water until a mucilage is formed, add 5 mL of

hydrochloric acid and again boil for 5 minutes; no pink or red colour develops. Sterculia. A.
Shake 0.2 g of the powdered material with 10 mL of ethanol (60 per cent) in a 10-mL stoppered
cylinder; any gel formed occupies not more than 1.5 mL.

B. Shake 1 g of the powdered material with 100 mL of water and titrate with 0.01 M sodium
hydroxide, using methyl red solution as indicator. Not more than 5.0 mL is required to change
the colour of the solution.

Foreign matter. Not more than 1.0 per cent, determined by the following method. To 2.0 g of
the powdered material in a 250 mL round-bottomed flask add 95 mL of methanol, swirl to
moisten the powder and add 60 mL of 7 M hydrochloric acid. Add a few glass beads and heat
under a reflux condenser in a water-bath for 3 hours, shaking occasionally. Remove the glass
beads and filter the hot suspension under reduced pressure through a sintered-glass crucible
(porosity No. 1). Rinse the flask with a small quantity of water, passing the rinsings through the
filter. Wash the residue on the filter with about 40 mL of methanol and dry to constant weight at
110º.
Arsenic Mix 3.3 g with 3 g of anhydrous sodium carbonate, add 10 mL of bromine solution and
mix thoroughly.
Evaporate to dryness on a water-bath, gently ignite, and dissolve the cooled residue in 16 mL of
brominated.
hydrochloric acid AsT and 45 mL of water. Remove the excess of bromine with 2 mL of
stannous chloride AsT. The resulting solution complies with the limit test for arsenic (3 ppm).

Heavy metals 0.5 g complies with the limit test for heavy metals.

Ash Not more than 4.0 per cent, determined on 1.0 g.

Microbial contamination 1 g is free from Escherichia coli and 10 g is free from salmonellae.

Storage. Store protected from moisture.

VII. Ashwagandha
Indian Ginseng; Withania
Ashwagandha consists of the dried mature roots of
Withania somnifera Dunal (Fam Solanaceae).
Ashwagandha contains not less than 0.02 per cent of total
withanolide A and withaferin A, calculated on the dried
basis.

Description
Buff to greyish-yellow roots. Taste, slightly
mucilaginous, bitter and acrid.

Identification
A. Macroscopic — Primary roots are straight, conical or finger like in shape, variable in
thickness with the age. Secondary roots are thin and fibrous. Surface buff to greyish-yellow with
longitudinal wrinkles.

B. Microscopic — Vessels with bordered pits and horizontal perforations. Fibres aseptate with
pointed ends. Wood elements lignified. Starch grains abundant, simple, mostly spherical,
reniform – oval with central hilum. Microcrystals in parenchyma cells.

Mobile phase.A mixture of 9 volumes of chloroform and 1 volume of methanol.

Test solution. Reflux 3 g of coarsely powdered substance under examination with 50 mL

methanol for 15 minutes, cool and filter.

Reference solution. Reflux 0.6 g of coarsely powdered ashwagandha RS with 10 mL methanol

for 15 minutes, cool and filter. Apply to the plate 10 μl of each solution as bands 10 mm by 2
mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air,
spray with solution of anisaldehyde reagent. Heat at 100º for 5-10 minutes and examine the plate
in day light. The chromatographic profile of the test solution is similar to that of the reference
solution.
Tests
Foreign organic matter Not more than 2.0 per cent.
Ethanol-soluble extractive Not less than 10.0 per cent.
Water-soluble extractive Not less than 15 per cent.
Ash Not more than 7.0 per cent.
Acid-insoluble ash Not more than 1.2 per cent.
Heavy metals 1.0 g complies with the limit test forheavy metals.

Loss on drying Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105º.

Microbial contamination Complies with the microbial contamination tests.

Assay. Determine by liquid chromatography

Test solution. Reflux about 5 g of the coarsely powdered substance under examination with 50
mL of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue further with
methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and
concentrate to 50.0 mL.

Reference solution (a). A solution containing 0.02 per cent w/v each of withanolide A RS and
withaferin A RS in methanol, prepared by heating gently on a water bath.

Reference solution (b).A 0.02 per cent w/v solution of withanolide A RS in methanol, prepared
by heating gently on a water bath, for peak identification.

Chromatographic system
A stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica
(10 μm), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by
dissolving 1.36 g of potassium dihydrogen phosphate in 900 mL water, adjust pH to 2.8 with
dilute phosphoric acid and diluting it to 1000 mL with water, flow rate. 1.5 mL per minute,
spectrophotometer set at 227nm, a 20 μl loop injector.
Time Buffer solution Acetonitrile
(min) (per cent v/v) ( per cent v/v)
0 90 10
18 55 45
25 20 80
27 90 10
37 90 10

Inject the reference solution (b) and determine the retention time of withanolide A. Inject the
reference solution (a). Identify the retention time of withaferin A. The test is not valid unless the
relative standard deviation for the replicate injections for both the analyte peaks corresponding to
withanolide A and withaferin A is not more than 2.0 per cent and resolution is not less than 2.0.
Inject the test solution. Calculate the contents of withanolide A and withaferin A.
Storage. Store protected from heat, moisture and against attack by insects and rodents.
VIII. Belladonna Leaf
Belladonna Leaf consists of the dried leaf and flowering tops of
Atropa belladonna Linn. or of A. acuminata Royle ex Lindley
(Fam. Solanaceae)‘ or a mixture of both species. Belladonna
Herb contains not less than 0.30 per cent of total alkaloids,
calculated as hyoscyamine with reference to the material dried
at 100º to 105º.

Description
Green to greenish-brown leaves, slightly darker on the upper
surface, often crumpled and rolled and partly matted together in
the drug. When whole, the lamina is 5 to 25 cm long and 3 to
12 cm wide, elliptical to ovate; acuminate at the apex, narrowing at the base; margin entire.
Petiole 0.5 to 4 cm in length. The young leaves are highly pubescent, the older leaves are slightly
pubescent along the veins. In the flowering tops, the stems are hollow and flattened, with leaves
in pairs of unequal size, in the axils of which are single flowers with campanulate corolla, about
2 cm long and 1.5 cm wide, purple or yellow-brown in colour, with five short, reflexed lobes,
five epipetalous stamens and one bilocular ovary with numerous ovules.

Identification
A. When examined under a microscope it shows epidermal cells with sinuate anticlinal walls and
cuticle which is often striated and furrowed. Covering and glandular hairs infrequent, though
more frequent in the young leaves and around the veins; covering hairs, multicellular, uniseriate,
with thin smooth walls; glandular hairs; short clavate glands with multicellular heads and glands
with a long uniseriate stalk and ovoid unicelluar head. Stomata, anisocytic, more frequent on the
lower epidermis. The midrib is characterised by an open arc of vascular bundles with isolated
groups of perimedullary phloem. Mesophyll dorsiventral with a single palisade layer.
Throughout the parenchyma and particularly just below the palisade layer are cells containing
microsphenoidal crystals of calcium oxalate or, very rarely, cluster crystals. The stems show
pericyclic fibres and perimedullary bundles of phloem, few trichomes; the cortical
parenchymatous cells and the pith cells contain microsphenoidal crystals of calcium oxalate.
B. Powder 1 g and shake for 2 minutes with 10 mL of 0.05 M sulphuric acid. Filter and add to
the filtrate 1 mL of strong ammonia solution and 5 mL of water. Extract with 15 mL of ether,
taking care to prevent the formation of an emulsion. Dry the ether extract over anhydrous sodium
sulphate and filter. Evaporate the filtrate to dryness, add 0.5 mL of fuming nitric acid and
evaporate to dryness. Add 10 mL of acetone and, dropwise, a 3 per cent w/v solution of
potassium hydroxide in ethanol (95 per cent); a deep violet colour develops.

C. Determine by thin-layer chromatography

Coating the plate with silica gel G..
Mobile phase.A mixture of 90 volumes of acetone, 7 volumes of water and 3 volumes of strong
ammonia solution.
Test solution. Add 15 mL of 0.05 M sulphuric acid to 0.6 g of the material under examination, in
fine powder, shake for 15 minutes, filter and wash the filter with 0.05 M sulphuric acid until 20
mL of filtrate is obtained; add 1 mL of strong ammonia solution to the filtrate, extract with two
quantities, each of 10 mL, of peroxide-free ether, separate the ether layer, by centrifugation if
necessary, dry the combined ether extracts over anhydrous sodium sulphate, filter, evaporate to
dryness on a water-bath and dissolve the residue in 0.5 mL of methanol.
Reference solution. Dissolve 50 mg of hyoscyamine sulphate in 9 mL of methanol (solution A)
and dissolve 15 mg of hyoscine hydrobromide in 10 mL of methanol (solution B); mix 8 mL of
solution A with 1.8 mL of solution B.
Apply to the plate 10 μl and 20 μl of each solution as bands.
After development, dry the plate at 105o for 15 minutes, allow to cool and spray with 10 mL of
modified potassiumiodobismuthate solution until the bands become visible as orange or brown
on a yellow background. The bands in the chromatogram obtained with test solution have similar
Rf values to those in the chromatograms obtained with reference solution (hyoscyamine in the
lower third of the chromatogram; hyoscine in the upper third) and are similar in colour and
atleast equal in size. Faint secondary bands may appear, particularly in the middle of the
chromatogram obtained with 20 μl of the test solution or near the line of application in the
chromatogram obtained with 10 μl of test solution. Spray the plate with a freshly prepared 10 per
cent w/v solution of sodium nitrite until transparent and examine after 15 minutes.
The colours due to hyoscyamine in the chromatogram change from brown to reddish-brown but
not to greyish-blue (atropine); any secondary bands are no longer visible.

Foreign organic matter Not more than 3 per cent.

Acid-insoluble ash Not more than 3 per cent.
Assay. Powder 50 g and determine the loss on drying by drying 2 g, accurately weighed, in an
oven at 105º. From the remaining sample, weigh accurately about 10 g, moisten with a mixture
of 5 mL of dilute ammonia solution, 10 mL of ethanol (95 per cent) and 30 mL of ether and mix
thoroughly. Transfer the mixture to a percolator with the aid of an extracting solvent mixture
consisting of 3 volumes of ether and 1 volume of chloroform. Allow to macerate for 4 hours and
percolate with the solvent mixture until complete extraction of the alkaloids is effected,
Concentrate the percolate to about 50 mL by distilling off the solvent mixture on a water-bath,
and transfer to a separator, previously rinsed with ether. Add a quantity of ether at least equal to
2.1 times the volume of the percolate and extract with three quantities, each of 20 mL, of 0.5 M
sulphuric acid. Transfer each acid extract to another separating Funnel. Combine the acid
extracts, make the solution alkaline with dilute ammonia solution and extract with chloroform
until complete extraction of the alkaloids has been effected. Wash the combined chloroform
extracts with 10 mL water, discard the water, evaporate the chloroform layer to dryness and heat
the residue for 15 minutes on a water-bath. Redissolve the residue in successive small quantities
of chloroform, evaporating to dryness on a water-bath each time before adding the solvent. Heat
for 15 minutes on a water-bath and dissolve the residue in 5 mL of chloroform. Add 20.0 mL of
0.01 M sulphuric acid, remove the chloroform by evaporation on a water-bath and titrate the
excess of acid with 0.02 M sodium hydroxide using methyl red solution as indicator.
1 mL of 0.01 M sulphuric acid is equivalent to 0.005788 g of total alkaloids calculated as
hyoscyamine.
Calculate the content of total alkaloids with reference to the dried material.
Storage. Store protected from light and moisture.
IX. Brahmi
Bacopa
Brahmi consists of the dried whole plant, preferably leaves
and stem of Bacopa monnieri (Linn.) Pennell (Fam.
Scrophulariaceae).
Brahmi contains not less than 2.5 per cent of bacoside A.

Description
A brown to reddish brown colour when completely dried or
green colour when partially dried with slightly bitter taste.

Identification

A. Macroscopic — Herbaceous comprising of stems, runner stems and leaves. Stems glabrous,
leafless towards the base; internodes long.Leaves spatulate-obovate, sessile, and glabrous.

B. Microscopic — Cortex in stem composed of parenchyma cells enclosing large air spaces;
xylem vessels radially arranged xylem rays uniseriate; pith parenchyma collapsed. In leaf, midrib
indistinct, mesophyll isobilateral of spongy cells, a few prismatic crystals of calcium oxalate in
mesophyll; stomata anomocytic on both the surfaces of leaf.

C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.

Mobile phase.A mixture of 70 volumes of chloroform and 30 volumes of methanol.

Test solution. Reflux 2 g of coarsely powdered substance under examination with 25 mL

methanol for 15 minutes, cool and filter. Reflux the residue further with 2 x 25 mL of methanol,
cool and filter. Combine all the filtrates and concentrate under vacuum to 25 mL.
Reference solution.Reflux 0.4 g of coarsely powdered brahmi RS with 5 mL methanol for 15
minutes, cool and filters.
Apply to the plate 10 μl of each solution as bands 10 mm by 2 mm. Allow the mobile phase to
rise 8 cms above the line of application. Dry the plate in air, spray with 20 per cent v/v sulphuric
acid in methanol. Heat at 100º for 5-10 minutes and examine the plate in day light. The
chromatographic profile of the test solution is similar to that of the reference solution. D. In the
Assay, the chromatogram obtained with test solution corresponds to the chromatogram obtained
with reference solution.
Tests
Foreign organic matter Not more than 2.0 per cent.
Ethanol-soluble extractive Not less than 6.0 per cent.
Water-soluble extractive Not less than 22 per cent.
Ash Not more than 18 per cent.
Acid-insoluble ash Not more than 6.0 per cent.
Heavy metals. 1.0 g complies with the limit test for heavy metals.
Loss on drying Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105º.
Microbial contamination Complies with the microbial contamination tests.

Assay. Determine by liquid chromatography

Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50
mL of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue further with
methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and
concentrate to 100.0 mL.

Reference solution.A 0.2 per cent w/v solution of bacoside ARS in methanol, prepared by heating
gently on a water bath.

Chromatographic system
A stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5
μm), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by
dissolving 0.5 g phosphoric acid in 800 mL of water, adjust pH to 2.8 with dilute phosphoric
acid and dilute to 1000 mL with water, flow rate. 1.5 mL per minute, spectrophotometer set at
205 nm, a 20 μl loop injector.

Time Buffer (pH 2.8) Acetonitrile

(min) ( per cent v/v) ( per cent v/v)
0 70 30
25 60 40
35 40 60
36 70 30
45 70 30
Inject the reference solution. The relative retention times are 1.00 for bacoside A3, about 1.04 for
bacopaside II, 1.13 for jujubogenin isomer of bacopasaponine C and 1.19 for bacopasaponine C
as bacoside A. The test is not valid unless the relative standard deviation for the replicate
injections is not more than 2.0 per cent.

Inject the test solution and reference solution.

Calculate the content of bacoside A.
Storage. Store protected from heat, moisture and against attack by insects and rodents.
X. Haridra
Haldi; Turmeric; Curcuma
Haridra consists of the dried rhizomes of Curcuma longa
Linn (Fam. Zingiberaceae).
Haridra contains not less than 1.5 per cent of curcumin,
calculated on the dried basis.

Description
Externally yellowish to yellowish brown with root scars
and annulations.Odour, aromatic; taste, warmly aromatic and bitter.

Identification
A. Macroscopic — Rhizome oblong, conical or cylindrical to elongate, finger-like; internally
orange yellow. Texture hard and heavy; fracture short.
B. Microscopic — Ground tissue of parenchyma cells; cells filled with gelatinized starch grains
and yellow pigment. Fibrovascular bundles and oil cells scattered throughout ground tissue.
C. Determine by thin-layer chromatography coating the1 plate with silica gel GF 254.

Mobile phase. A mixture of 94 volumes of chloroform, 5 volumes of ethanol and 1 volume

glacial acetic acid.

Test solution. Extract 1 g of the coarsely powdered substance under examination with 5 mL
methanol for 10 minutes with slight warming. Filter and use the filtrate.

Reference solution. Reflux 1 g of coarsely powdered haridra RS with 5 mL methanol for 15

minutes, cool and filter.
Apply to the plate 10 μL of each solution as bands 10 mm by 2 mm. Allow the mobile phase to
rise 8 cms above the line of application. Dry the plate in air and examine in daylight and ultra-
violet light 365 nm. The chromatographic profile of the test solution is similar to that of the
reference solution.
Tests

Foreign organic matter Not more than 2.0 per cent.

Ethanol-soluble extractive Not less than 6.0 per cent.

Water-soluble extractive Not less than 12 per cent.

Ash Not more than 10 per cent.

Acid-insoluble ash Not more than 2.0 per cent.

Heavy metals 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).

Water Not more than 12.0 per cent, determined on 0.2 g.

Microbial contamination Complies with the microbial contamination tests.

Assay. Determine by liquid chromatography

Test solution. Reflux about 1 g of the coarsely powdered substance under examination with 50
mL of methanol on a water bath for 15 minutes cool and filter. Reflux the residue further with 5
x 25 mL of methanol, cool and filter. Combine all the filtrates and concentrate to 100.0 mL.
Reference solution.A 0.01 per cent w/v solution of curcumin RS in methanol.

Chromatographic system
A stainless steel column 25 cm x 4.6 mm packed with silicagel consisting of porous spherical
particles with chemically bonded nitrile group, mobile phase: 35 volumes of tetrahydrofuran and
65 volumes of a buffer solution prepared by dissolving 10 g of citric acid in 1000 mL of water,
adjusting the pH to 3.0 with dilute ammonia solution,flow rate. 1.2 mL per minute,
spectrophotometer set at 430 nm, a 20 μl loop injector.
Inject the reference solution. The test is not valid unless the relative standard deviation for the
replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution.
Calculate the content of curcumin.

Storage. Store protected from moisture.

XI. Sarpgandha
Rauwolfia Root

Serpgandha consists of the dried roots of Rauwolfia

Serpentina Bentham ex Kurz (Fam Apocynaceae).

Serpgandha contains not less than 0.15 per cent of

reserpine and ajmalicine, calculated on the dried basis.

Description. Taste bitter, odour indistinct.

Identification
A. Macroscopic — Roots are sub cylindrical to tapering, tortuous or curved, rarely branched.
Occurs as segments usually from 5 to 15 cms in length and 3 to 20 mm in diameter. Externally
grayish yellow to brown, wood pale yellow. Roots tough with longitudinal marking & slightly
wrinkled surface. When scraped, bark separates readily from the wood. Fracture is short and
irregular.

B. Microscopic — In transverse section, cork cells in 2 to 8 alternating bands of radically narrow

and broader cells, thin, lignified up to 75 μm in tangential width, broader cells up to about 90 μm
in radial length, phelloderm, tangentially elongated to isodimetric parenchyma cells containing
starch and short latex cells with brown resinous matter; secondary cortex consists of parenchyma
cells, heavily packed with starch grains secondary phloem contains phloem parenchyma and
sieve elements, parenchyma contains starch and angular crystals of calcium oxalate 3 to 20 μm in
length. Xylem is about 4/5 of the diameter of the root, wood is transversed by medullay rays 1 to
5 cells in width. Xylem consists of vessels, tracheids, wood parenchyma & wood fibers. Xylem
vessels are elongated up to 350 μm in length and 50 μm in width and contains simple or bordered
pits; tracheids lignified, pitted; wood parenchyma with moderately thick, lignified and pitted
walls containing starch; wood fibres highly thickened with pointed ends, stone cells absent.
C. Determine by thin-layer chromatography

Coating the plate with silica gel G..

Mobile phase. A mixture of 70 volumes of chloroform and 30 volumes of acetone.

Test solution. To 250 mg of the coarsely powdered substance under examination, add 5 mL
methanol, shake for 10 minutes, and filter. Wash the residue with 5 mL of methanol and add the
washing to the filtrate.
Reference solution. To 250 mg of sepgandha RS, add 5 mL methanol, shake for 10 minutes, and
filter. Wash the residue with 5 mL of methanol and add the washing to the filtrate.

Apply to the plate 10 μL of each solution as bands 10 mm by 2 mm. Allow the mobile phase to
rise 12 cm. Dry the plate in air, spray with anisaldehyde solution and heat at 100° for 5 minutes
and examine the plate in day light. The chromatogram obtained with test solution shows two
pinkish-violet bands corresponding to the bands in the chromatogram obtained with reference
solution. A dark brown band may also appear at the line of application in the chromatogram
obtained with test solution.

Tests

Foreign organic matter Not more than 2.0 per cent

Ethanol-soluble extractive Not less than 2.0 per cent.

Water-soluble extractive Not less than 5.0 per cent.

Ash Not more than 8.0 per cent.

Acid-insoluble ash Not more than 2.0 per cent.

Heavy metals 1.0 g complies with the limit test for heavy metals,
Loss on drying Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105°.

Microbial contamination limits Complies with the microbial contamination tests.

Assay. Determine by liquid chromatography.

Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50
mL of ethanol (95 per cent), on a water bath for 15 minutes, cool and filter. Reflux the residue
further with ethanol (95 per cent), till the last extract turns colorless, cool and filter. Combine all
the filterates and concentrate to 100.0 mL.

Reference solution (a).A solution containing a 0.002 per cent w/v reserpine RS and 0.002 per
cent w/v ajmalicine RS in ethanol (95 per cent).

Reference solution (b).A 0.002 per cent w/v reserpine RS in ethanol (95 per cent) for peak
identification.

Chromatographic system
A stainless steel column 25 cm × 4.6 mm packed with octadecylsilane bonded to porous silica
(5μm), mobile phase: a mixture of 35 volumes of acetonitrile and 65 volumes of a buffer
solution prepared by dissolving 6.80 g of potassium dihydrogen phosphate in 1000 mL water,
adjust the pH to 3.0 with dilute orthophosphoric acid, flow rate. 1 mL per minute,
spectrophotometer set at 268 nm, a 10 μl loop injector.
Inject the reference solution (a). The test is not valid unless the relative standard deviation for the
replicate injections is not more than 2.0 per cent. Inject reference solution (b) for peak
identification.
Inject the test solution and reference solution (a).
Calculate the content of reserpine and ajmalicine.
Storage. Store protected from heat, moisture and against attack by insects and rodents.
XII. Senna Leaf

Cassia leaf
Senna leaf consists of the dried compound leaves of
Cassia angustifolia or Cassia senna Vhal. (Family:
Leguminosae).

Description
Pale yellowish green coloured leaflets with
mucilaginous and faint odour.

Identification

A. Macroscopic — Leaflets,2.5 to 8 cm long and 5-15 mm wide at centre, pale yellowish green,
elongated lanceolate, slightly asymmetric at base; margins entire, flat, apex acute with a sharp
spine; both surface smooth with sparce trichomes; odour, faint but distinctive; taste,
mucilaginous and disagreeable but not distinctly bitter.

B. Microscopic — Transverse section shows outer single layered mucilaginous epidermal cells.
Unicellular hairs presents. Stomata paracytic, numerous on both surface. Mesophyll consists of
upper and lower palisade layers with spongy layer in between, primatic crystals of calcium
oxalate present on larger veins.

C. In the Assay, the chromatogram obtained with test solution corresponds to the chromatogram
obtained with reference solution.

D. Determine by thin-layer chromatography coating the plate with silica gel F254.

Mobile phase. A mixture of 40 volumes of n- propyl alcohol, 40 volumes of ethyl acetate, 29

volumes of water and 1 volume of glacial acetic acid.
Test solution. Take 1 g of the dried leaves powder substance under examination. Add 25 mL of
methanol, reflux for 10 minutes, cool and filter. Reflux the residue with another 20 mL of
methanol, cool and filter. Combine all the filtrates and concentrate to 10 mL.

Reference solution.A 0.001 per cent w/v solution of Sennoside A and B in methanol. Apply to
the plate 10 μl of each solution as bands 10 mm by 6 mm. Allow the mobile phase to rise 8 cm.
Dry the plate in air, spray with 20 per cent v/v of nitric acid solution. Heat at 1000 to 105° for
about 10 minutes and immediately examine the plate. The chromatographic profile of the test
solution is similar to that of the reference solution.

Tests

Foreign organic matter Not more than 1.0 per cent.

Ash Not more than 14.0 per cent.

Acid-insoluble ash Not more than 2.5 per cent.

Loss on drying Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105°.

Microbial contamination Complies with the microbial contamination tests.

Assay. Determine by liquid chromatography.

Test solution. Weigh accurately 1.0 g of the coarsely powdered substance in a round bottom
flask, add about 10 mL of 1 per cent v/v acetic acid and 25 mL of methanol and reflux on a
water bath for about 30 minutes. Cool to room temperature; make up the volume up to 50.0 mL
with methanol and filter.

Reference solution.A 0.004 per cent w/v solution of sennosides RS in methanol.

Chromatographic system
A stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5
μm), mobile phase: a mixtures of 82 volumes of 1 per cent v/v acetic acid in water and 18
volumes of acetonitrile, flow rate. 1 mL per minute, spectrophotometer set at 350 nm, a 20 μl
loop injector.

Inject the reference solution. The test is not valid unless the relative standard deviation for the
replicate injections is not more than 2.0 per cent.

Inject the test solution and reference solution.

Calculate the content of sennoside A and B.

Storage. Store protected from light and moisture.

XIII. Shatavari
Asparagus racemosus root.
Shatavari consists of the tuberous roots of Asparagus
racemosus willd. (Fam .Liliaceae).

Shatavari contains not less than 0.1 per cent of shatavarin

IV, calculated on the dried basis.

Description
The tuberous root bits are dirty white in color,
longitudinally wrinkled with yellow hard central core. It is starchy and slightly bitter followed by
sweet taste.

Identification

A. Macroscopic — The tuberous roots are borne in a compact bunch and are fleshy, and spindle
shaped. They are marketed in pieces 5-15 cm in length and 2 cm in thickness. They are silvery
white or ash-colored externally and white internally more or less smooth when fresh, developing
longitudinal wrinkles when dry.

B. Microscopic — The inner parenchymatous zone of cortex is composed of 18-24 layers in

upper and 42-47 layers in the middle tuberous portion of the roots. The cells are thin - walled
cellulosic, with circular to oral outlines and distinct inter cellular spaces. In some roots 3-4 layers
of cortex immediately adjacent to the endodermis are modified into a sheath of stone cells
round the endodermis. The number of vascular bundles is 30- 35 in the upper levels and 35-45 in
the middle tuberous portions of the roots.

C. Determine by thin-layer chromatography coating the plate with silica gel F254.

Mobile phase.A mixture of 13 volumes of chloroform, 10 volumes of methanol and 2 volumes of

water.
Test solution. Reflux 1 g of the coarsely powdered substance under examination with 30 mL of
methanol for 30 minutes, cool and filter. Reflux the residue further with 2 × 30 mL of methanol,
cool and filter. Combine all the filtrates and concentrate under vacuum to 10.0 mL.

Reference solution. Reflux 1 g of shatavari RS with 30 mL of methanol for 30 minutes, cool and
filter. Reflux the residue further with 2 × 30 mL of methanol, cool and filter. Combine all the
filtrates and concentrate under vacuum to 10.0 mL.

Apply to the plate 5 μL of each solution as bands 10 mm by 2 mm. Allow the mobile phase to
rise 8 cm. Dry the plate in air, spray with vanillin sulphuric acid reagent prepared by mixing 1
per cent w/v vanillin in ethanol (95 per cent) and methanolic sulphuric acid (5 per cent) before
spraying. Heat at 120º for 10 minutes and examine the plate in day light. The chromatographic
profile of the test solution is similar to that of the reference solution.

Tests

Foreign organic matter Not more than 2.0 per cent.

Ethanol-soluble extractive Not less than 15.0 per cent.

Water- soluble extractive Not less than 20.0 per cent.

Ash Not more than 15.0 per cent.

Acid-insoluble ash Not more than 3.0 per cent.

Heavy metals 1.0 g complies with the limit test for heavy metals.

Loss on drying Not more than 15.0 per cent, determined on 5 g by drying in an oven at 105º.

Microbial contamination Complies with the microbial contamination tests.

Assay. Perform the Assay by following method I or by method II. The result of the method II
will be official in case of dispute.

Method I

Determine by thin-layer chromatography coating the plate with silica gel GF 254.

Mobile phase.A mixture of 6.5 volumes of chloroform and 3.5 volumes of methanol.

Test solution. Reflux 4 g of the coarsely powdered substance under examination with 50 mL of
methanol on a water-bath for 30 minutes, cool and filter. Reflux the residue further with
methanol till the last extract turns colourless, cool and filter. Combine all the filtrates and
concentrate to 50 mL.

Reference solution.A 0.008 per cent w/v solution of shatavarin IV RS in methanol.

Apply to the plate 5 μl of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise
8 cm. Dry the plate in air and spray with 10 per cent v/v sulphuric acid in methanol.

Heat the plate at 100º for 5 minutes, scan the plate in absorbance mode at 500 nm. Record the
chromatograms and measure the responses for the analyte peak.

Calculate the content of shatavarin IV.

Method II
Determine by liquid chromatography

Test solution. Reflux 5 g of the coarsely powdered substance under examination with 50 mL of
methanol on a water-bath for 30 minutes, cool and filter. Reflux the residue further with
methanol till the last extract turns colourless, cool and filter. Combine all the filtrates and
concentrate to 50.0 mL.

Reference solution.A 0.01 per cent w/v solution of shatavarin IV RS in methanol. Dilute suitably
to prepare 0.0075- 0.075 per cent w/v solution.

Chromatographic system
A stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5
μm), mobile phase: a mixture of 60 volumes of acetonitrile and 40 volumes of water, flow rate. 1
mL per minute, use evaporative light scattering detector, temperature evaporator 110º, nebulizer
90º, nebulizer gas nitrogen and gas flow 1 SLM, a 20 μl loop injector.

Inject the reference solution. The test is not valid unless the regression coefficient is not more
than 0.9.
Inject the test solution and reference solution.

Calculate the content of shatavarin IV.

Storage. Store protected from heat, moisture and against attack by insects and rodents.
 Experiment No. 7
Aim: Determination of Aldehyde Content.

Requirements:
Chemicals:
Benzene, hydroxylamine hydrochloride reagent, alcohol, methyl orange indicator, potassium
hydroxide.
Apparatus:
Stoppered tube, burette, pipette, burette stand, measuring cylinder.

Procedure:
Take accurately weighed quantity of 10 gm. of oil sample into stoppered tube approximately 25
mm in diameter and 150 mm in length. To this add 5 mL of benzene and 10 -15 mL of
hydroxylamine hydrochloride reagent in alcohol (60%) and a drop of methyl orange as indicator.
Stopper the tube and shake vigorously and titrate immediately with 0.5 N alcoholic KOH, until
red colour changes to yellow. Continue shaking and neutralization, still full yellow colour of the
indicator is permanent in lower layer. Shake vigorously for 2 minutes and allow the layer to
separate.
The reaction is complete in about 15 minutes(each mL of 0.5 N KOH in alcohol(60%) is
equivalent to 0.070672 g of citral).This procedure gives an approximate determination of citral in
volatile oil.Then carry out the second determination in the same manner using the colour
standard of first experiment with addition of 0.5mL of 0.5 N alcoholic potassium hydroxide
solution.Note the second reading and calculate the content(each mL of 0.5 N alcoholic potassium
hydroxide(60%) is equivalent to 0.06661 g of cinnamic aldehyde) .

Questions
Q. 1. What is the significance of aldehyde content?
Q. 2. Give any two drugs which having aldehyde as main chemical compound.
 Experiment No. 8
Aim: Determination of Phenol Content.

Requirements:
Chemicals:
Clove oil, sulphuric acid, water, potassium hydroxide solution, potassium hydroxide.
Apparatus:
Cassia flask, water bath, separating Funnel, burette, pipette, burette stand, measuring cylinder.

Procedure:
For determination of eugenol in clove oil, take cassia flask of 150 mL capacity with long neck
graduated from 0-10 mL. Clean the flask with sulphuric acid, followed by water and dry.
Introduce accurately 10 mL of the sample, add 75 mL of potassium hydroxide solution and shake
for 5 minutes and then heat on a boiling water bath shaking about 3 times during heating. Allow
to cool to room temperature and when layers are separated completely add sufficient potassium
hydroxide solution, to raise the lower level in the graduated portion of flask. Allow it to stand for
18-24 hrs and read the volume of oily layer and calculate the eugenol content.

Questions
Q. 1. What is the biological source of clove?
Q. 2. What is the significance of determination of phenol content?
Q. 3. Give any two drugs having phenolic active chemical constituent.
 Experiment No. 9
Aim: Determination of Total Alkaloids.

Requirements:
Apparatus:
Digital weighing balance, beaker, measuring cylinder, separating Funnel, heating mental, water
bath, conical flask, separating Funnel, pipette, burette.
Chemicals:
Ammonia solution, chloroform, sulphuric acid, ammonia solution, chloroform, sodium
hydroxide, methyl red.

Principal:
The alkaloid components present in given sample are liberated by using an alkali and it is extract
by using an organic solvent such as chloroform. The unstable alkaloids are converted in to its
salts, which are further estimated by titrating against the alkali by using methyl red as indicator.
Determination of total alkaloid is performed for the purpose of standardization,proof of purity,
commercial evaluation, and pharmacological purposes.
The amount of alkaloid that occur in crude alkaloid drug are subjected to considerable variation
in different sample of same drug due to; the age of plant when it it is get collected, the season of
the year when the drug is get harvested, the soil and the climate in which the drug is grown, the
condition under which the drug is get collected, how it is get dried and stored.solution

Procedure:
1) Weigh 10 gm of drug sample and add ammonia solution, mix thoroughly ensures that
complete liberation of basic alkaloids.
2) Macerate the content with organics solvent (chloroform) and allow macerate for four
hours.
3) Concentrate the extract about 50 mL. and transfer the content to separating Funnel, and
extract with dilute acid 0.5 N sulphuric acid.
4) The acid alkaloid is made alkaline with dilute ammonia and extract with chloroform.
 5) Wash the chloroform extract with 10 mL of water and evaporate the chloroform part to
dryness.
6) Dissolve the residue in 5 mL of chloroform and add 20 mL of 0.02N sulphuric acid and
evaporate it on water bath to remove chloroform.
7) Titrate the content against 0.02N sodium hydroxide using methyl red as indicator.

Calculations:
The total alkaloid present in drug = NaOH volume consume × normality of silphuric acid ×
equivalent weight factor (0.005788) × 100/ ( weight of the grug taken)

Questions
Q. 1. The principle of determination of total alkaloid depends on which physical characteristic of
alkaloids.
Q. 2. What is the ultimate use of determination of total alkaloids?