Philadelphia University

Faculty of Pharmacy

Manual of Practical
Pharmacognosy &Phytochemistry
Lab.
2016/2017

1
Course/ academic calendar

Week Basic and support material to be covered

16-20/10 Safety rules

23-27/10 Introduction to the microscopy

30/10-3/11 Microscopical identification for starch + calcium

oxalate

6-10/11 Microscopical identification for ginger root + cinnamon

barks

13-17/11 Microscopical identification for senna leaves +

chamomile flower

First exam

27/11-1/12 Microscopical identification for Anise fruit +linseed

4-15/12 Extraction and identification for Anthraquinone

glycosides
18-22/12 Extraction and identification for cardiac glycosides

25-29/12 Identification for alkaloid by general test and specific

test (first part).

Second exam

8-12/1 Identification for alkaloid by microcrystalline test

(second part)+ TLC for rutin

Final exam

2
 Experiment (1)

Introduction

Pharmacognosy is the study of starting material and substance

intended for therapeutics, and biological origin, in other words obtained
from plants animals, or by fermentation from microorganism. The word
pharmacognosy is derived from the Greek pharmakon, a" drug" and
gignosco," to acquire knowledge of" pharmacognosy is closely related to
botany and plant chemistry and indeed both originated from the earlier
scientific studies on medicinal plants.

Preparation of drugs for microscopical examination and general use

of reagents
The following aims should be kept in mind for the microscopical
examination of advanced crude drugs:
1- The determination of the size, shape and relative positions of the
different cell and tissues.
2- The determination of chemical nature of the cell walls.
3- The determination of the form and chemical nature of cell content.
Disintegration serves for the isolation of the specific tissues and
bleaching and defatting techniques for observing deeply colored
materials and fatty seeds respectively. Almost certainly, clearing
reagents will be required together with the range of suitable stains for
cell wall and cell contents.

Reagent used as mounting reagent:

Chloral hydrate reagent: dissolve 80 gm of chloral hydrate in 20 ml

water, a valuable and widely used reagent, heating at water bath and
maceration of the plant powder well give a better result since
defatting and clearing action of the reagent well be better, this
reagent well dissolve many cell content as fat, resin starch and
calcium oxalate.

Phloroglucinol/ HCl reagent: a 1% solution in 90% ethanol with

conc. HCl as a test for lignin. Lignified structured well be colored
pink/red.
Hydrochloric acid is a powerful clearing agent and it will dissolve
many cells content as ca oxalate and starch.

3
Diluted ethanol reagent: different strength are used for preserving
material and for hardening, alcohol acts as a clearing reagent by
dissolving oils, resins, chlorophyll, etc., while it does not dissolve gum
and mucilage (therefore it is useful for plant powder containing them).

Iodine reagent: this gives blue color with starch and hemicelluloses.
Smith's reagent: is suitable mounting reagent for starch, it is composed
of equal amount of water, glycerin and 50% acetic acid.

4
5
 Experiment (2)
Starch

Use of starch in pharmaceutical industry:

1- Dusting powder (in which adsorbent properties are important).

2- Antidote in treatment of iodine poisoning.
3- Skin emollient, basis for enemas.
4- Tablet disintegrant.
5- Lubricant for surgeons gloves (sterilizable maize starch).

Macroscopical character:

Starch occurs in irregular, angular masses or as a white powder. It

is insoluble in cold water but forms a colloidal solution on boiling with
about 15 times it weight of water, the solution forming a translucent jelly
on cooling. A starch mucilage is colored deep blue with solution of iodine,
the color disappear on heating but reappear on cooling, when starch is
heated with water the granules first swells and then undergo
gelatinization, maize starch is neutral but other commercial starches show
an acid ( wheat and potato) or alkaline (rice) reaction.

Microscopical character:

Starch can be identifies by microscopical examination, they should

be mounted with water or smith's reagent.the size, shape and structure of
starch granules from any particular plant varies within definite limits, so
that it is possible to distinguish between the starch derived from different
species starch granules may be simple or compound, and the description
of starch granules as 2-, 3-, 4-, 5-compound refers to the number of
component granules present in the compound granules.

Hilum (the starting point of the granules in the amyloplast) is

another character that can be used to distinguish different types of starch.
On microscopical examination, hilum takes the form of a rounded dot or
simple or multiple cleft.

Striation: starch granules are built up of the deposition of

successive layers around the hilum and concentric rings or striation are
often clearly visible in large granules as potato starch.

6
Rice:
Origin: obtained from the grains of the fruits of Oryza sativa Poaceae
Shape: polyhedral or subspherical.
Hilum: small central points.
Striation: absent.
Aggregation: mainly compound.

Maize:
Origin: obtained from the grains of the fruit of Zea mays Poaceae
Shape: polyhedral or subspherical
Hilum: central cleft with three to five rays.
Striation: not visible.
Aggregation: simple.

Potato:
Origin: obtained from the tubers of Solanum tuberosum Solanaceae
Shape: ovoid to subspherical.
Hilum: eccentric point near the narrower end of ovoid granules.
Striation: well marked concentric.
Aggregation: mostly simple occasionally compound.

7
8
 Report sheet (2)

Title:
Student name :
Objective:

Botanical source Drawing Regent used

Genus:

Species:

Family:

Genus:

Species:

Family:

Genus:

Species:

Family:

9
 Experiment (2)

Calcium Oxalate

Calcium oxalate are found in plants as a result of interaction of

oxalic acid (a metabolic product) with calcium salt; e.g. Ca-sulphate and
the consequent precipitation upon super saturation of the cell sap with
this salt. Different types of calcium oxalate crystals with various shapes
can be used as a diagnostic element for plant identification. It is usually
sufficient to describe the general form and size of the crystals, without
reference to a crystallographical class, the most common form
encountered are prisms (senna, hyoscyamus, liquorices); rosettes (rhubarb,
senna, clove); bundles of acicular crystals (squill); microsphenoidal or
sandy crystal (belladonna). When calcium oxalate is present, it is
important to record the type of the crystal, shape and distribution. The
cells containing calcium oxalate differ from those which don’t contain
calcium oxalate in size, form or content, and are often called idioplast.

Procedure: Clear different plant powders using chloral hydrate

solution and examine the various types of calcium oxalate crystals.
The crystals can be identified as calcium oxalate if they are insoluble in
acetic acid and caustic alkali, but soluble in hydrochloric acid and sulfuric
acid without effervescent.

10
 Crystal layer

11
 Report sheet (2)

Title:
Student name:
Objective:

Type of Ca-oxalate Drawings Reagent used

12
 Experiment (3)

Roots and Rhizomes

Root: is the descending portion of the plant axis which

usually grows below the soil, and serves as an anchor and acts to
absorb water and nutrients to be transported to the upper portion of
the plant.

Rhizome: a fleshy, elongate non-erect stem, often, but not

always. It is subterranean.

Peeled & Unpeeled root and rhizomes: Peeled rhizomes and

roots will have the outermost layer removed (cork layer will be
absent), on the contrary of the unpeeled one which will preserve the
outermost layer.

Sclerenchymal cells: Sclerenchyma is a hard supporting

tissue with heavy secondary thickening. Sclerenchymatous cells are
usually divided into two categories according to their aspect ratio:

Scleride (stone cells): are typically roughly isodiametric. Although

elongated and branched forms also occur. They may be found singly, in-
group or as a complete layer. Pitting and stratification may occur

Fibers are typified by high length to width ratio. They are usually
thick walled and have a narrow lumen and pointed ends. Fibers are
usually classified according to the area in which they occur as a pericyclic,
xylem or phloem fibers. A crystal sheath is sometimes formed around
sclerenchyma and this features, together with the size, frequency, and the
distribution of the cells, is often of diagnostic significance.

Cork: as the plant axis increases in diameter, a cork cambium

or phellogen usually arises which by its activity, produces a new
protective tissues known collectively as periderm, which replaces
the epidermis and part or all of the primary cortex. Cork tissues is
built up of a compact masses of cells, the mature cork cell is dead,
impermeable to water and often filled with dark reddish-brown
content rich in tannins and related substances.

Starch: occurs as granules in almost all organs in plant but

most abundantly in roots, rhizomes, fruit and seeds.

13
 Ginger Rhizome

Origin: Dried rhizomes of Zingiber officinalis Zingiberaceae

Constituent: volatile oil, resin, starch
Uses: carminative, aromatic stimulant, motion sickness, antiemetic.

Physical Characters:
Colour: pale yellow to creamy.
Odor: pleasant aromatic.
Taste: pungent.
Shape: Pieces called hand, buffy with scars on the outer cork
layer ,on drying loose the buffy shape due to shrinking of cells.

14
15
 Report sheet (3)
Student name:

I- Objective:
-------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------

II- Scientific name of the plant (Botanical source):

Genus Species Family

III- Macroscopical characters:

Color

Odor

Touch

Taste

Size

Shape

IV-Microscopical character:

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

16
 Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

17
 Experiment (3)

Barks
Xylem: It is the principal water-conducting tissue of the plant.
Xylem is a compound tissue, the structural elements of which are:
tracheid, vessels, xylem fibers and xylem parenchyma.
Tracheid and vessels are elongated structures that are non-
living at maturity and are concerned in water conduction. Secondary
wall thickening may give rise to annular spiral scaliform or reticulate
lignified walls. The main difference between vessels and tracheids is
that vessels have pores at each end and are connected to form
continuous tube while trachieds have no pores.

Phloem: is a compound tissue and is responsible for the

transport of food. It contains parenchyma, sclerenchyma and sieve
element. The sieve tubes are the most highly specialized cell
composed of a vertical series of elongated cells, interconnected by
perforation in their walls in areas known as sieve plates. Sieve
elements are usually broken during powdering while phloem scleride
are important in the identification of certain barks.
Cork: see the cork in root experiment
Scleride: see the cork in root experiment

Cinnamon Bark

Origin: dried bark of Cinnamomum zeylanicum Lauraceae

Constituent: Volatile oil, tannins, starch, Ca-oxalate.
Uses:, carminative, antifungal,astringent,antimicrobial.

Physical Characters:
Color: reddish brown.
Odor: pleasant and aromatic.
Taste: pleasant.
Chemical test: test for tannins with FeCl3
Shape: channeled or single quills, decorticated.
The outer surface is brown with longitudinal shiny wavy lines, and
with occasional scars and holes. Fractures are short.

18
19
 Report sheet (3)

Student name:

I-Objective:
-------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------

II-Scientific name of the plant (Botanical source):

Genus Species Family

III-Macroscopical characters:

Color

Odor

Touch

Taste

Size

Shape

IV-Microscopical character:

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

20
 Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

21
 Experiment (4)
Leaves
Epidermis: this is the outermost layer of the plant structure and is
usually one cell thick. Important diagnostic features include the
shape of the anticlinal (vertical) and the periclinal (horizontal) walls
(e.g. straight or wavy), the presence of thickening (such as beading),
and the occurrence of striations of the surface cuticle.
Distributed among the relatively non specialized epidermal
cells are a number of highly specialized and characteristic structures
mainly stomata and trichomes.
Trichomes: Most leaves and many herbaceous stems, flowers fruits,
and seeds possess hairs or trichomes of one kind or another. Many
show hair of more than one type. The trichomes (hair) may be
grouped into:

1. Non-glandular (clothing or protective) hairs:

These may be unicellular which vary from small papillose
outgrowth to large structures, or multicellular which may be uniseriate,
biseriate or multiseriate.

2. Glandular (secretory) hairs:

A glandular hair usually has a stalk and a head. The stalk may be
either unicellular or multicellular, and likewise, the head may be either
unicellular or multicellular. Trichomes serve a number of functions,
which include physical and chemical protection for the leaf against
aphide and insects, and the maintenance of a layer of still air on the leaf
surface, thus preventing excess water loss by transpiration. Glandular
trichrome secretions are highly important in perfumery, food and
pharmaceiutical industry.

Stomata: Stomata (Singular: stoma) most frequently occur on the

young leaves and stems, but can also be found on other organs like
flowers. Stomata allow gas exchange between the surrounding air
and the photosynthetic cells inside the leaves. Also, they are the
major avenues for the loss of water from the plant by evaporation, a
process called transpiration.

The arrangement of epidermal cells around the stoma falls into one
of four main types:

Anomcytic stomata: the stomata are surrounded by cells resembling

the other epidermal cells with no particular arrangement.

22
Anisocytic stomata: the stoma is surrounded by three or four
subsidiary cells one of which is smaller or larger than the others.

Diacytic stomata: the stoma has two subsidiary cells with their long
axis at right angles to the pore of the stoma.

Paracytic stomata: the stoma has two subsidiary cells with their
long axis parallel to pore of the stoma.

Senna Leaves

Origin: dried leaves of Cassia acutifolia, Cassia angustifolia fam.

Fabaceae.
Constituent: anthraquinone glycoside
Uses: purgative, laxative.

Physical characters:
Color: grayish-green to yellowish-green.
Odor: faint.
Taste: mucilaginous slightly bitter
Shape: Leaflets: ovate to lanceolate in shape, 1.5-6cm, entire margin
with brittle and thin texture, pinnate reticulate venation.

23
24
 Report sheet (4)

Student name:

I-Objective:
-------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------

II-Scientific name of the plant (Botanical source):

Genus Species Family

III-Macroscopical characters:

Color

Odor

Touch

Taste

Size

Shape

IV-Microscopical character:

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

25
 Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

26
 Experiment (4)

Flowers

Flower: a determinate axis with spore-bearing appendages

(and usually sterile appendages) and short internodes occurring in
the angiosperm.
Pollen grain: a microspore wall containing a mature
or immature male gametophyte in seed plant.
Epidermis: see underlying epidermis in leaf experiment.
Stomata: see underlying stomata in leaf experiment
Trichomes: see underlying trichomes in leaf experiment

Chamomile Flowers

Origin: dried flower heads of Matricaria chamomilla, Fam.

Asteraceae.
Constituent: Sesquiterpene (chamazulene), flavones.
Uses: anti-inflammatory, spasmolytic, carminative, insomnia.

Physical Characters:
Color: light brown to puff.
Odor: characteristic strong aromatic.

27
Taste: slightly bitter aromatic.
Shape: Small flowers with oval buffy receptacle, white tubular
ligulate florate (petals), stalked with bracet.

28
 Report sheet (4)

Student name:

I-Objective:
-------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------

II-Scientific name of the plant (Botanical source):

Genus Species Family

III-Macroscopical characters:

Color

Odor

Touch

Taste

Size

Shape

IV-Microscopical character:

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

29
 Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

30
 Experiment (5)

Fruit

Fruit: the mature ovary or ovaries of one or more flowers

and sometimes associated structures.
Endocarp: the innermost layer of the ovary wall in the fruit.
Epicarp: the outermost layer of the ovary wall in the fruit.
Mesocarp: the layer in the fruit between exocarp
and endocarp.

Anise fruit:

Origin: dried ripe fruit of Pimpinella anisum Umbelliferae.

Constituent: volatile oil, fixed oil.
Uses: aromatic stimulant, carminative.

Physical characters:
Color: medium brown.
Odor: characteristic aromatic.
Taste: aromatic.
Shape: Entire cremocarp with 5 ridges, which split into mericarps
and carpophores.

31
 Report sheet (5)

Student name:

I-Objective:
-------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------

II-Scientific name of the plant (Botanical source):

Genus Species Family

III-Macroscopical characters:

Color

Odor

Touch

Taste

Size

Shape

IV-Microscopical character:

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

32
 Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

33
 Experiment (5)

Seed

Endosperm: the nutritive tissue for the embryo in

angiosperms.
Endosperm and perisperm are tissues containing food reserve
and are formed respectively inside and outside the embryo sac.
Embryo: ovules are developed into embryo. size and position
of embryo are important. Likewise, size, shape and number of
venation of the cotyledon are also significant. For the radicle, size
and shape are also important.
Oil: fixed oil and fat are widely distributed and occur in both
vegetative and reproductive structures. They often occur in seeds
where they may replace carbohydrate as a reserve food material.
Starch: see starch from underlying starch in root experiment.

Linseed

Origin: Dried ripe seed of Linum usitatissimum Linaceae

Constituent: Fixed oil, mucilage, cyanophoric glycoside.
Uses: Demulcent, treatment of constipation.

Physical characters:
Color: Yellowish-brown with distinct darker brown fragments.
Odor: Slight.
Taste: Oily and mucilaginous.
Shape: Glossy brown flattened shaped.5mm length, the seed coat
called testa with hilum at the lower end and raphe near the upper end.

34
 Report sheet (5)

Student name:

I-Objective:
-------------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------------

II-Scientific name of the plant (Botanical source):

Genus Species Family

III-Macroscopical characters:

Color

Odor

Touch

Taste

Size

Shape

IV-Microscopical character:

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

35
 Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

36
 Experiment (6+7)

Detection of Anthraquinone Glycosides in Plant Extract

Objectives:
1. To extract and detect free anthraquinone and glycosidic anthraquinone.
2. To hydrolyze both O- and C- glycoside and to detect the presence of
free aglycones by chemical and chromatographic method.

Natural sources:
Powdered Rhubarb.
Powdered Aloe.
Powdered Senna.
Pharmaceutical Preparation.

Principle:
Anthraquinones are phenolic compounds naturally occurring in
free form, C-glycoside and O-glycoside. The principle of the test is based
on the ability of free anthraquinone to form a colored adduct upon the
addition of standard alkali (e.g. KOH, NaOH or ammonia). The reaction
involves the formation of phenolate-type ions which are visibly colored.
This fact is used to distinguish between the O- and the C-glycoside. The
O-glycoside is hydrolyzed to free anthraquinone by heating with dilute
HCl acid, while the C- glycoside releases the free anthraquinone only
after oxidative cleavage as illustrated in figure 1 and 2. Anthraquinone C-
glycoside and dimeric anthraquinone are hydrolyzed using oxidative
cleavage in the presence of FeCL3 in acidic medium releasing free
anthraquinone (or rhein in the case of anthraquinone dimmers) and the
sugar moiety.

Identification of Anthraquinone Glycosides from Plant Extract

Procedure:

Prepare and outline the TLC plate before you start the practical work.

I. Extraction of the free anthraquinone:

1. In a test tube add 10 ml Petroleum ether to 2 gm of powdered drug.
2. Shake for 10 minutes and filter through a filter paper into a test tube
and keep marc on the filter paper, spot the filtrate on TLC plate.
3. Add 5 ml of the standard alkali (KOH or 10% ammonia).
4. Observe and record the colour which develops immediately or upon
standing for a few minutes.

37
 II. Extraction of the anthraquinone glycoside:
5. Transfer the marc on the filter paper in step 2 into conical flask or a
beaker and add to it 20 ml of 50% ethanol.
6. Boil for 5 minutes on a water bath.
7. Filter while warm through cotton wool into graduated beaker, spot
the filtrate into the TLC plate then wash with hot alcohol to adjust the
volume to 20 ml.
III. Testing for anthraquinone O- glycoside
8. Transfer 10 ml of the glycosidic extract powder produced in step 7
into a conical flask or a beaker and add to it 10 ml of 25% HCl.
9. Boil for 15 minutes over a boiling water bath.
10. Cool the solution and transfer into a separating funnel.
11. Shake the solution with 10 ml petroleum ether in the separating
funnel.
12. Separate the organic layer into a test tube, spot the petroleum ether
extract onto the TLC plate then shake the organic layer with 5 ml of
the standard alkali.
13. Observe and record the colour produced on standing for few
minutes and observe any change in colour

IV. Testing for the anthraquinone C- Glycosides:

14. Transfer 10 ml of the glycoside extract produced in step 7 into a
conical flask and adds 1 gm of FeCl3 and heat for 20 minute on a
boiling water bath.
15. Cool the solution down and then transfer into a separatory funnel.
16. Extract the solution with 10 ml chloroform and separate the
aqueous layer from the chloroform layer.
17. Wash the organic layer with water and transfer the chloroform
layer into a test tube, spot the organic layer onto the TLC plate.
18. Add 5 ml of the standard alkali then observe and record the colour
formed immediately and on standing for a few minutes.

V. Thin layer chromatography:

19. Develop the TLC on the mobile phase
20. Examine the plate under day and both long and short UV lights
21. Spray the plate with alcoholic KOH spraying reagent.
22. Heat the plate for 10 minutes to intensify the colors and examine
the chromatogram under the day and UV lights.
23. Record your findings.

38
 Report sheet (6+7)

Student name:

Objective:

-----------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------

Principle:

-----------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------

Results (Observed color):

Type of plant Free Anthraquinone O-glycoside C-glycoside

Answer the following questions in the report and record your findings in
tabular form.

Q1. What is the botanical source of test anthraquinone?

39
Q2. draw the chemical structure(s) of the main anthraquinone for the
tested plant source showing the effect of acid and alkali on both the
free and the glycosidic anthraquinone,

Q3. Sketch the hydrolytic process of the glycosidic anthraquinone with

labeled chemical structures

Q4. Explain why did we use aqueous ethanol to extract glycosidic

anthraquinone and chloroform to extract hydrolyzed anthraquinone
glycoside?

40
 Experiment (8+9)
Alkaloid Part 1: Specific Test for Alkaloid

Vitali’s Test:

1. Evaporate 0.5 ml of alkaloid solution to dryness in a porcelain dish.

2. Add 2 drops of conc. HNO3 .
3. Evaporate again on water bath to dryness (leaving a yellow residue)
4. Dissolve yellow residue with acetone.
5. Add a few drops of KOH.
(Formation of violet color indicates a positive result)

Fluorescence Test:

1. In a test tube add 1 ml of alkaloid solution

2. Add 1 ml of dilute sulfuric acid.
3. Examine the test tube at 365 nm UV.
4. Formation of blue fluorescence under UV indicates positive result.

Murexide Test:

1. Evaporate 0.5 ml of alkaloid solution to dryness in a porcelain dish.

2. Add 0.5 ml of conc. HCl and 0.1 g Potassium chlorate
Take care of bubbling and fire
3. Evaporate to dryness over a water bath to form a yellow residue….
Crimson color.
4. Add 2 drops of diluted ammonia (Purple color indicates positive result)

Chen’s Test:

1. In a test tube add 0.5 ml of the alkaloidal solution.

2. Add a few drops of diluted HCl.
3. Add 2 drops of 5% CuSO4.
4. Add 1 ml of 20% NaOH and shake.
5. Watch for reddish-purple color.
6. Add 1 ml of ether and shake.
7. Watch for the formation of the upper purple layer and the aqueous
blue lower layer.

41
 Report sheet (8)

Student name:

Objective:

-----------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------

Results of general chemical test (observed colors):

Reagents Atropine Papaverine Quinine Cinchonidine Ephedrine Caffeine

Mayer’s

Hager

Dragendroff

Results of specific test:

1-Vitali’s test:
-Procedure:

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Name of alkaloid:

Observed result (color):

42
2-Flurescense test:

-Procedure:

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Name of alkaloid(s):

Observed result (color):

3-Murexide test:

-Procedure:

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Name of alkaloid:

Observed result (color):

43
4-Chen’s test:

-Procedure:

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Name of alkaloid:

Observed result (color):

44
 Alkaloid Part 2: Microchemical (microcrystalline) test for
alkaloids

Objectives: to introduce the student to the methods of alkaloid

identification utilizing microchemical techniques and crystal formation
using heavy metal and other reagents.

Introduction:
Alkaloids have been among the most popular group of chemical
entities used for poisoning, either for suicide or homicide, down through
history. Advances in the science of toxicology have discouraged most
murders from using poisons to gain their goal. Since these compounds are
potent poison and only small amount can be isolated from the victim,
microchemical method lend themselves well to this type of qualitative
analysis. Another striking example of the use of this test is for the
detection of spermine in victims of rape of sexual assaults. The alkalinity
of the seminal fluid is due to the number of the amines of which spermine
is the major one. Placing a drop of saline extract of the suspect stain in a
slide and addition of a drop of saturated aqueous solution of picric acid
would produce insoluble needle-like crystals of spermine picrate which
can be observed under the microscope.

Microchemical testing techniques for identifying alkaloids have

proved to be accurate and specific techniques and they are more useful
than color test which are rarely specific. They are based on the
characteristic crystalline shapes of alkaloidal derivatives produced by the
addition of certain reagents. The shape of the crystal obtained after the
addition of certain reagent(s) to a given alkaloid is generally specific for
that alkaloid. However, one should not only rely on the identification
using one reagent.

The technique is applicable for a minute amount of alkaloid and

alkaloidal preparation; hence, it would be useful in identifying an alkaloid
in a pharmaceutical preparation. Also, it is important in toxicological
detection as mentioned above.

45
Identification:
Place a drop of alkaloid solution on each of clean glass slide. Add a
drop of each reagent specific for the alkaloid on the slide without stirring
or covering. Examine under the microscope using low power. Examine
the crystals within 15 minutes. They should not be allowed to dry up.
Compare with the reference shown on the chart.

The following are specific reagents for the identification of alkaloids:

1-Wagner’s: Iodine solution.
2- Mayer’s reagent: potassium mercuric iodide solution.
3- Dragendroff: potassium bismuth iodide.
4- Saturated solution of picric acid: Hagger’s reagent.
5- Mercuric chloride.
6- Disodium phosphate solution.
7- Kraut’s reagent: Bi (NO3)2 + Nitric acid +KI.
8- ZnCl2.
9- Sodium benzoate.

46
 SCHEME FOR IDENTIFICATION OF ALKALOIDS

Test with Mayer’s reagent on a A watch glass

Mayer’s Positive Mayer’s –ve

(ppt or turbidity) (no ppt or turbidity)

Caffeine Ephedrine
1. Murexide Test
(+ ve Yellow residue Crimson
Purple with ammonia)
If -ve carry out TEST TUBE Chen’s Test

(+ve Purple upper ethereal

layer and Blue aqueous layer)
Indicates EPHEDRINE

In Test Tube Confirm with Microcrystalline test on slide

*1ml of alkaloid + 1ml dil. H2SO4
*Examine under the for fluorescence UV365 light (Test tube)

-ve (No fluorescence) +ve (Blue Fluorescence)

Quinine or Cinchonidine
Differentiate by Microcrystalline test on slide

Evaporate 1ml of alkaloid in a porcelain dish

Add drops of Conc. HNO3

-Ve (No Yellow residue) +Ve (Yellow residue)

Carry out Vitali’s Test Carry out Warren’s Test

+ve (Green-Blue Colour with KMnO4

Brown upon addition Marqui’s)
Papaverine
Confirm Microcrystalline
Permanent Violet colour Other Colours on slide

Atropine Nicotine

Confirm with microcrystalline reagents on slide

47
48
 Report sheet (9)

Student name:

I-Objective:

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II-Principle:

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Mounting reagent used:

---------------------------------------------
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Label:-------------------------------------

Mounting reagent used:

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Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

49
 Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
----------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

Mounting reagent used:

---------------------------------------------
---------------------------------------------
Label:-------------------------------------

50
 Experiment (9)

Detection of Cardiac Glycosides

Objectives:

1. To extract cardiac glycosides from their natural sources or to prepare

them in their form.

2. To detect the presence of butenolide ring on cardinolides and the

presence of deoxy sugars and the steroidal nucleus.

Procedure:

Extraction:

Extract 5 gm of powdered drug by boiling in a beaker for 5 min with 50

ml of 70 % alcohol and filter.

Purification:

Dilute the filtrate with equal volume of water and 1 ml of strong lead
acetate solution and filter off the ppt. Shake the filtrate with 50 ml of
chloroform and take 18 ml of the chloroform extract after being dried on
anhydrous sodium sulphate. Divide into four equal portions and
evaporate off the solvent in porcelain dish using water bath under fume
cupboard.

I. Test for the Butenolide Ring:

a. Kedde's test: re-dissolve one portion of the residue in 2 ml of

freshly prepared 3,5-dinitrobenzoic acid solution (0.1 gm of 3,5-
dinitrobenzoic acid dissolved in 10 ml methanol) and add 1 ml of 1
N NaOH solution. Allow standing for a few minutes, and observe
the change in color and record the time required for color
development.
b. Baljet test: re-dissolve the second portion of the residue in 5ml
methanol and add equivalent volume of freshly prepared Baljet's
reagent (9.5 ml of 1% picric acid mixed with 0.5 ml of 10 %
NaOH). Allow standing for a few minutes. Observe the change in
color and record the time required for color developments.
c. Legal’s test: re-dissolve the third portion of the residue in pyridine
and add few drops of 2% Na nitoprusside with few drops of 20%

51
 NaOH. Allow standing for a few minutes, and observe the change
in color and record the time required for color development.

II. Test for deoxy sugars:

a. Keller-Kiliani test:

Re-dissolve the forth portion of the residue in 3 ml glacial acetic acid

containing 2 drops of ferric chloride solution, stir and pour the
solnution carefully into a dry test tube containing 2 ml of conc.
sulfuric acid so that a two-phase system is produced. Observe the
color developed immediately at the interphase and record the change
in the color of the upper phase which will take place and the time
required.

b. Xanthydrol-HCl test:

Re-dissolve the fifth portion of the residue in 3 ml Xanthydrol-HCl

(100 ml 96% acetic acid and 10 ml 37% conc. HCl mixed with 0.1 ml
of 10 % Xanthydrol soln.). Heat the mixture for 3 minutes in water
bath. Allow standing for a few minutes and observe the change in the
color and record the time required for the color development.

III. Test for the steroidal nucleus:

Re-dissolve the last portion of the residue in 2 ml acetic anhydride and

cool with ice. Add carefully 1 drop of conc. sulfuric acid
(Leibermann- Burchard’s test) allow standing for a few minutes,
and observe the change in the color, and record the time required for
the color development.

52
 Identification of Cardiac Glycoside from Pharmaceutical
Preparation

Student number:
Report no.:
Group:
***********************************************************

Principle of Experiment:

Objectives of Experiment:

Material:

Experimental Procedure and Result for the Following Test:

procedure Result
Baljet’s test

Keller's keliani 's

test

Xanthydro-HCl test

Kedde's test

**Discussion and conclusion (support with available structures and

equation)

53