Plant analysis: Extraction, separation, chromatography, types of plant constituents, comparative phytochemistry and chemotaxonomy.

2. Extraction of plant constituents
Extraction is the first step in the analysis
of plant constituents.
It is a process of removing or taking out
or separating the chemical constituents
from the plant tissues with or without the
aid of solvents.
Extraction methods:
A. Methods of Extraction without the
use of solvent include the following:
1. Expression
2. Sublimation
3. Distillation
The extraction of natural
products progresses through the
following stages:
(1) the solvent penetrates into
the solid matrix;
(2) the solute dissolves in the
solvents;
(3) the solute is difused out of
the solid matrix;
(4) the extracted solutes are
collected.
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3. 1. Expression:
➢ In this method the fresh or dry material is subjected to
hydraulic pressure with or without the application of
heat.
➢ When no heat is used the method is called cold
expression and
➢ Any extraction done with the simultaneous application of
heat is called hot expression.
➢ Example: Plant constituents like fixed oils and fats are
extracted by this method.
2. Sublimation:
➢ In this process, the powdered plant material is heated in
a wide mouthed container covered with a glass sheet.
➢ The sublimable constituent is first vaporized and then
condensed on the lower surface of the cover.
➢ Example: extraction of caffeine from tea leaves.
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4. 3. Distillation:
Distillation is also a method of hot extraction specifically used for the
extraction of volatile oils.
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5. B. Method of extraction with the use
of solvents are of two types:
1. Extraction with aqueous solvents:
In this method fresh or dried materials
are extracted by pouring hot water on
the material.
i. Decoction
2. Extraction with organic solvents:
i. Maceration
ii. Percolation
iii. Soxhlet
extraction
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6. Extraction with aqueous solvents
1. Decoction:
➢ In this process, the crude drug is boiled in a
specified volume of water for a defined time;
➢ it is then cooled and strained or filtered.
➢ Application:
➢ This procedure is suitable for extracting water-
soluble, heat stable constituents.
➢ This process is typically used in preparation of
Ayurvedic extracts called “quath” or “kawath”.
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7. Extraction with organic solvents
1. Maceration:
➢ In this process, the whole or coarsely powdered
crude drug is placed in a stoppered container with
the solvent and allowed to stand at room
temperature for a period of at least 3 days with
frequent agitation until the soluble matter has
dissolved.
➢ The mixture then is strained, the marc (the damp
solid material) is pressed, and the combined liquids
are clarified by filtration or decantation after
standing.
➢ Advantage: This is a very simple extraction
method.
➢ Disadvantage: Long extraction time and low
extraction efficiency.
➢ Application: It could be used for the extraction of
thermolabile components.
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8. 2. Afterwards the mass is packed and the top of the percolator is closed.
3. Additional menstruum is added to form a shallow layer above the mass,
and the mixture is allowed to macerate in the closed percolator for 24 h.
4. Then the outlet of the percolator is opened and the liquid contained
therein is allowed to drip slowly.
5. Additional menstruum is added as required, until the percolate
measures about three-quarters of the required volume of the finished
product.
6. The marc is then pressed and the expressed liquid is added to the
percolate.
7. Sufficient menstruum is added to produce the required volume.
8. Finally the mixed liquid is clarified by filtration or by standing followed
by decanting.
Application: This is the procedure used most frequently to extract active
ingredients in the preparation of tinctures and fluid extracts.
Percolation
(For this process A percolator (a narrow, cone-shaped vessel open at both ends) is generally used. )
1. The solid ingredients are moistened with an appropriate amount of the specified menstruum and allowed to
stand for approximately 4 h in a well closed container.
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9. In this method, the finely ground crude drug is placed in a porous bag or
“thimble” made of strong filter paper, which is placed in chamber E of the
Soxhlet apparatus (Figure).
The extracting solvent in flask A is heated, and its vapors condense in
condenser D.
The condensed extractant drips into the thimble containing the crude drug, and
extracts it by contact.
When the level of liquid in chamber E rises to the top of siphon tube C, the liquid
contents of chamber E siphon into flask A.
This process is continuous and is carried out until a drop of solvent from the
siphon tube does not leave residue when evaporated.
Advantages:
Large amounts of drug can be extracted with a much smaller quantity of solvent.
This process is beneficial in terms of time, energy and finance.
It becomes much more economical and viable when converted into a continuous
extraction procedure on medium or large scale.
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10. Separation Techniques
✓ Volatile constituents having different boiling points
when occur in mixtures are conveniently separated from
each other by distilling the mixture at different
temperatures.
✓ This method is used for the separation of hydrocarbons
from oxygenated volatile oil e.g. citral, eucalyptol
After extraction from the plant material the mixtures
of the extracted chemical constituents are separated
by various separation techniques and methods for
further analysis, isolation. purification and
identification of the individual compounds present in
the mixture.
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11. ✓ The mixture is then gradually
basified in increasing order with
the addition of an aliquot of
ammonia solution at a time and
✓ After each addition the mixture is
shaken with the organic solvent
and fractions are collected
separately.
✓ In this way the alkaloids are
fractionally liberated depending on
their degree of basicity.
✓ Mixtures of basic plant constituents, like alkaloids are often separated by this
method depending on the degree of basicity.
✓ For the separation of a mixture of alkaloids with different degree of basicity;
✓ At first the mixture is first slightly acidified and shaken with a strong organic
solvent (e.g. Chloroform).
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12. ✓ Therefore, this separation technique exploits the
differential solubility of the components of a
mixture in a particular solvent.
✓ The least soluble one crystallizes out first from
the mixture on cooling when it is dissolved in a
minimum quantity of the hot solvent.
✓ On separation of these crystals the others
crystallize out in descending order depending on
their degree of solubility, the most soluble one
being the last.
3. Fractional Crystallization:
✓ It is the process of separating the components of a solution on the
basis of their different solubilities, by means of evaporating the solution
until the least soluble component crystallizes out.
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13. Chromatography
It is one of the most important analytical tools.
It serves as a means for the resolution of mixtures and for the isolation and partial
detection of the separated substances.
It permits the separation and partial description of unsuspected and unknown
substances.
It is among the most selective and most Widely applicable separatory techniques yet
devised.
Chromatography is also very suitable for the extraction of components from plant
extracts, especially when a small amount of material is available & when the components
closely resemble with each other (e.g. alkaloids, anthraquinones).
Chromatography is widely used for the separation &
identification of components of a mixture.
Chromatography comprises a group of methods for separating
compounds from mixtures, that depends on the differential
affinities of the solutes between two immiscible phases.
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14. In this separation the stationary phase serves as
the carrier or holder of the mixture to be
separated.
The mobile phase moves through the stationary
phase pushing or carrying the components of
the mixture over it, at different speeds
depending on the affinity of the individual
components for the two phases.
The whole chromatographic process depends
on the activities of these two phases.
Principle of Chromatography:
In chromatography there area two phases
Mobile phase: Moves through the stationary phase.
Mobile Phase can be two types: Liquid & Gas
Stationary Phase: Fixed phase/static phase
Stationary Phase can be two types: Solid & Liquid
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15. Types of Chromatography:
Chromatographic process may be broadly
divided into the following major groups.
Adsorption chromatography
Partition chromatography
lon-exchange chromatography
Electro chromatography or Electrophoresis
Gel filtration chromatography
Depending on the specific medium used or
specific technique employed chromatographic
methods are also variously named.
Column chromatography
Thin-layer chromatography
Paper chromatography
Gas chromatography
High performance liquid chromatography
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16. Plant constituents
During normal metabolic activities the living
plant produces or synthesis a large variety
chemical substances (carbohydrates,
proteins, fats) that are utilized as food by man
and other animals; while there are multitude
of compounds (glycosides, alkaloids,
terpenes) that exert physiologic effects.
The usual term used to refer these various
chemical substances present in plants is
constituents.
Phytochemistry is concerned with the
chemical study of these plant
constituents.
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17. Those compounds that are responsible for the therapeutic effect are called active
constituents.
Those constituents which also occur in plant and animal drugs, have no definite
pharmacologic activity are considered as inert constituents.
Active constituents may be divided into 02 classes: Pharmaceutically active and
Pharmacologically active.
Pharmaceutically active constituents may cause precipitation or other chemical
changes in a medicinal preparation.
e.g. Cinchotannic acid present in cinchona bark or its extracts, cause precipitation when
used with iron salts.
Pharmacologically active constituents are responsible for the therapeutic activity of the
drug. They may be either single chemical substances or mixtures of principles.
The single chemicals are sugars, starches, plant acids, enzymes, glycosides, steroids,
alkaloids, proteins, hormones, and vitamins.
The mixtures include fixed oils, fats, waxes, volatile oils, resins, oleoresins, oleo-gum-
resins, and balsams.
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18. Chemotaxonomy
Chemotaxonomy or biochemical systematics is composed of nearly equal
parts of chemistry and biology.
This discipline attempts to utilize chemical facts to obtain a more exact
understanding of biologic evolution and natural relationships.
Its principles and findings are applied to determine potential sources of known
drugs or to explore those areas of the biologic kingdom in which new ones are
most likely to be discovered.
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19. Thank
You
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20. Natural
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