EXTRACTION

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EXTRACTION
Extraction: is the release/transfer of natural products from the natural
material or biomass (plants, animals or microbes) into the solvent.

Extraction is the process of isolation of soluble material from an insoluble

residue which may be liquid or solid, by treatment with solvent.

 Is the separation of medicinally active portions of plant (and animal)

tissues using selective solvents through standard procedures.

 The products so obtained from plants are relatively complex mixtures

of metabolites, in liquid or semisolid state or in dry powder form (after
removing the solvent), & are intended for oral or external use.
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Terminologies
Menstrum: Solvent used for extraction (ex. water, alcohol,
ether, acetone, ethyl acetate)
Marc: The inert fibrous and other insoluble materials
remaining after extraction
Extractives: Concentrated preparations of vegetable or
animal drugs obtained by removal of the active constituents
of the respective drug with suitable menstrum, evaporation of
all or nearly all solvent.
Tinctures: are alcoholic or hydro alcoholic solutions
prepared from vegetable material or from chemical
substances. E.g. belladona tincture
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Galenicals
These include classes of preparations viz.,
decoctions
infusions
fluid extracts
tinctures
pilular (semisolid) extracts
powdered extracts.

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STANDARDIZED
EXTRACTION
The purpose of standardized extraction procedures
for crude drugs (medicinal & aromatic plant parts)

To attain the therapeutically desired portions

To eliminate unwanted material by treatment with
a selective solvent known as “menstrum”

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CHOICE OF SOLVENTS

Successful determination of biologically active compounds

depends on the type of solvent used in the extraction
procedure.

The choice of solvent is influenced by what is intended with

the extract.

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PROPERTIES OF A GOOD SOLVENT IN PLANT
EXTRACTIONS

low toxicity

ease of evaporation at low heat

promotion of rapid physiologic absorption of the extract

preservative action

inability to cause the extract to complex or dissociate.

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SOLVENTS USED FOR ACTIVE COMPONENT
EXTRACTION

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WATER

Water is universal solvent.

used to extract plant products with antimicrobial activity.
Traditional healers use primarily water & consistent
antimicrobial activity is obtained.
Plant extracts: organic solvents >>> water extract.
Water soluble flavonoids (mostly anthocyanins) have no
antimicrobial significance.
only water soluble phenolics are important as antioxidant
compound.
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ACETONE

Dissolves many hydrophilic and lipophilic

components.
a very useful extractant, especially for antimicrobial
studies (phenolic group extract).
extraction of tannins + phenolics:
aqueous acetone >>> aqueous methanol
Both acetone and methanol were found to extract
saponins  antimicrobial activity.

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ALCOHOL
The identified components from plants (antimicrobial) =
aromatic or saturated organic compounds most often
obtained through initial ethanol or methanol extraction.
Ethanol, found easier to penetrate the cellular membrane to
extract the intracellular ingredients(polyphenols) from the plant
material.
Methanol is more polar than ethanol but due to its cytotoxic
nature.
The higher concentrations of more bioactive flavonoid
compounds were detected with 70% ethanol
due to its higher polarity than pure ethanol.
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Chloroform
Used to obtain tannins and terpenoids.
Terpenoid lactones successive extractions of dried barks with
chloroform.
Ether
Commonly used selectively for the extraction of coumarins
and fatty acids.
Dichloromethanol
Specially used for the selective extraction of only terpenoids.
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Steps Involved in the Extraction of Medicinal Plants

1.Size reduction
2. Extraction
3. Filtration
4. Concentration
5. Drying

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1. Size Reduction
Objective:
To rupture plant organ, tissue & cell structures so that its
medicinal ingredients are exposed to the extraction solvent.
Size reduction maximizes the surface area, which in turn
enhances the mass transfer of active principle from plant
material to the solvent.
The 30-40 mesh size is optimal.
Hammer mill or a disc pulverizer which has built in sieves
controlled by varying the speed of the rotor clearance b/w the
hammers & the lining of the grinder.
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2. Extraction
By using both modern extraction Traditional extraction method
method and traditional extraction
method
Modern extraction method Macération
 Counter-current Percolation
 Microwave assisited Infusion
Ultrasound extraction (sonication), Décoction
Supercritical fluid extraction,
Hot continuos extraction (Soxhlet)

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Parameters influencing the quality of an extract

Plant part used as starting material

Solvent used for extraction
Extraction procedure

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Effect of extracted plant phytochemicals depends
on

The nature of the plant material

Its origin
Degree of processing
Moisture content
Particle size

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Selection of Plant
Plant based natural constituents can be derived
from any part of the plant like bark, leaves, flowers,
roots, fruits, seeds, etc.
Plants are usually air dried to a constant weight
before extraction.
 oven drying: every part were cut into pieces dried
in an oven @ 60°C for 9 hrs. & pulverized.
Other method for drying the plants is the oven
drying at about 40°C for 72 h.
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3. FILTRATION
The extract so obtained is separated out from the
marc (exhausted plant material) by allowing it to
trickle into a holding tank through the built-in false
bottom of the extractor, which is covered with a filter
cloth.
The marc is retained at the false bottom, and the
extract is received in the holding tank.
From the holding tank, the extract is pumped into a
sparkler filter to remove fine or colloidal particles from
the extract.
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 4. CONCENTRATION
The enriched extract from percolators or extractors is fed into
a wiped film evaporator where it is concentrated under
vacuum to produce a thick concentrated extract.
The concentrated extract is further fed into a vacuum
chamber dryer to produce a solid mass free from solvent.
The solvent recovered from the wiped film evaporator and
vacuum chamber dryer is recycled back to the percolator or
extractor for the next batch of plant material.
The solid mass thus obtained is pulverized and used directly
for the desired pharmaceutical formulations or further
processed for isolation of its phytoconstituents.
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5. DRYING
The filtered extract is subjected to spray drying with a high pressure pump
at a controlled feed rate and temperature to get dry powder.

The desired particle size of the product is obtained by controlling the inside
temperature of the chamber and by varying the pressure of the pump.

The dry powder is mixed with suitable diluents or excipients and blended in
a double cone mixer to obtain a homogeneous powder that can be straight
away used (for example, for filling in capsules or making tablets).

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VARIATION IN EXTRACTION
METHODS
Length of the extraction period
Solvent used
pH of the solvent
Temperature
Particle size of the plant tissues
The solvent-to-sample ratio.

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PARAMETERS FOR SELECTING AN
APPROPRIATE EXTRACTION METHOD
Authentication of plant material by botanist.
Use the right plant part + the age of plant + the time,
season & place of collection.
The nature of its chemical constituents.
Grinding methods & powdering techniques.
Nature of constituents (polar/nonpolar).
The quality of water / menstruum.
The design & material of fabrication of the extractor.
Analytical parameters of the final extract,(TLC/HPLC).

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Methods of extracton

Solvent extraction Distillation Expression

(Mechanical extraction)
Mostly for non-volatile For volatile plant
plant constuituents and constuituents For both volatile and
sometimes for voilatile ones non-volatile plant
constituents

Volatile solvent Non-volatile

extraction solvent
extraction
> pet. ether
> hexane > petrolatum
> methanol > silicon polymers
> butter
> chloroform
> acetone etc. > fixed oils

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1. MACERATION

The whole / coarsely powdered crude drug is placed in a

stoppered container with the solvent.
Allow to stand @ room temperature for a period of at least 3 days
with frequent agitation until the soluble matter gets dissolved.
The mixture then is strained, the marc (the damp solid material) is
pressed,
The combined liquids are clarified by filtration or decantation after
standing.
This method is best suitable for use in case of the thermolabile
drugs.
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 Advantage of maceration
Heat labial drug extracted
Any apparatus can be used.

Disadvantage of maceration

Quite time-consuming, taking from a few hours up to

several weeks.

Consume large volumes of solvent.

Some compounds may not be extracted efficiently if they

are poorly soluble at room temperature.

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2. INFUSIONS
Fresh infusions are prepared by macerating the
crude drug for a short period of time with cold or
boiling water.
These are dilute solutions of the readily soluble
constituents of crude drugs.

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3. DIGESTION
This is a form of maceration in which gentle heat is used
during the process of extraction.
It is used when moderately elevated temperature is not
objectionable.
The solvent efficiency of the menstruum is thereby increased.
Image=microwave digestion system

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4. DECOCTION
In this process, the crude drug is boiled in a specified volume of
water (1;4) for a defined time.
Volume is reduced to 1/4th the original
It is then cooled and strained / filtered.
This procedure is suitable for extracting water-soluble, heat-
stable constituents.
Typically used in preparation of Ayurvedic extracts = “quath” /
“kawath”

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 5. PERCOLATION
 Discontinuous type of extraction
 Cold or hot extraction
 Adequate for both initial and large-scale
extraction
Powdered plant material is
soaked initially in a solvent
in a percolator (a cylindrical
or conical container with a
tap at the bottom).
Additional solvent is then poured on top of
the plant material and allowed to
percolate slowly (drop wise) out of the
bottom of the percolator.

Additional filtration of the extract is not required

because there is a filter at the outlet of the
percolator.
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Used most frequently to extract active ingredients in the preparation of
tinctures and fluid extracts.

The solid ingredients are moistened with an appropriate amount of the

specified menstruum,

Allowed to stand for approximately 4 hours in a well closed container,

After stand time, the mass is packed & the top of the percolator is closed.

the mixture is allowed to macerate in the closed percolator for 24 h.

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Additional menstruum is added as required, until the percolate measures
about three-quarters of the required volume of the finished product.

The marc is then pressed and the expressed liquid is added to the
percolate.

Sufficient menstruum is added to produce the required volume.

The mixed liquid is clarified by filtration or by standing followed by

decanting.

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 Factors affecting efficiency of percolation includes
 Extent to which the material is ground.

 Contact time between the solvent and the plant (i.e., the percolation
rate) and
 Temperature of the solvent

Disadvantages of percolation
 Large volumes of solvents are required and

 Time-consuming.

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6. HOT CONTINUOUS EXTRACTION (SOXHLET
EXTRACTION)

 Soxhlet extraction is the process of continuous extraction in which the

same solvent can be circulated through the extractor several times. The
process involves extraction followed by evaporation of the solvent.

The vapours of the solvent are taken to a condenser and the condensed
liquid is returned to the drug for continuous extraction.

The finely ground crude drug is placed in a porous bag or “thimble”

made of strong filter paper, which is placed in chamber of the Soxhlet
apparatus.
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The extracting solvent in flask is heated, and its vapors condense in
condenser.
The condensed extract drips into the thimble containing the crude
drug & extracts it by contact.
When the level of liquid in chamber rises to the top of siphon tube,
the liquid contents of chamber siphon into flask
This process is continuous and is carried out until a drop of solvent
from the siphon tube does not leave residue when evaporated.

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SOXHLET
 Components of Soxhlet extraction set up

◦Heat source
◦Flask containing solvent
◦Extraction chamber
◦Condenser connected to a flowing water
◦Extraction timble (made of filter paper)

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 SOXHLET (CONTINUOUS
EXTRACTION) APPARATUS

Water outlet

Condenser
Water inlet

Extraction Timble
Extraction
chamber

Flask

Continuous hot extraction technique

(Soxhlet extraction process)

Heat source

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Advantage:

Less solvent is needed for yielding more concentrated products.

The extraction can be continued until complete exhaustion of the drug.

Disadvantage:
•It is restricted to pure boiling solvents .
 Requires special apparatus

 Not applicable for thermolabile plant constituents

 No direct contact between plant material and heat source

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 Extraction of volatile oils

Distillation: is a method of separating mixtures based on differences

in volatilities of components in a boiling liquid mixture. Distillation
is a unit operation, or a physical separation process, and not a
chemical reaction. Coo
Heat Vapo l
Liquid Liquid
ur

Most economical method of preparation of volatile oils

Hydro distillation

Steam distillation

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 Water distillation (hydro distillation)

 The material is completely immersed in water, which is boiled by applying heat by

direct fire, steam jacket etc. The main characteristic of this process is that there is
direct contact between boiling water and plant material.

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 Steam distillation
This process involves bubbling steam through a heated mixture of the raw material.

The vapor mixture is cooled and condensed, usually yielding a layer of oil and a
layer of water.

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 ISOLATION AND
PURIFICATION

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ISOLATION AND
PURIFICATION
The physical methods used for the isolation and purification of active
constituents are:
 Classical methods: e.g. fractional crystallisation, fractional distillation,
fractional libration, sublimation
 Chromatographic methods

Fractional crystallisation:

It is depends upon the inherent character of the compound which forms crystals
at the point of super saturation in the solvent in which it is soluble.

Compounds such as sugars, glycosides,. shows crystalline nature.

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Processes such as concentration, slow evaporation and
refrigeration are used for crystallising the product.
Fractional distillation:
For distillation compound should have volatile nature.
It is used for separation of essential oil components.
This process widely used for separation of hydrocarbons from
oxygenated volatile oil components.
Components such as citral, citronellal and eucalyptols.

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Fractional Liberation

In this process, groups of compounds having tendency of

precipitation comes out of the solution.

Alkaloids, is modified by converting it to its salt form or free

base.

This process is used for separation of cinchona alkaloid

quinine, isolation of morphine and many other alkaloids.
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Sublimation:

In this process the compound, if subjected to heat, changes

from solid to gaseous state directly without passing through the
liquid stage.

The process is traditionally used for the separation of camphor

from the chips of wood of Cinnamomum camphor to obtain
solid sublimate of camphor.
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CHROMATOGRAPHY

Chromatography is usually introduced as a

technique for separating and/or identifying the
components in a mixture.

The basic principle is that components in a mixture

have different tendencies to adsorb onto a surface or
dissolve in a solvent.
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Chromatography is a physical method of separation in which
the components to be separated are distributed between two
phases (KD/P = Distribution/partition constant)
one of which is stationary (stationary phase) while the other
(the mobile phase) moves through it in a definite direction.
The chromatographic process occurs due to differences in the
distribution constant of the individual sample components.
 The sample under go repeated equilibration
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BASIC CHROMATOGRAPHIC TERMINOLOGY

Chromatograph: Instrument employed for a chromatography.

Stationary phase: Phase that stays in place inside the column
Mobile phase: Solvent moving through the column, either a liquid
in LC or gas in GC.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through the
column.
Chromatogram: Graph showing detector response as a function of
a time.
Flow rate: How much mobile phase passed / minute (ml/min).
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TYPE OF CHROMATOGRAPHY

1. Paper chromatography

2. Thin layer chromatography (TLC)

3. Gas chromatography (GC)

4. liquid chromatography (LC)

5. High performance liquid chromatography (HPLC)

6. Ion exchange chromatography

7. Gel filtration chromatography.

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All chromatographic methods require one static part (the
stationary phase) and one moving part (the mobile phase).
The techniques rely on one of the following phenomena:
adsorption; partition; ion exchange; or molecular exclusion.
Adsorption versus Absorption:
 In absorption one substance penetrate in to the bulk of
another substance.
 A sponge and water
 Adsorption is a surface phenomenon where interaction
takes place only on the surface of one substance.
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Mobile phase - flows through column, carries analyte.
Gas = Gas Chromatography (GC)
Liquid = Liquid Chromatography (LC), Thin Layer
Chromatography (TLC)
Supercritical fluid = Supercritical Fluid Chromatography (SFC)
Stationary phase - stays in a place, does not move.
GC, LC placed inside of the column
TLC – layer of a sorbent on the plate
The SEPARATION is based on the partitioning between the
mobile and stationary phase.
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CLASSIFICATION OF CHROMATOGRAPHY

2. Based on the Configuration of the instrument (the way the stationary

phase is dispersed)
a. Planar Chromatography: - the SP is immobilized in the plane. Ex..
TLC, PC
b. Columnar Chromatography: - the SP is immobilized in the column
Ex. GC, CC, HPLC
3. Based on the nature of the Mobile phase: -
A. Liquid Chromatography
B. Gas Chromatography

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4. Based on the nature of the Stationary Phase
A. Normal phase Chromatography: - stationary
phase is polar, mobile phase is non polar
B. Reverse phase Chromatography: - stationary
phase is non- polar, mobile phase is polar

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5.Based on column type
1. Closed column chromatography
• stationary phase is packed inside a column
• mobile phase + solute flows through the column ->
separation
2. Open column chromatography
 Paper chromatography- sheet of paper is used to support the
stationary phase
 Thin-layer chromatography- adsorbent is spread evenly over the
surface of a flat sheet of glass
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Adsorption-Chromatography
It has a solid stationary phase and a liquid or gaseous
mobile phase
Each solute has its own equilibrium between adsorption
onto the surface of the solid and solubility in the solvent
The least soluble or best adsorbed ones travel more
slowly.
The result is a separation into bands containing
different solutes.
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One of the oldest types of Separation is based on
chromatography around adsorption strength
It involves
a stationary solid phase
a liquid or gaseous mobile
phase
Not used as widely as partition
chromatography
 used mainly on TLC & CC
packed with silica gel/alumina

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PARTITION
In partition chromatography the stationary phase is a non-volatile liquid
which is held as a thin layer (or film) on the surface of an inert solid.

The mixture to be separated is carried by a gas or a liquid as the mobile

phase.

The solutes distribute themselves between the moving and the stationary
phases, with the more soluble component in the mobile phase reaching the
end of the chromatography column first

Paper chromatography is an example of partition chromatography.

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Based on a thin film formed on the
surface of a solid support by a liquid
stationary phase.
Solute equilibrates between the mobile
phase and the stationary liquid.
Partition - based on the relative
solubility of analyte in mobile and
stationary phases
Reversed phase chromatography (GC,
HPLC)
Paper chromatography

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SELECTION OF THE SOLID SUPPORT

The support material should:

 adsorb & retain the liquid stationary phase.
 be mechanically stable.
 be easy to pack.
 not retard the solvent flow
 expose as large surface as possible to the mobile phase
Examples of solid supports:
Silica gel, diatomaceous earth (as kieselguhr, celite etc.) &
cellulose.
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1. PURPOSE OF
CHROMATOGRAPHY

Analytical :
 For identification and analysis purpose
 Chemical profile (composition) of a sample
 To check the purity of the compound
 for screening purpose
 A very thin layer of stationary phase coated on
supporting materials
 Deals with analytes in micrograms quantities
eg. TLC; HPLC; GC
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Preparative :
 vital for Isolation of compounds
 purify and collect one or more components of a
sample Relatively thicker layer of stationary
phase coated on supporting materials
 Deals with analytes in milligrams or large
quantities
eg. PTLC; Prep-HPLC; Prep-GC

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PAPER CHROMATOGRAPHY

A chromatographic analytical separation technique for

complex mixtures involving the progressive adsorption of the
dissolved component onto a special grade of paper.
SP: Water adsorbed on cellulose
The SP can be modified in to reversed phase or ion exchange
MP: A binary mixture of incompletely miscible components
MoS: Partition

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 PRINCIPLE

 It is an example of a partition chromatography

 Liquid-Liquid chromatography
 Cellulose fibers strips serve as inert solid support.
 The factor governing separation of mixtures of
solutes on filter paper is the partition between two
immiscible phases.
 One is usually water adsorbed on cellulose fibers in
the paper (stationary phase).
 The second is the organic solvent flows past the
sample on the paper (stationary phase).
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What are some limitations of paper
chromatography?

Paper chromatography is a very simple and cost effective method for

separation of components.
But it has some major disadvantages:-
 paper chromatography gives better sample resolution, but it is a very slow
technique.
 Paper chromatographic techniques can not be used in separation of
volatile substances such as hydrocarbons and volatile fatty acids.
 The lower limit for the detection of most compounds is 1-5 mg.
 In paper chromatography utmost care has to be taken while hanging the
paper in the chromatography chamber. If the paper is tilted, the solvent front
will be uneven due to which calculating the correct Rf value will be almost
impossible. A similar problem arises when the paper is cut unevenly.
 Paper chromatography also requires some skill in sample spotting.

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Theretention factor, or Rf, is defined as the distance traveled by the
compound divided by the distance traveled by the solvent

The Rf value of a compound in a particular solvent system is constant

under identical conditions of the experiment, e.g. temperature, pH, etc.
For example, if a compound travels 2.1 cm and the solvent front travels
2.8 cm, the Rf is 0.75:

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SPOT DETECTION

- Color spot  observed by naked eye

- Non – color spot  color reagent will give specific
colors for different compound.
Example :
 Ninhydrin – a.amino
 Iodine–alkaloid

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Thin layer chromatography(TLC)

Overview of TLC
TLC—one form of solid-liquid chromatography
Adsorbent—solid phase; it won’t dissolve in the
associated liquid phase
Ex: Silica Gel (Si02) & Alumina (Al2O3)
Eluting—liquid phase
Solvated substances are spotted onto a TLC
plate coated with the adsorbent and allowed to
elute up the plate by capillary action
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 Capillary action occurs because molecules of a pure substance is
sticky, thanks to the forces of cohesion (similar molecules of a
pure substance like to stay close together) and adhesion
(molecules of a given pure substance are attracted and stick to
other substances).
 Adhesion of a liquid to the walls of a vessel will cause an upward
force on the liquid at the edges and result in a meniscus which
turns upward. The surface tension acts to hold the surface intact.
 Capillary action occurs when the adhesion to the walls is stronger
than the cohesive forces between the liquid molecules. The height
to which capillary action will take the liquid in a uniform circular
tube (picture to left) is limited by surface tension and, of course,
gravity.

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Four Purposes of Performing TLC

Isolation components in a mixture (preparative

TLC)
Assess purity (how many spots are present)
Identify a substance (analytical TLC)
Assess compound polarity

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TLC Development Setup

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This technique manipulates POLARITY

More polar substances bind strongly to the adsorbent
and elute SLOWER
Less polar substances bind weakly to the adsorbent
and elute FASTER

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Overview of TLC—The R f Value

A given compound will always travel a fixed distance relative

to the distance the solvent travels
This ratio is called the Rf value and is calculated in the
following manner: . distance traveled by substance .
distance traveled by solvent front

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TLC Visualization Methods

 Ultraviolet Light—some organic compounds illuminate or fluoresce

under short-wave UV light
 Iodine Vapor—forms brown/ yellow complexes with organic
compounds
 Fluorescent Indicators—compounds fluoresce when placed under
UV light
 Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon
exposure to light
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Plate Preparation
Adsorbent + Calcium sulfate (gypsum) + Water = thick slurry
Spread, pouring, dipping and spraying done on glass,
aluminum foil, or plastic.
analytical TLC 0.1–0.25 mm
preparative TLC around 1–2 mm
30 minutes at 110 °C. “Activation”
Stored over desiccant.

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Advantages Disadvantages
 Convenient and simple.  Lack of automation
 Simple detection of separated  HPTLC (automated TLC)
bands  the problems of reproducibility
 Fast screening of raw materials which sometimes occur
 Inexpensive  Lack of accuracy in
 Consumes smaller amounts of quantitation
solvents than HPLC.
 Simultaneous application and
determination of samples on a
single TLC plate

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Column chromatography
It is used for the separation and isolation of different
constituents of extracts.
Column chromatographic grade adsorbents of choice
are used are used for packing the column and various
organic solvents are used as eluting solvents.
A glass column is used in this technique.
Silica gel is used as a packing column.
Sample applied on the top of the column, in such a way
that narrow band is formed, for further elution.
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The column is subsequently eluted with various proportions of
solvents in the order of increasing polarity.
The fractions collected from the bottom are distilled to
recover the solvent .

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HPLC (High Performance Liquid
Chromatography)
It utilises a column of packing material of particle of small
size and regular shape.
As the column finely packed, pressure up to 5000 lb in-2 can be
employed to achieve acceptable flow rate.
This technique has advantages of quantitatively analysing the
components of the mixture at a high level of resolution and
sensitivity.
The disadvantages of this method include the cost of the
instrument and the requirement of injecting only highly pure,
particle free solution on the column to avoid blockage.

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