Extraction of Plant Material and

Chromatography (TLC, HPTLC)

 Extraction of plant material
• The commonly employed technique for separation of
active substance from crude drugs is called
‘Extraction’ which involves the use of different
solvent.
• Whether samples are plants, microbes, marine
animals or insects they are referred to as biomass.
• All plant material used should be properly
authenticated, as much time and money can be
wasted on the examination of material of doubtful
origin.
• Dried materials are usually powdered before extraction,
whereas fresh plants (e.g. leaves, etc) can be homogenized
or macerated with a solvent such as alcohol.
• Dried biomass is ground into small particles using either a
blender or a mill. Plant material is milled twice, first using
a course mill and then a fine mill to generate a fine
powder.
• The grinding process is important as effective extraction
depends on the size of the biomass particles; large
particles will be poorly extracted, whereas small particles
have a higher surface area and will therefore be extracted
more efficiently.
• Selection of the solvent extraction approach is very
important.
• If a plant is under investigation from an
ethnobotanical perspective, then extraction should
mimic the traditional use.
• For example; if indigenous people use a specific
extraction protocol such as a water extract, a
cold/heat tea, alcohol or alcohol-water mixtures,
then an identical or at least a very similar method
should be used in the laboratory so that the same
natural products are extracted.
• Failure to extract biomass properly may result in
loss of access to active compounds.
• Additionally, using an inappropriate extraction
method, such as strong heating of biomass with a
solvent, may result in degradation of natural
products and consequent loss of biological activity.
• The choice of extraction procedure depends
on the nature of the plant material and the
components to be isolated.
• Alcohol is general solvent for many plant
constituents
• Water-immiscible solvents are widely used;
Light petroleum (essential and fixed oil,
steroids),
• Ether and chloroform (alkaloids, quinines).
• Special method for volatile oil , such as Clavenger
and sometimes Enfleurage process.(Enfleurage: is
the process of extracting fragrance from flowers
by using odorless fats or oils to capture the
essential oils. The perfumes of plants like jasmine
could only be extracted by enfleurage).
 cold extraction
• Numerous extraction methods are available,
the simplest being cold extraction (in a large
flask with agitation of the biomass using a
stirrer) in which the ground dried material is
extracted at room temperature sequentially
with solvents of increasing polarity: first
hexane (or petroleum ether), ethyl acetate,
acetone, methanol and finally water.
• The major advantage of this protocol is that it is a soft
extraction method as the extract is not heated and
there is little potential degradation of natural
products.
• The use of sequential solvents of increasing polarity
enables division of natural products according to their
solubility (and polarity) in the extraction solvents.
This can greatly simplify an isolation process.
• Cold extraction allows most compounds to be
extracted, although some may have limited solubility
in the extracting solvent at room temperature.
 Hot percolation
• In hot percolation, the biomass is added to
round-bottomed flask containing solvent and
the mixture is heated gently under reflux.
• Typically, the plant material is ‘stewed’ using
solvents such as ethanol or aqueous ethanol
mixtures. The technique is sometimes referred
to as total extraction and has the advantage
that, with ethanol, the majority of lipophilic
and polar compounds is extracted.
• Heating the extracts for long periods may also
degrade labile compounds; therefore a pilot
experiment should first be attempted and
extracts assessed for biological activity to
ascertain whether this extraction method
degrades the bioactive natural products.

• Care should be taken, as extraction is never

truly total; for example, some highly lipophilic
natural products are insoluble.
 Decoction
• The decoction is used for active ingredients
that doesn´t modify with temperature. In this
process the drug is boiled in water for 15 to 60
minutes depending on the plant or the active
ingredient to extract.
 Supercritical fluid extraction
• Supercritical fluid extraction utilizes the fact that some
gases behave as liquids when under pressure and have
solvating properties. The most important example is
carbon dioxide which can be used to extract biomass and
has the advantage that, once the pressure has been
removed, the gas boils off leaving a clean extract.

• Carbon dioxide is a non-polar solvent but the polarity of

the supercritical fluid extraction solvent may be increased
by addition of modifying agent, which is usually another
solvent (e.g. methanol or dichloromethane).
 Soxhlet extraction
• The most widely used method for
extraction of plant natural products is
Soxhlet extraction. This technique uses
continuous extraction by solvents of
increasing polarity.
• The biomass is placed in a Soxhlet
thimble constructed of filter paper,
through which solvent is continuously
refluxed.
• The Soxhlet apparatus will empty its
contents into the round-bottomed flask
once the solvent reaches a certain level.
• As fresh solvents enters the apparatus by a reflux
condenser, extraction is very efficient and
compounds are effectively drawn into the solvent
from biomass due to their low initial concentration
in the solvent.
• The method suffers from the same drawbacks as
other hot extraction methods (possible degradation
of products), but it is the best extraction method for
the recovery of a big yields of extract. Moreover,
providing biological activity is not lost on heating,
the technique can be used in drug lead discovery.
• In general terms, regardless of the extraction method
used, extracts are of two types: lipophilic (‘fat-loving’),
resulting from extraction by non-polar solvents ( e.g.
petroleum, ethyl acetate, chloroform, dichloromethane)
and hydrophilic (‘water-loving’), produced by extracting
biomass with polar solvents (e.g. acetone, methanol,
water).

• The value of using solvents of different polarities is that

the chemical complexity of the biomass is simplified when
taken into the extract, according to the solubility of the
components. This can greatly simplify the isolation of an
active compound from the extract.
• Additionally, certain classes of compounds
may have high solubilities in a particular
solvent (e.g. monoterpens in hexane), which
again can simplify the chemical complexity
of an extract and help with isolation process.

• Regardless of the extraction technique used,

extracts are concentrated under vacuum
using rotary evaporators for large volumes of
solvent (> 5 ml) or ‘blown down’ under
nitrogen for small volumes (1-5 ml), ensuring
that volatile components are not lost.
• Removal of solvent should be carried out
immediately after extraction, as natural
products may be unstable in the solvent.
• Dried extracts should be stored at freezing
temperatures prior to screening for biological
activity as this will decrease the possibility of
degradation of bioactive natural product.
 Distillation
• Fractional distillation has been traditionally used for
separation of the components of volatile mixtures; in
phytochemistry it has been widely used for isolation
of components of volatile oils.

• On a laboratory scale it is not easy by this method to

separate minor components of a mixture in a pure
state and gas chromatography is now routinely used.

• Steam distillation is much used to isolate volatile oils

and hydrocyanic acid from plant material.
TLC
 Principle:
 The sample is dissolved in in a volatile solvent
 Sample is applied with the help of capillary on to the base line
drawn on the solid adsorbent.
 The plate is dipped in to the solvent working as mobile phase.
 As the mobile phase rises up the TLC plate by capillary action, the
components dissolve in the solvent and move up the TLC plate.
 In adsorption separation will occur on the basis of differences in
adsorption (preferential adsorption). Weekly adsorbed
component will separate first and strongly later on.
 In partition , if the component is soluble in the mobile phase it will
move with the solvent and vise versa .
 The movement of the solute is measured relative to that of solvent
is expressed as the retardation factor.
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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
• The strength of interactions between the adsorbent
and eluting components vary approximately in this
order:

Salt formation > coordination > H-bonding >

More Less
Polar dipole-dipole > van der Waals Polar

Polarity decreases 22
 Less
Polar
More
Polar
Adsorbs weakly
and separate
very fast

Adsorbs stronger
and separate
very slowly

Sample to be applied on
this area 23
 The Rf 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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 Materials used in TLC

• Glass Plate
• Adsorbents
• Oven for activation of plate
• Developing chamber
• Mobile Phase
• A device for applying the adsorbent layer
• Storage facility for the prepared plate

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 Materials used in TLC

Hooper
A device for applying
Glass the adsorbent layer
Mobile
phase
Plate

Developing chamber

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 Stationery phase
Stationery phase Description Application

Silica gel G Silica gel with average Used in wide range

particle size 15µm pharmacopoeial test
containing ca 13%
calcium sulfate binding
agent

Silica gel G254 Silica gel G with Same application with Silica
fluorescence added gel G where visualization is to
be carried out under UV light.

Alumina

(Al2O3)
Cellulose Cellulose powder of less Identification of tetracycline's
than 30µm particle size. 27
 MOBILE PHASE
 TLC Solvents or Solvent Systems.
 A single solvent or mixture of two solvents can work
as mobile phase in TLC .Ex. petroleum ether, carbon
tetrachloride, chloroform, ethyl acetate, hexane can
used as mobile phase.
 The ability of mobile phase to move up is depend on
the polarity itself
 Volatile organic solvents is preferably used as
mobile phase.

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 MOBILE PHASE
SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0
Water

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 HPTLC
•  The process steps of HPTLC are identical to classical
TLC. The main difference between them is in the
characteristics of the separation plate. HPTLC plates
are based on optimized silica gel 60 with a significantly
smaller particle size than used for classical TLC.
• This allows a higher packing density and a smoother
surface. Hence, sample diffusion is reduced, resulting
in compact bands or spots. Furthermore, the smaller
particle size and thinner layer significantly increase
detection sensitivity and analysis speed.
 HPTLC Benefits

• Faster analysis, only 3 to 20 minutes for

optimal separation
• 5 to 10 times better detection sensitivity than
classical TLC
• Highly reproducible, sharp bands for
quantitative analysis.
 Advantages of HPTLC over TLC
• Visual chromatogram
• simplicity
• Multiple sample handling
• Low running and maintenance costs and
disposable layer etc
• Highly precised

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Thanks!