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EXTRACTION, SEPARATION &

ISOLATION OF PLANT CONSTITUENTS



EXTRACTION OF PLANT
MATERIAL
All plant material used 1
st
has to be
properly authenticated (so time &
money is not wasted by
examining material of doubtful
origin).
The choice of extraction procedure
depends on the nature of the
plant material & the components
to be isolated.
Dried material is normally powdered
before extraction.
Fresh plants (leaves, flowers) can
be homogenized or macerated
with a solvent (alcohol).
Alcohol is also useful for stabilising
fresh leaves.

SOLVENTS USED FOR PLANT
EXTRACTION
Alcohol is a general
solvent for many plant
constituents (except?)
Water immiscible
solvents are also
commonly used (e.g.
light petroleum
volatile & fixed oils,
steroids), ether &
chloroform (alkaloids,
quinones).
EXTRACTION OF ALKALOIDS &
PHENOLS
Before extracting
alkaloids (organic
bases), basification of
the plant material is
necessary (if a water
immiscible solvent is
used).
If aromatic acids &
phenols are going to
be extracted,
acidification may be
required.
METHODS OF EXTRACTION
Extraction may be performed
by repeated maceration
with agitation, percolation
or by continuous extraction
(e.g. Soxhlet extractor).
Special methods are used to
extract volatile oils
(Enfleurgage).
Ultrasound may be used to
enhance the extraction
process for some plant
material BP uses this
method to assay the
alkaloids of opium.
SPROUTED BED EXTRACTION
Sometimes by removing the pigment layer of seed-
coats leads to a better product than methods of
solvent extraction.
This type of method can involve the use of a ball mill or
a sprouted bed unit.

METHOD: The sprouted bed method consists of a
cylinder tapered at both ends & contains the seeds at
the lower end through which a jet of hot air is forced.
Seeds & pigment loaded fine particles are propelled
into the space above where the seeds fall back to be
re-circulated & the fine powder moves to a cyclone
from which it is collected.

E.g. Annatto powder Bixa orellana
Bixa orellana

SUPERCRITICAL FLUID
EXTRACTION
The use of supercritical fluids for the extraction of a
range of materials including plant products of
medicinal, flavouring & cosmetic interest has become
of increasing economic & research interest.
SUPERCRITICAL STATE: Above a certain T & P,
single substances do not condense or evaporate but
exist as a fluid. Under these conditions the gas/liquid
phases have the same density & no division exists
between the 2 phases.
Water: Critical conditions for Temperature (tc) and
Pressure (pc) are 374C and 220 atmospheres.
CO2: tc = 31, Pc = 74 atm
THE SUPERCRITICAL STATE

SUPERCRITICAL FLUID
EXTRACTION
In phytochemistry these properties can be
exploited to maximize the extraction of plant
contituents.
For industrial purposes supercritical fluid CO2 has
an environmental advantage over many
common organic solvents & leaves no solvent
residues in the product. It also allows a low
temperature process and has proved of value for
the extraction of labile expensive fragrances &
medicinal phytochemicals.

EXAMPLES OF PHYTOCHEMICALS
EXTRACTED WITH SUPERCRITICAL
CO2
1. ALKALOIDS
- Decaffeination of
green tea

2. FIXED OILS

3. VOLATILE OILS &
RESINS

SUPERCRITICAL FLUID
EXTRACTION
DISADVANTAGE: The
high pressures & high
temperatures (for
some substances).

SEPARATION & ISOLATION OF
CONSTITUENTS
The most difficult operation in phytochemical
research is to isolate & purify plant
constituents.

TECHNIQUES OF SEPARATION &
ISOLATION
- Sublimation
- Distillation
- Fractional liberation
- Fractional crystallization

SUBLIMATION
Sublimation is
sometimes possible
on whole drugs (e.g.
isolating caffeine from
tea).
Modern equipment uses
low pressures with a
strict control of
temperature.

DISTILLATION
i. FRACTIONAL
DISTILLATION: traditional
method of separation of
constituents of volatile
mixtures (isolation of
components of volatile
oils).

ii. STEAM DISTILLATION:
used to isolate volatile oils
and hydrocyanic acid from
plant material.

FRACTIONAL & STEAM DISTILLATION

FRACTIONAL LIBERATION
Some groups of compounds
can be fractionally isolated
(liberated) from a mixture.
E.g. A mixture of alkaloid salts
in aqueous solution
Treat with aliquots of alkali
liberates the weakest
base first, followed by the
liberation of other bases in
an ascending order of
basicity.

IF: The mixture is shaken with
an organic solvent after each
addition, then a fractionated
series of bases will be
obtained.

FRACTIONAL LIBERATION
A similar scheme can
be used for organic
solvents soluble in
water-immiscible
solvents. Here you
would start with a
mixture of acid salts,
making it possible to
fractionally liberate
the acids by the
addition of mineral
acids.

FRACTIONAL CRYSTALLIZATION
This method is commonly used
in traditional isolations & is
still valuable for the
resolution of mixtures which
would otherwise be
intractable.
The method exploits the
differences in solubility of the
components of a mixture in a
particular solvent.
Often, derivatives of the
particular components are
employed (e.g. picrates of
alkaloids, osazones of
sugars).

CHROMATOGRAPY
Classical methods for the separation of compounds in a
mixture are not always adequate for complete
separation, and often times require large amounts of
material (e.g. fractional distillation, crystallization).
Chromatography is widely used for the separation &
identification of components of a mixture.
Chromatography is also very suitable for the extraction
of plant extracts, especially when a small amount of
material is available & when the components closely
resemble each other (e.g. alkaloids, anthraquinones).
In the earliest form, chromatography was applied only to
coloured compounds, and the results of separation could
be followed by visual observation.
ADSORPTION
CHROMATOGRAPHY
In its simplest form, this
method of
chromatography consists
of passing a solution of
the mixture of
compounds needing to
be separated, through a
hollow glass column,
packed with a finely
divided absorbent
powder, and collecting
the solution (eluate).

The surface phenomenon of
adsorption is utilized. The
finely-divided solids are
capable of selective
adsorption of other
substances. The
components of a mixture
introduced onto a column of
adsorbent are more or less
strong adsorbed. Those
which are least strongly
adsorbed are carried down
the column by the passage
of solvent, & are the 1
st
to
be eluted from the bottom
of the column.

ADSORPTION
CHROMATOGRPAHY
The passage of the
more strongly
adsorbed substances
is slower, & these are
the last to elute.
The fractions of eluant
containing each
component of the
original mixture can
then be separately
analyzed.

Alternatively, the entire
adsorbent core may
be extruded, after
bands containing the
compounds of the
mixture have
developed, & each
band is removed by
cutting the cylinder
into sections.
Individual bands can
then be extracted with
a suitable solvent to
obtain each
compound.


THIN LAYER
CHROMATOGRAPHY (TLC)
TLC is an e.g. of adsorption
chromatography, the stationary phase
being a thin layer adsorbent held on a
suitable backing. Separation of the
compounds present in the plant extract
depends on the differences in their
adsorptive/desorptive behaviour in respect
of the stationary phase.
TLC involves a thin
layer of adsorbent,
mixed with a binder
such as CaSo4,
which is spread on a
glass plate & allowed
to dry.
The plant mixture to be
separated is applied
as a spot near the
base of the plate,
which is then placed
in a closed glass tank
containing a a layer of
developing solvent.

The solvent moves up
the plate by capillary
action, carrying with it
the less strongly
adsorbed
components of the
mixture, while the
more strongly
adsorbed compounds
remain near the base
of the plate. When
the solvent has
reached 1-2 cm from
the top of the plate,
the plate is removed
from the tank & dried.
The now separated
components of the
mixture appear as
spots on the finished
plate (chromatogram),
corresponding to the
bands of the
adsorbent column.

TLC THE ADSORBENT
With adsorption TLC, different substances have
different adsorptive capacities & any one
material can vary in its activity according to the
pre-treatment of the TLC plate.
The absorbent must be chosen in relation to the
properties of the solvent & the mixture to be
separated.
In general: if a highly active adsorbent is used,
then a solvent with a corresponding high power
of elution for this substance will be required.
E.g. Aluminium (acidic, neutral or basic) with
different activity grades is commonly used.
To produce a film with reasonable handling
properties, the adsorbent may be mixed with
12% of its weight of calcium sulphate to act as
a binder.
Ready-mixed powders are commercially available
they need to be mixed with a certain amount
of water and may be spread onto the plate with
a glass rod.
The film sets within a few minutes & is then
activated by heating at a suitable temperature.
Solutions to be examined are applied to the film
with the help of capillary tubes or microsyringes
/micrometer pipettes.
TLC THE SOLVENT
Solvents used for TLC must be pure.
Commonly used solvents
- Methanol
- Ethanol
- And other alcohols
- Chloroform
- Ether
- Ethyl acetate
VISIBILITY OF SPOTS
(COMPOUNDS)
If invisible, the spots may be
made visible by
- Heating for a specific period
- Examining under U.V light (if
substances are florescent).
- Spraying the finished
chromatogram with a suitable
reagent e.g. iodine &
Dragendorffs reagent are used
as sprays for the general
detection of iodine (although
they are not specific for
alkaloids).
ADVANTAGES OF TLC OVER
PAPER CHROMATOGRAPHY
- Separation of compounds can be
achieved more rapidly & with less plant
material.
- The separated spots are more compact &
clearly demarcated from one another
- Reagents such as concentrated H2SO4
would destroy a paper chromatogram, but
ma be used to locate the separated
substances on a TLC plate.

ADVANTAGES OF TLC
- Simple, inexpensive
- Quick results can be achieved from
between 30 minutes to a few hours
- Good separation of spots (compounds)
- Very sensitive
- The chromatogram is resistant to the
action of chemicals used for the
visualization of compounds.
COMPONENTS OF THE TLC
SYSTEM
3 components

i. THE ADSORBENT Stationary Phase
ii. THE ELUENT (THE DEVELOPING
SOLVENT) Mobile Phase
iii. THE SUBSTANCE REQUIRING
SEPARATION Plant Sample
THE ADSORBENT
The adsorptive capacities of the adsorbents are
related to their polarity.
The more polar the adsorbent material is, the
better it will separate compounds (e.g.
aluminium & silica gel).
Under normal conditions, these adsorbents hold
little water & adsorbed organic material (these
decrease the adsorptive capacity of the
material).
Heating may activate absorbent.
THE ELUENT
The more polar the
solvent, the better its
eluting (desorbing)
properties. Therefore
water is a better
eluant than ether,
which in turn is better
than cyclohexane.
THE SUBSTANCES REQUIRING
SEPARATION
Polarity also plays an
important role.
The least polar
substances (e.g.
saturated
hydrocarbons) are
least strongly
adsorbed, while the
highly polar
compounds (e.g.
acids) are strongly
adsorbed.
In practice, only 2
adsorbents are widely
used
- Silica gel
- Aluminium

When using these
adsorbents, the rule is
to match the polarity
of the developing
solvent with that of
the compounds of the
mixture to be
chromatographed.
SEPARATION OF ALKALOIDS
Alkaloids need a moderately polar
solvent for good separation (e.g.
ether/ethanol: 95/5).

A more polar solvent (e.g. pure
methanol), would be preferentially
adsorbed, & the alkaloids would
be carried along by the passage of
the solvent resulting in poor
separation.

On the other hand, a non-polar
solvent (e.g. cyclohexane) would
be unable to displace the alkaloids
from the adsorbent layer & they
would then remain at or near the
base of origin.
ADDITIONAL FACTOR FOR
SEPARATING ALKALOIDS
If using an aluminium thin
layer (neutral), a neutral
solvent should be used.

If using Si-gel (slightly
acidic due to the
method of preparation),
and alkaline solvent
such as
acetone/water/25%am
monia: 90/7/3 makes
for good separation.
SEPARATION OF VOLATILE OILS
The constituents of volatile
oils are mainly non-polar
(terpenes) & are
therefore best separated
with corresponding non-
polar solvents such as
chloroform/benzene
mixtures.

Certain oils may need
more polar solvents (e.g.
clove oil phenolic).
SEPARATION OF SUGARS &
SUGAR ACID MIXTURES
These mixtures are
produced by
hydrolysing starch &
gums. They are
strongly polar & are
therefore strongly
adsorbed onto silica &
aluminium layers.
Although strongly polar
solvents are used,
separation on these thin
layers is not generally
satisfactory & better
results are obtained using
weakly polar adsorbents
such as cellulose & a
polar solvent such as
butanol/ethanol/water:
5/4/1. Alternatively, paper
chromatography could be
used.

GENERAL RULE FOR TLC
TLC is best used for
moderately or weakly
polar mixtures.
PARTITION CHROMATOGRAPHY
Partition chromatography is based on the
differences in partition co-efficients of the
compounds of a mixture which have to be
separated, between an aqueous &
immiscible organic liquid.

The stationary phase is normally aqueous,
which is mixed with an inert carrier powder
& packed into a glass column.

The mixture to be separated is dissolved in an
organic solvent, & is introduced onto the
column & the chromatogram developed with
more solvent, on different solvents of
increasing eluting power.
Diffusion of the mobile phase through the
stationary phase occurs, & the different rates
of travel of the constituents of the mixture are
directly related to their partition coefficients
between the mobile organic & stationary
aqueous phase.
The finished chromatogram exhibits separated
zones similar to those seen an adsorbent
column.
PARTITION CHROMATOGRAPHY
ON PAPER
Although it has been for
a large part been
replaced by TLC, it
still remains the
method of choice for
the separation of
some types of
compounds.


METHOD OF PAPER
CHROMATOGRAPHY
The solution to be
separated is applied as a
spot near one end of a
prepared filter-paper
strip The paper is then
supported in an airtight
chamber which has an
atmosphere saturated
with solvent & water, and
a supply of the water-
saturated solvent.

The best solvents are those which are partially
miscible with water (phenol, n-butanol & amyl
alcohol).

Either the paper may be dipped in the solvent
mixture so that the solvent travels up the
paper (ascending technique) or a trough of
solvent may be supported at the top of the
chamber so it travels down the paper
(descending technique).
As the solvent moves,
the compounds also
move along the paper
at varying rates,
depending on the
differences in their
partition coefficients
between the aqueous
& organic phases.

VISIBILITY OF COMPOUNDS
After the filter-paper strips have been dried, the
positions of the separated components can be
revealed by the use of suitable developing
agents:
- Ninhydrin solution amino acids
- Iodine solution/vapour or modified Dragendorffs
reagent alkaloids
- Ferric chloride solution phenols
- Alkali anthraquinones
- Antimony trichloride in chloroform steroids &
some volatile oil components
- Aniline hydrogen phthalate reagent - sugars

The positions of the compounds & the size of the
spots depend on the solvent.

The solvent should be selected to give good
separation of the compounds with well-
defined, compact spots.

Improved separation can be attained by
adjusting the acidity of the solvent with
ammonia, acetic acid, or hydrochloric acid, or
by impregnating the paper with a buffer
solution or formamide solution.
SEPARATION OF SUBSTANCES
For the separation of
substances, it is
necessary to use a 2-D
chromatogram.

First one solvent is run in
one direction, then,
after drying of the
paper, a 2
nd
solvent is
run in a direction at
right angles to the 1
st

This is especially
applicable to mixtures
of amino acids.

PAPER CHROMATOGRAPHY
The ratio between the distance travelled on
the paper by a component of the test
solution & the distance travelled by the
solvent is termed the RF value. Under
standard conditions, this is a constant for
the particular compound.

In practise, however, variations of the RF
value often occur & it is best to run a
reference compound alongside the
unknown mixtures.
PAPER CHROMATOGRAPHY:
ADVANTAGES & DISADVANTAGES

ADVANTAGES
i. Simple & inexpensive
ii. Sensitive gives good separation of very
small amounts, of especially water-soluble
compounds, e.g. sugars.

DISADVANTAGES
i. Fragile chromatogram may be destroyed by
chemicals used for visualization
ii. May be time-consuming.
RF VALUES
Rf (rate of flow) values are
used as a way of
identifying compounds
on a chromatogram.
In theory, the Rf value is
constant in a constant
set of chromatographic
conditions. In practice, it
is difficult to achieve
because of the many
factors that may affect
the Rf value.
RF VALUES & MOISTURE
Very NB:= amount of moisture adsorbed on the
thin layer. The presence of significant amounts
of moisture decrease the number of sites
available for active adsorption. The compounds
undergoing separation will therefore have a
higher than normal Rf value.

The moisture content of thin layer plates is difficult
to control & is therefore a major factor in non-
reproducibility of Rf values.
RF & TANK SATURATION
In a non-saturated tank, the solvent
evaporates as it travels up the plate
producing a lower solvent front than in a
saturated tank.

Rf values of substances being separated will
therefore be higher than these obtained
using a saturated tank.
SOLUTION
These difficulties may be overcome by
spotting reference solutions of known
compounds alongside the mixtures to be
separated.
In this way, both the known & the
unknown compounds are subject to the
same variables. If the Rf values may be
used as a means of identification.

ALTERNATIVE SOLUTION
Alternatively, an Rx or R standard value may
be calculated from:

Rf unknown compounds / Rf reference
compound
RECAP - TLC
Separations in TLC involve
distributing a mixture of two or
more substances between a
stationary phase and a mobile
phase. The stationary phase is a
thin layer of adsorbent (usually
silica gel or alumina) coated on a
plate. The mobile phase is a
developing liquid which travels up
the stationary phase, carrying the
samples with it. Components of the
samples will separate on the
stationary phase according to how
much they adsorb on the stationary
phase versus how much they
dissolve in the mobile phase.









Equipment used in a thin
layer chromatography
experiment.

Preparing the Chamber
To a jar with a tight-fitting lid add
enough of the appropriate
developing liquid so that it is 0.5 to 1
cm deep in the bottom of the jar.
Next, place a piece of filter paper into
the jar so that it lines the walls and is
immersed in the liquid. (The filter
paper absorbs liquid and helps the
atmosphere of the chamber become
saturated with the solvent. A solvent-
saturated atmosphere helps the TLC
experiment proceed more quickly).
Close the jar tightly, and let it stand for
about 30 minutes so that the
atmosphere in the jar becomes
saturated with solvent


Preparing the Plates for Development
With a pencil, etch two small notches into the
adsorbent about 2 cm from the bottom of the
plate. The notches should be on the edges of
the plate, and each notch should be the same
distance up from the bottom of the plate. The
notches must be farther from the bottom of the
plate than the depth of the solvent in the jar.
Using a drawn-out capillary tube, spot the
samples on the plate so that they line up with the
notches you etched. If more sample is needed
on the plate for the experiment, the sample may
be re-spotted
Developing the Plates

After preparing the development
chamber and spotting the
samples, the plates are ready for
development. Be careful to handle
the plates only by their edges, and
try to leave the development
chamber uncovered for as little
time as possible.

When the plates are removed from
the chamber, quickly trace the
solvent front (the highest solvent
level on the plate) with a pencil.

Identifying the Spots
If the spots can be seen, outline them with a
pencil.

If no spots are obvious, the most common
visualization technique is to hold the plate
under a UV lamp (CAUTION: Do not look
directly into the lamp.) Many organic
compounds can be seen using this
technique, and many commercially made
plates often contain a substance which aids
in the visualization of compounds.
Identifying the Spots










Commercial TLC plate after
development
in normal lighting










Same TLC plate held under
a UV lamp -
Note the appearance of
additional spots
Interpreting the Data
The Rf value for each spot
should be calculated. Rf
stands for "ratio of fronts"
and is characteristic for any
given compound on the
same stationary phase
using the same mobile
phase for development of
the plates. Hence, known
Rf values can be compared
to those of unknown
substances to aid in their
identifications.


Interpreting the Data
(Note: Rf values often depend on the temperature
and the solvent used in the TLC experiment; the
most effective way to identify a compound is to
spot known substances next to unknown
substances on the same plate).

In addition, the purity of a sample may be
estimated from the chromatogram. An impure
sample will often develop as two or more spots,
while a pure sample will show only one spot.
RECAP - PC
Paper chromatography is
one method for testing
the purity of compounds
and identifying
substances. Paper
chromatography is a
useful technique because
it is relatively quick and
requires small quantities
of material.
PC - DESCRIPTION
Separations in paper chromatography involve the same
principles as those in thin layer chromatography. In
paper chromatography, like thin layer
chromatography, substances are distributed between
a stationary phase and a mobile phase. The stationary
phase is usually a piece of high quality filter paper.
The mobile phase is a developing solution that travels
up the stationary phase, carrying the samples with it.
Components of the sample will separate on the
stationary phase according to how strongly they
adsorb to the stationary phase versus how much they
dissolve in the mobile phase.
Preparing the Chamber
Choose a developing chamber that can be
sealed well. The chamber should be large
enough to hold the paper that is to be
developed.
The chamber should be clean and dry before
use.
Add the mobile phase to the chamber so that
it is about 2 cm deep. Seal the chamber
tightly and let the chamber stand overnight if
possible (Allowing the chamber to stand
permits its atmosphere to become saturated
with the developing solvent. A saturated
atmosphere allows for more effective
development of the chromatograms. The
larger the chamber, the longer it should
stand )
Preparing the Chamber
The larger the
chamber, the longer it
should stand

Preparing the Stationary Phase
Cut a square piece of
high-quality filter
paper to fit into your
development
chamber. With a
pencil, draw a straight
line about 3 cm from
the bottom edge of
the paper.

Spotting the Samples
First, each sample should
be dissolved in an
appropriate solvent to
make about a one
percent solution (0.01 g
sample/1 g solvent). Less
than one milliliter of
solution will be needed
for the experiment. Then
the dissolved samples
may be spotted to the
paper.

Spotting the Samples
If a larger quantity of
sample is needed for the
experiment than is
provided by one
application, the solution
may be re-spotted.
All spots on the
chromatogram should be
2 to 2.5 cm away from the
edges of the paper and
from each other.


Developing the Chromatograms
After preparing the chamber and spotting the
samples, the paper is ready for development. Be
careful to handle the paper only by its edges,
and try to leave the development chamber
uncovered for as little time as possible.
Initially, the chromatogram should be suspended
in the chamber without touching the solvent. To
suspend the chromatogram, to the top of the
paper and thread a piece of string throught the
paper clip. Then tape the string to the outside of
the chamber to hold the chromatogram in place.
The paper should hang in the development
chamber overnight, if possible.
Developing the Chromatograms
After the
chromatogram has
hung in the chamber,
immerse the paper's
bottom edge into the
developing solvent.
Allow the
chromatogram to dry
in a well-ventilated
area.
Identifying the Spots
If the spots can be seen, outline them with a
pencil.
If the spots are not obvious, the most
common visualization technique is to hold
the paper under an ultraviolet lamp. (Caution:
Do not look directly into the lamp!) Many
organic compounds can be seen using this
technique. Outline the spots with a pencil.