WELCOME

GENERAL METHODS OF
ISOLATION AND SEPERATION
OF PLANT CONSTITUENTS

RAHUL B S
M PHARM PART 1
 CONTENTS

INTRODUCTION

EXTRACTION PROCESS
* Types of extraction * Solvents used
* Process of extraction * Types of extracts
SEPERATION AND IDENTIFICATION OF PLANT
CONTISTITUENTS
* Fractional crystallization * Fractional distillation * TLC
* Fraction liberation * Sublimation * Column chromatography
* Counter - current extraction * Paper chromatography
QUALITATIVE REACTIONS FOR THE DETECTION OF
PLANT CONSTITUENTS
APPLICATION OF GLC
APPLICATION OF HPLC
CONCLUSION
REFERENCE
INDRODUCTION

• A natural product is a chemical compound or substance

produced by a living organism. They may be extracted from
tissues of terrestrial plants, marine organism or micro - organism
fermentation.
• In that respect any biological molecule is a natural product, but
in general the term is reserved for secondary metabolites
(carotinoids, phytosterines, saponines, phenolic compounds,
alkaloids, glycosinates, terpenes etc).
• The extracts from plant tissue are a rich source of lead
compounds for nutraceutical or pharmaceutical applications
 Extraction

PLANT Steam
Distillation MATERIAL Distillation

Pressing

Methods for recovery of secondary metabolites.

 EXTRACTION PROCESS
Extraction may be defined as the treatment of the plant or animal
tissues with solvent, whereby the medicinally active constituents
are dissolved, and most of the inert matter remains undissolved.

The solvent used for extraction is known as Menstruum and the

inert insoluble material that remains after extraction is called
Marc
The various process used for extraction are
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.
2. Infusion

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.

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.
4. 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.
This procedure is suitable for extracting water-soluble, heatstable
constituents. The starting ratio of crude drug to water is
fixed, e.g. 1:4 or 1:16; the volume is then brought down to one-
fourth its original volume by boiling during the extraction
procedure. Then, the concentrated extract is filtered and used as
such or processed further.
 5. Percolation
Percolation is a continuous flow of the solvent through the bed
of the crude drug material to get the extract.
In this process, the powdered drug is moistened with an
appropriate amount of the specified menstruum and allowed to
stand for approximately 4 h in a wellclosed container, after
which the mass is packed and the top of the percolator is
closed.
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.
The outlet of the percolator then is opened and the liquid
contained therein is allowed to drip slowly.
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, and the mixed liquid is clarified by filtration or by
standing followed by decanting.
 MODIFIED PERCOLATION
The conventional percolation process is modified to include
evaporation for the production of more concentrated products,
especially when the solvent is dilute alcohol

In simple percolation
Drug imbibition maceration
In conventional percolation
Drug imbibition maceration
maceration
maceration
The process is continued in case the drug is not completely
exhausted.
percolation and collect the
percolate
percolation and collect the
1000 ml of percolate
percolation and collect the
1000 ml of percolate
percolation and collect the
1000 ml of percolate
Hot Continuous Extraction (Soxhlet)
In this method, the finely ground crude drug is placed in a
porous bag or “thimble” made of strong filter paper, of the
Soxhlet apparatus.

The extracting solvent in flask is heated, and its vapors

condense in condenser . The condensed extractant drips into the
thimble containing the crude drug, and 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.

The advantage of this method, compared to previously described

methods, is that large amounts of drug can be extracted with a
much smaller quantity of solvent.
SOXHLET APPARATUS
 Aqueous Alcoholic Extraction by Fermentation

It involves soaking the crude drug, in the form of either a

powder or a decoction for a specified period of time, during
which it undergoes fermentation and generates alcohol in situ;
this facilitates the extraction of the active constituents contained
in the plant material.
The alcohol thus generated also serves as a preservative.
Some examples of such preparations are
karpurasava, kanakasava, dasmularista..
COUNTER-CURRENT EXTRACTION

In counter-current extraction (CCE), wet raw material is

pulverized and produce a fine slurry.

Here the material to be extracted is moved in one direction

within a cylindrical extractor where it comes in contact with
extraction solvent.

The further the starting material moves, the more concentrated

the extract becomes.

Complete extraction is thus possible when the quantities of

solvent and material and their flow rates are optimized.

Finally, sufficiently concentrated extract comes out at one end of

the extractor while the marc falls out from the other end.
Advantages

Smaller volume of solvent as compared to other methods

like maceration, decoction, percolation.

CCE is commonly done at room temperature, which

spares the thermolabile constituents from exposure to heat
which is employed in most other techniques.

As the pulverization of the drug is done under wet

conditions, the heat generated during comminution is
neutralized by water. This again spares the thermo labile
constituents from exposure to heat.

The extraction procedure has been rated to be more

efficient and effective than continuous hot extraction.
Ultrasound Extraction (Sonication)

The procedure involves the use of ultrasound with requencies

ranging from 20 kHz to 2000 kHz;this increases the
permeability of cell walls and produces cavitation.

The process is useful in some cases, like extraction of

rauwolfia root, its large-scale application is limited due to the
higher costs.

Disadvantage

The deleterious effect of ultrasound energy (more than 20 kHz)

on the active constituents of medicinal plants through
formation of free radicals and consequently undesirable
changes in the drug molecules.
Supercritical Fluid Extraction
The critical point represents the highest temperature and
pressure at which the substance can exist as a vapour and
liquid in equilibrium. The phenomenon can be easily explained
with reference to the phase diagram for pure carbon dioxide
A super-critical fluid is a substance, mixture, or element,
which under certain operative conditions of pressure and
temperature, and mechanical operations, is above its critical
point but below the pressure needed to condense it into a solid.

Extraction via super-critical fluids is better for the

environment than conventional methods of extraction, because
it uses gases such as CO 2 at high pressure, in a liquid or
super-critical state, instead of chlorinated solvents which
produce toxic waste.

carbon dioxide is the preferred fluid for SFE, They are

powerful solvents and have a great capacity of penetration in
solids, which allows a rapid and almost complete exhaustion
of extractable solids.

They can easily be completely separated from extracts,

simply by modifying pressure or temperature, up to the point
advantages:
i) The extraction of constituents at low temperature, which
strictly avoids damage from heat and some organic solvents.
ii) No solvent residues.
iii) Environmentally friendly extraction procedure.

The main drawback is the time of extraction, which is

usually long. In fact, in some cases, it can take as much as 24
hours. With normal fluids, extraction can be speeded up by
mechanical shaking, but this presents problems when using
super-critical fluids, which limits industrial use.
Types of solvent
The solvents used in extraction are capable of penetrating the
tissues of the drug and dissolve the active principles contained in
its cell.
The various solvents used are water, propene, butane,
ethylacetate, ethanol, methanol, CO 2 , N 2 O, acetone etc.
Water
It is the cheap, non toxic, non inflammable and has wide solvent
action. Eg; proteins, glycosides, enzymes, sugar etc.
Disadvantages
•Water may promote growth of mould and bacteria, hence
requires a preservative.
•It may leads to hydrolysis.
•Large amount of heat is required to concentrate the aqueous
preparations.
•It promote fermentation or decomposition of the preparation
Alcohol
It is the important solvent for dissolves alkaloids, alkaloidal
salts, glycosides etc. it also dissolves many colouring
matter, tannins, etc. it doesn’t dissolve gums waxes, fats etc.

Merits
It doesn’t allow the growth of mould and bacteria in above 20%
of alcohol
It is nontoxic in the concentration mostly present in the
preparations.
Small amount of heat is requiered for concentration

Demerits
Cost
Inflammable, volatile etc
Solvents such as ether, chloroform, light petroleum are rarely
used.
factors considered when selecting a solvent

• Solvent power (selectivity). Only the active, desired constituents should be

extracted from the plant material, which means that a high selectivity is
required.
• Boiling temperature. The boiling point of the solvent is as
low as possible in order to facilitate removal of the solvent
from the product.
• Reactivity. The solvent should not react chemically with the
extract, nor should it readily decompose.
• Viscosity. A low viscosity of the solvent leads to low pressure
drop and good heat and mass transfer.
• Safety. The solvent should be non-fl ammable and non-corrosive, and should
not present a toxic hazard; its disposal should not imperil the environment.
• Cost. The solvent should be readily available at low cost.
• Vapor pressure. To prevent loss of solvent by evaporation, a
low vapor pressure at operating temperature is required.
• Recovery. The solvent has to be separated easily from the
extract to produce a solvent-free extract.
Types of extract

Aqueous extracts
The medicinal preparations intended to be used immediately after
preparation or to be preserved for use, solvent used is water. The methods
used for their preparation are decoction, infusion, and digestion.

Hydro alcoholic or Alcoholic

These are prepared by the methods of maceration and percolation eg
tintures, here the solvent using is alcohol

Soft extracts
They are extracts with semisolid or syrup consistency Can be used in a
variety of dosage form like ointments and suppositories
Eg; glycerriza extracts

Dry extracts
They powdered extracts or dry powder Extract obtained from suitable
process is filtered and get concentrated under vacuum, dried completely
by spray or tray drying.
Eg; belladona used in dossage forms such as capsules, tablets etc.
 SEPARATION AND ISOLATION OF CONSTITUENTS

The instrumentation for the structure for the structure

elucidation of organic compounds becomes effective and
allows the use of increasingly.

The most difficult operation in phytopharmacetical research

is the isolation and purification of plant constituents.

The physical methods used are chromatographic techniques

and methods such as fractional crystallisation, fractional
distillation, fractional liberation.

Chemical method is based on groups or moieties present in

the compound and chemical reactions.
FRACTIONAL CRYSTALLISATION

It is an important method for the purification of

compounds from mixture.

It depends upon the compound which form crystals at

the point of super saturation in the solvent in which it is
soluble

Many natural products are crystaline nature even in

mixture, process such as concentration, slow
evaporation, refrigeration are using for crystalisation
FRACTIONAL DISTILLATION

This method is used for the separation of

the components from volatile mixtures
Largely using in the separation of
hydrocarbons from oxygenated volatile
oil eg citral, eucalyptol

FRACTIONAL LIBERATION

In this proces the groups of compounds

having the tendency of precipitation from
the solution.
Incertain cases the compounds may
modified by converting to its salt form.
This proces is often used in separation of
cinchona alkaloids, morphine etc.
 SUBLIMATION

Here the compound is

heated the solid state
changes to gaseous state
without passing via liquid
state. Such compounds get
deposited in form of
crystals or cake.

This method is traditionally

used for the separation of
camphor from chips of
cinnamomum camphora.
 CHROMATOGRAPY

Chromatography is widely used for the separation &

identification of components of a mixture.

Separation of chemical compounds is carried out by

mobile phase and stationary phase.

Chromatography can be classified according to mechanism of

separation as:
adsorption chromatography,
partition chromatography,
ion exchange chromatography,
size exclusion chromatography and affinity chromatography.
PAPER CHROMATOGRAPHY

The principle is partition

Mainly the stationary phase is
moisture present in the cellulose
fibers and mobile vary as we using.
The components separated based
on their solubility

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.
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.
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 layer of developing solvent.
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.
COLUMN CHROMATOGRAPHY

It is a method used to purify

individual chemical
compounds from mixtures of
compounds the principle of
separation is adsorption.

The classical preparative

chromatography column, is a
glass tube with a diameter from
5 mm to 50 mm and a height of
5 cm to 1 m with a tap and some
kind of a filter (a glass frit or
glass wool plug – to prevent the
loss of the stationary phase) at
the bottom.
GAS CHROMATOGRAPHY (GC)
It is an analytical technique for separating compounds based primarily on
their volatilities.
GC provides both qualitative and quantitative information for individual
compounds present in a sample.
Compounds move through a GC column as gases, either because the
compounds are normally gases or they can be heated and vaporized into a
gaseous state.
The differential partitioning into the stationary phase allows the compounds
to be separated in time and space.
 APPLICATIONS
Quality control
contamination of plant and plant based products with pesticides, herbicides and many
other materials that are considered a health risk, all such products on sale today must
be carefully assayed

Identification of Source /Origin

The source of many plants (herbs and spices) can often be identified from the
peak pattern of the chromatograms obtained.
Technique of fingerprint could really identify the false herbal products.
The fundamental reason of quality control of herbal medicines is based on
the concept of phytoequivalence of herbs, and then to use this conception to
identify the real herbal medicine and the false one, and further to do
quality control.

Qualification and Quantification of Phytoconstituents

Alkaloids
Capillary gas chromatography (GC), often coupled with a mass spectrometer
as a detector (GC-MS), is a well established technique for analyzing complex
mixtures of alkaloids.
Terpenes

A qualitative comparative study was performed for terpenes

from volatile oils by GC and GC-MS technique
Flavanoids and Flavones

Flavonoids receive considerable attention in the literature,

specifically because of their biological and hysiological
importance. Gas Chromatography Coupled to Mass
Spectrometry GC-MS is established as a routine technique
for the analysis of flavonoid aglycones.
Essential Oils /Volatile oils

Many pharmacologically active components in herbal

medicines are volatile chemical compounds. Thus, the
analysis of volatile compounds by gas chromatography is
very important in the analysis of herbal medicines
 High-performance liquid chromatography (HPLC)

High performance liquid chromatography is a powerful tool in

analysis. This page looks at how it is carried out and shows
how it uses the same principles as in thin layer chromatography
and column chromatography.
 Application of HPLC
1- Isolation and purification of biologically active natural products
2- Control of synthetic reactions
Identification of intermediates and target compound.
3- Biosynthesis study
Detection of biogenetic intermediates and enzymes involved.
4-Control the microbiological process
Used for separation of antibiotic from broth mixture
5- Pharmacokinetics study
Pharmacokinetic study comprises the measurement of drug metabolites
concentration in body fluids, absorption, bioavailability and elimination of
drugs
HPLC determines the drug and its metabolites in one step.
6- Stability test
Rapid method of analysis in stability test.
7- Quality control
HPLC is used to know the identity, purity and content of the ingredients
(drugs, raw and pharmaceutical products,
8- Drugs metabolisms
 9- Purification
refers to the process of separation or extraction the target
compound from other compounds or contaminants
10- Quantification of compounds by HPLC
Quantitative (assay) and qualitative determination of natural
products
11- Is the process of determination of the unknown
concentration of a compound in a known solution.
12- Identification of compound by HPLC through :
- Comparison of retention time with authentic
- Comparison of UV spectrum of the compound with that of
the authentic.
- Comparison of the Mass spectrum with that of the authentic.
Qualitative Reactions For The Detection Of Plant Constituents

Test for alkaloids

1. Dragendorff’s test
1 ml of extract, add 1 ml of Dragendroff’s reagent (potassium bismuth iodide
solution). An orange-red precipitate indicates the presence of alkaloids.

2.Mayer’s test
1 ml of extract, add 1 ml of Mayer’s reagent (potassium mercuric iodide
solution). Whitish or cream colored precipitate indicates the presence of
alkaloids.

3.Hager’s test
1 ml of extract, add 3 ml of Hager’s reagent (saturated aqueous solution of
picric acid). Yellow colored precipitate indicates the presence of alkaloids

4. Wagner’s test
1 ml of extract, add 2 ml of Wagner’s reagent (iodine in potassium iodide).
Reddish brown colored precipitate indicates the presence of alkaloids
Test for glycosides

Bontrager's test
In this test boil test sample with 1ml of sluphuric acid in a test
tube for 5min,filter while hot. Cool the filterate and shake with
equal volume of dichloromethane or chloroform then seperate
the lower layer of chloroform and shake it with half volume of
dilute ammonia. A rose pink to red colour is produced in the
ammonical layer.

Modified Borntragor’s Test:

To 1 gm of drug add 5 ml dilute HCl followed by 5 ml

ferricChloride (5% w/v). Boil for 10 minutes on water bath, cool
and filter, filtrate was extracted withcarbon tetrachloride or
benzene and add equal volume of ammonia solution, formation
of pink tored colour due to presence of anthraquinone moiety.
This is used C-type of anthraquinoneglycosides
Chemical tests for steroid and triterpenoid glycosides
Libermann Bruchard test:
Alcoholic extract of drug was evaporated to dryness and
extracted with CHCl 3 , add few drops of acetic anhydride
followed by conc. H2SO4 from sidewall of test tube to the
CHCl3extract. Formation of violet to blue coloured ring at
the junction of two liquid, indicate the presence of steroid
moiety.

Salkovaski test:
Alcoholic extract of drug was evaporated to dryness and
extracted withCHCl3, add conc. H2SO4 from sidewall of test
tube to the CHCl3 extract. Formation of yellow colored ring at
the junction of two liquid, which turns red after 2 minutes,
indicate the presence of steroid moiety
Chemical tests for cardiac glycosides
Keller Killiani test:
To the extract of drug equal volume of water and 0.5 ml of strong lead acetate
solution was added, shaked and filtered. Filtrate was extracted with equal
volume of chloroform. Chloroform extract was evaporated to dryness and
residue was dissolved in 3 ml of glacial acetic acid followed by addition of few
drops of FeCl3 solution. The resultant solution was transferred to a testube
containing 2 ml of conc. H2SO4. Reddish brown layer is formed, which turns
bluish green after standing due to presence of digitoxose.

Legal test:
Treat the test solution with 2ml of pyridine and sodium nitropruside 2 ml was
added followed by addition of NaOH solution to make alkaline. Formation of
pink colour in presence of glycosides or aglycon moiety.

Baljet test:
Treat the test solution with picric acid or sodium picrate solution, it forms
yellow to orange colour in presence of aglycones or glycosides
 Tests for tannins

Goldbeater’s skin test: Goldbeater’s skin is a membrane produced from the

intestine of Ox. It behaves just like untanned animal hide. A piece of
goldbeaters skin previously soaked in 2% hydrochloric acid and washed with
distilled water is placed in a solution of tannin for 5 minutes. It is then washed
with distilled water and transferred to 1 % ferrous sulphate solution. A change
of the color of the goldbeater’s skin to brown or black indicates the presence
of tannin. Hydrolysable and condensed tannins both give the positive
goldbeater’s test while pseudo tannins show very little color or negative test.

Phenazone Test: To 5 ml of aqueous solution of tannin containing drug, add

0.5 g of sodium acid phosphate. Warm the solution, cool and filter. Add 2
% phenazone solution to the filtrate. All tannins are precipitated as bulky,
colored precipitate.

Gelatin Test: To a 1 % gelatin solution, add little 10 % sodium chloride. If a

1 % solution of tannin is added to the gelatin solution, tannins cause
precipitation of gelatin from solution.
Tests for flavonoids:

Shinoda Test: To the test solution, and few drops of conc.

HCl. To this solution 0.5 g of magnesium turnings were
added. Observance of pink coloration indicated the presence
of flavonoids.

With Lead Acetate: To the small quantity of test solution lead

acetate solution was added. Formation of yellow precipitate
showed the presence of flavonoid.

With Sodium Hydroxide: On addition of an increasing

amount of sodium
hydroxide, the ethanolic extract showed yellow coloration,
this decolorized after addition of acid.
 Tests of protein
Biuret test

On adding 1% copper sulphite to alkaline solution (4% NaOH solution) of

protein, a violet colour is developed. This test is due to the presence of peptide
linkage.
Xanthoproteic Test:
To 5ml test solution add 1ml of Con.HNO3 and boil, yellow ppt is formed.
On addition of NH4OH, yellow ppt. turned orange.
Nihydrin test
When protein is boiled with a dilute solution of ninhydrin, a violet colour is
produced.
CONCLUSION

Extraction, as the term is used pharmaceutically, involves the

separation of medicinally active portions of plant or animal
tissues from the inactive or inert components by using
selective solvents in standard extraction procedures.

The products so obtained from plants are relatively impure

liquids, semisolids or powders intended only for oral or
external use.

There are several techniques for the separation and

identification of natural products. Selection of method is
important in result.
Reference