Continuous Hot 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.
Continuous Hot Extraction (Soxhlet)
 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
SEPARATION AND ISOLATION OF
CONSTITUENTS
 SEPARATION AND ISOLATION OF CONSTITUENTS

The instrumentation 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 methods used for separation are as follows

• Fractional crystallization
• Fractional distillation
• Fractional liberation
• Sublimation
• Chromatographic techniques
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 crystalline nature

even in mixture, process such as
concentration, slow evaporation,
refrigeration are using for crystallization
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.

FRACTIONAL DISTILLATION
FRACTIONAL LIBERATION
 In this process, the groups of
compounds having the tendency of
precipitation from the solution can
be separated.
 In certain cases the compounds
may modified by converting to its
salt form.
 This process is often used in
separation of cinchona alkaloids,
morphine etc.
Separation of alkaloidal mixture through fractional liberation and
crystallization
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.
CHROMATOGRAPY

 Chromatography’ is an analytical technique commonly used for separating a mixture

of chemical substances into its individual components, so that the individual
components can be thoroughly analyzed.
 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.
 Typically, the stationary phase is a porous solid (e.g., glass, silica, or alumina) that is
packed into a glass or metal tube or that constitutes the walls of an open-tube capillary.
 The mobile phase flows through the packed bed or column.
CHROMATOGRAPY
 Chromatography can be classified as follows:
• Paper chromatography
• Thin layer chromatography
• Column chromatography
• High performance liquid chromatography
 CHROMATOGRAPY
 Let’s first familiarize ourselves with some terms that are commonly used in the context of chromatography:
 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.
 Normal phase thin layer chromatography (The stationary phase i.e. silica is very
polar in nature, while the solvent is less polar compared to silica) is performed on a
piece of glass plate that is coated with a thin layer of silica. Here, silica acts as the
stationary phase and the solvent in which the plate is dipped and that runs up the plate
by capillary action is the mobile phase.
 THIN LAYER CHROMATOGRAPHY (TLC)

 TLC involves a thin layer of

Watch glass
adsorbent (Silica), mixed with a
binder 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. Pencil line
THIN LAYER CHROMATOGRAPHY (TLC)
 The polar components of the analyte will adhere to the silica tightly and thus travel
slowly up the plate, while the less polar or non-polar components will not adhere
that strongly to the silica and travel up the plate relatively fast with the solvent.
 Rule of thumb:
o The component that travels the least distance on the TLC plate is the most polar, since it
binds to the silica most tightly.
o The component that travels the maximum distance is the least polar; it binds to the silica
least tightly and is most soluble in the non-polar solvent (mobile phase), and hence
moves up the plate with the solvent.
THIN LAYER CHROMATOGRAPHY (TLC)

As show, the three components

A, B and C of the reaction
mixture travelled different
distances, as the solvent moved
up the TLC plate. Measured
from the origin (where we
spotted the reaction mixture):
component C travelled 1 cm,
component A travelled 2 cms
and component B travelled 3
cms. The solvent travelled 5
cms (distance from origin to
solvent front).
THIN LAYER CHROMATOGRAPHY (TLC)

Component Distance Distance Retention

travelled by travelled by factor Rf of
the the solvent the
component (cm) component
(cm)

C 1 5 = 1/5 = 0.2

A 2 5 = 2/5 = 0.4

B 3 5 = 3/5 = 0.6
THIN LAYER CHROMATOGRAPHY (TLC)

Significance of TLC:
 We can tell which component is more polar and which component is less polar
 Calculate the retention factor (Rf ) for every individual.
 Retention factor is defined as the distance travelled by the individual component
divided by the total distance travelled by the solvent. ‘Lower the Rf, more polar the
component.’
 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.
PRINCIPAL OF COLUMN CHROMATOGRAPHY
 The analyte is loaded over the silica bed (packed in the column) and allowed to
adhere to the silica. Here, silica acts as the stationary phase.
 Solvent (mobile phase) is then made to flow through the silica bed (under gravity
or pressure).
 The different components of the analyte exhibit varying degrees of adhesion to
the silica (see later), and as a result they travel at different speeds through the
stationary phase as the solvent flows through it, indicated by the separation of
the different bands. 
 The components that adhere more strongly to the stationary phase travel more
slowly compared to those with a weaker adhesion.
PRINCIPAL OF COLUMN CHROMATOGRAPHY
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

High performance liquid chromatography is a powerful tool in analysis. It uses the

same principles as in thin layer chromatography and column chromatography.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

 High Performance Liquid Chromatography (HPLC) is a form of column chromatography

that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high
pressure through a column with chromatographic packing material (stationary phase).
 HPLC has the ability to separate, and identify compounds that are present in any sample
that can be dissolved in a liquid in trace concentrations as low as parts per trillion.
 Because of this versatility, HPLC is used in a variety of industrial and scientific
applications, such as pharmaceutical, environmental, forensics, and chemicals.
HPLC Column
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
 APPLICATION OF HPLC

Isolation and purification of biologically active natural products

Control of synthetic reactions
 Used for separation of antibiotic from broth mixture
Pharmacokinetics study
Stability test
Quality control
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.
Characterization and identification

 UV Spectroscopy

 IR Spectroscopy
 NMR

 Mass Spectroscopy
Qualitative reactions for detection of Plant Constituents
 Tests for alkaloids 
1. Dragendorff’s test (potassium bismuth iodide solution)
2.Mayer’s test (potassium mercuric iodide solution)
3.Hager’s test (saturated aqueous solution of picric acid).
4. Wagner’s test (iodine in potassium iodide).
 Tests for glycosides
1. Bontrager's test
2. Modified Borntragor’s Test
 Tests for steroid and triterpenoid glycosides
1. Libermann Bruchard test
2. Salkovaski test
Qualitative reactions for detection of Plant Constituents
 Tests for cardiac glycosides
1. Keller Killiani test
2. Legal test
3. Baljet test
 Tests for tannins
1. Goldbeater’s skin test
2. Phenazone Test
3. Gelatin Test
 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.
Thank you all