Extraction and identification of phyto-constituents

EXTRACTION AND IDENTIFICATION OF
PHYTO-CONSTITUENTS
K.Sudheer Kumar,
Assistant professor.
Dept.of Pharmacognosy
Chilkur Balaji college of Pharmacy
Hyderabad.
E-mail:sudheer.y2k8@gmail.com
K.Sudheer Kumar,
Assistant professor.
Dept.of Pharmacognosy
Chilkur Balaji college of Pharmacy
Hyderabad.
E-mail:sudheer.y2k8@gmail.com

INTRODUCTION
Extraction is the process of efficiently dissolving and separating the desired constituent
from crude drug with the use of solvent
The choice of solvent depends on charachteristics of the secondary metabolites like
polarity, pH, thermal stability.
The solvent should non toxic,inflammable,easy to remove.
Factors effecting extraction:
a)Quantity and chemical nature
b)Size of powder of crude drug
c)Moisture content
d)Nature and volume of solvent
e)Mixing ration of solvent and sample
f)Temperature of extraction process
g)pH of extraction solvent
h)Lipophilicity of solvent mixture and sample
INTRODUCTION
Extraction is the process of efficiently dissolving and separating the desired constituent
from crude drug with the use of solvent
The choice of solvent depends on charachteristics of the secondary metabolites like
polarity, pH, thermal stability.
The solvent should non toxic,inflammable,easy to remove.
Factors effecting extraction:
a)Quantity and chemical nature
b)Size of powder of crude drug
c)Moisture content
d)Nature and volume of solvent
e)Mixing ration of solvent and sample
f)Temperature of extraction process
g)pH of extraction solvent
h)Lipophilicity of solvent mixture and sample

Types of extractions
Maceration :
Maceration is the extraction process of crude drugs from crushed or pressed
plant and animal material, which is soaked in a suitable solvent stirring at room
temperature.
Place solid material with whole of menustrum in a closed vessel and allow
stand for 7 days
Shaking occasionally/strain pressing marc
Mixing liquids obtained
Clarify by filtration
Seven days are consider to adequate period of equilibrium between solute and
solvent

PERCOLATION
Passage of menustrum under gravity column
Moisten the solid material with sufficient quantity of menustrum
Allow to stand for 4 hrs in well closed vessel
Packed in a percolator(24 hrs)
Allow percolation to proceed slowly until percolation measures about three
quarters of volume required for finished tincture
Press marc mix expressed liquid with percolate
Clarify by filtration

SOXHLET EXTRACTION
It is also known as counter current extraction
A piece of laboratory apparatus invented in 1879 by Franz von Soxhlet.
Originally designed for the extraction of a lipid from a solid material.
However, a Soxhlet extractor is not limited to the extraction of lipids.
Typically, a Soxhlet extraction is only required where the desired
compound has a limited solubility in a solvent, and the impurity
is insoluble in that solvent.
SOXHLET EXTRACTION
It is also known as counter current extraction
A piece of laboratory apparatus invented in 1879 by Franz von Soxhlet.
Originally designed for the extraction of a lipid from a solid material.
However, a Soxhlet extractor is not limited to the extraction of lipids.
Typically, a Soxhlet extraction is only required where the desired
compound has a limited solubility in a solvent, and the impurity
is insoluble in that solvent.

Drug is formed in to powder and the solvent is mixed in to it in equal
quantity place in apparatus and evaporate with heat condensation takes
place ,vapors are collected.
The thimble is loaded into the main chamber of the Soxhlete xtractor.
The extraction solvent to be used is placed in a distillation flask. The
flask is placed on the heating element. The Soxhlet extractor is placed
atop the flask. A reflux condenser is placed atop the extractor.

1. Stirrer bar
2. Still pot
3. Distillation path
4. Thimble
5. Solid
6. Siphon top
7. Siphon exit
8. Expansion adapter
9. Condenser
10. Cooling water in
11. Cooling water out
1. Stirrer bar
2. Still pot
3. Distillation path
4. Thimble
5. Solid
6. Siphon top
7. Siphon exit
8. Expansion adapter
9. Condenser
10. Cooling water in
11. Cooling water out
Parts of Soxhlet extractorParts of Soxhlet extractor

Continuous extraction of a component from a solid mixture.
Boiling solvent vapors rise up through the larger side-arm
Condensed drops of solvent fall into the porous cup, dissolving out the
desired component from a solid mixture
When the smaller side-arm fills to overflowing, it initiates a siphoning
action.
Residual solvent then drains out of the porous cup, as fresh solvent
drops continue to fall into the porous cup.
The solvent, containing the dissolved component, is siphoned into the
boiler below
Continuous extraction of a component from a solid mixture.
Boiling solvent vapors rise up through the larger side-arm
Condensed drops of solvent fall into the porous cup, dissolving out the
desired component from a solid mixture
When the smaller side-arm fills to overflowing, it initiates a siphoning
action.
Residual solvent then drains out of the porous cup, as fresh solvent
drops continue to fall into the porous cup.
The solvent, containing the dissolved component, is siphoned into the
boiler below
STEPS

INFUSION
Infusion is the process of extracting chemical compounds or flavors
from plant material in a solvent such as water, oil or alcohol, by allowing
the material to remain suspended in the solvent over time (a process
often called steeping).
An infusion is also the name for the resultant liquid. The process of
infusion is distinct from decoction, which involves boiling the plant
material, or percolation, in which the water passes through the material
This is very imp and simple method for extraction used for vitamins, volatile
ingredients and soft ingredients in which the powdered drug is extracted with
hot or cold water.
INFUSION
Infusion is the process of extracting chemical compounds or flavors
from plant material in a solvent such as water, oil or alcohol, by allowing
the material to remain suspended in the solvent over time (a process
often called steeping).
An infusion is also the name for the resultant liquid. The process of
infusion is distinct from decoction, which involves boiling the plant
material, or percolation, in which the water passes through the material
This is very imp and simple method for extraction used for vitamins, volatile
ingredients and soft ingredients in which the powdered drug is extracted with
hot or cold water.

Most people are familiar with an infusion which is what people do
when they put a tea bag or tea ball in a cup of hot water and allow it to
steep a couple minutes before drinking.
Infusions are used to extract vitamins and volatile ingredients from soft
ingredients like leaves, flowers, citrus peelings, etc. Green tea, black tea,
peppermint tea and chamomile tea are examples of an infusion.
The short brewing time helps to retain the volatile ingredients while
drinking.
Most people are familiar with an infusion which is what people do
when they put a tea bag or tea ball in a cup of hot water and allow it to
steep a couple minutes before drinking.
Infusions are used to extract vitamins and volatile ingredients from soft
ingredients like leaves, flowers, citrus peelings, etc. Green tea, black tea,
peppermint tea and chamomile tea are examples of an infusion.
The short brewing time helps to retain the volatile ingredients while
drinking.

METHOD
Powder drug is soaked in hot or Luke warm water
Filtered otter specified time
Press the marc and extract again with fresh water or
Luke warm water
METHOD
Powder drug is soaked in hot or Luke warm water
Filtered otter specified time
Press the marc and extract again with fresh water or
Luke warm water

DECOCTION
Decoction is a method of extraction by boiling of dissolved chemicals
from herbal or plant material, which may include stems, roots, bark and
rhizomes. Decoction involves first mashing and then boiling in water
to extract oils, volatile organic compounds and other chemical
substances. Decoction can be used to make herbal
teas, teas, coffees, tinctures and similar solutions.
Decoction means concentrate by boiling .this method is useful for the
wood,seed,type of drugs.
Powdered drug is boiled with water
Filtered

A decoction is used to extract primarily the mineral salts and bitter
principles of plants from hard materials such as roots, bark, seeds and
wood.
These hard materials generally require boiling for at least 10 minutes
and then are allowed to steep for a number of hours. The tea is boiled
down and concentrated so that water needs to be added before drinking.
The word "decoct" means to concentrate by boiling. Essiac tea and
taheebo bark tea are examples of a decoction.

WATER DISTILLATION
This method is useful for the extraction of essential oils
Distilled water is water that has had many of its impurities removed
through distillation. Distillation involves boiling the water and then
condensing the steam into a clean container.
Applications
In chemical and biological laboratories, as well as in industry, cheaper
alternatives such as deionized water are preferred to distilled water.
But if these alternatives are not pure enough, distilled water is used. If
exceptionally high purity water is required, double distilled water is
used.

Heating of plant material
with water
Vapor of volatile oil
component is passed to
wards condenser along
with steam
Oil and water layers are
separated on cooling
Heating of plant material
with water
Vapor of volatile oil
component is passed to
wards condenser along
with steam
Oil and water layers are
separated on cooling
Water distillation unitWater distillation unit

STEAM DISTILLATION
Steam distillation is a special type of distillation (a separation process)
for temperature sensitive materials like natural aromatic compounds. It
once was a popular laboratory method for purification of organic
compounds, but has become obsolete by vacuum distillation.
STEAM DISTILLATION
Steam distillation is a special type of distillation (a separation process)
for temperature sensitive materials like natural aromatic compounds. It
once was a popular laboratory method for purification of organic
compounds, but has become obsolete by vacuum distillation.

Steam distillation method
Steam passed through plant material suspended in water
Steam which can collected and cool it subsequently
In direct steam distillation meshes are used to separate steam from material and
vapor collected
Steam distillation method
Steam passed through plant material suspended in water
Steam which can collected and cool it subsequently
In direct steam distillation meshes are used to separate steam from material and
vapor collected

EXPRESSION OF OILS
When oils are taken from the rind of fruits they are called "expressed
oils".
There are many different ways to extract essential oils and they are
listed below:
a.Enfleurage
b.Expressed Oils
c.Steam Distillation
d.Solvent Extraction
e.Fractional Distillation and Percolation
f.Carbon Dioxide Extraction
g.Phytonic Process
EXPRESSION OF OILS
When oils are taken from the rind of fruits they are called "expressed
oils".
There are many different ways to extract essential oils and they are
listed below:
a.Enfleurage
b.Expressed Oils
c.Steam Distillation
d.Solvent Extraction
e.Fractional Distillation and Percolation
f.Carbon Dioxide Extraction
g.Phytonic Process

a)Enfleurage is one of the oldest methods of extracting essential
oils and is rarely used these days because of its high cost. It
involves placing the flower petals on a layer of glass that is first
spread with a thin layer of fat called "chassis". The volatile oil
diffuses into the fat, then the fat is collected and the oil is extracted
from the fat using alcohol.
b)When oils are taken from the rind of fruits they are
called "expressed oils". This method is cold and does not involve
any solvents or heat of any kind. Most of the citrus oils are
extracted in this manner including grapefruit, lime, orange
and lemon essential oils.

C)Steam distillation has been used for hundreds of years and today
remains one of the most favorably methods of extracting essential oils.
There are actually three different steam processes that are described
further in the distilling section. But in all of the methods, steam is used
to rupture the oil membranes in the plant and release the essential oil.
The steam carries the essential oil to a condenser and then as it re-
liquefies the lighter essential oil floats on top. The water and oil is then
separated and the water portion is referred to as the hydrosol, hydrolyte,
or flower or floral water.
C)Steam distillation has been used for hundreds of years and today
remains one of the most favorably methods of extracting essential oils.
There are actually three different steam processes that are described
further in the distilling section. But in all of the methods, steam is used
to rupture the oil membranes in the plant and release the essential oil.
The steam carries the essential oil to a condenser and then as it re-
liquefies the lighter essential oil floats on top. The water and oil is then
separated and the water portion is referred to as the hydrosol, hydrolyte,
or flower or floral water.

D)Solvent extraction is a method of extracting essential oils that
is dominated by the perfume industry. The oil particles are released
when the concrete substance is then mixed with alcohol. Once it is
separated out, the chemicals remain in the oil and the oil is used by the
perfume industry. These are perfume or aromatherapy grade oils.
E) Fractional distillation separates the volatile oil in different fractions or
portions at various boiling points. This is used in oil refineries for
distillation of petroleum products and is not suited for therapeutic grade
essential oils.
D)Solvent extraction is a method of extracting essential oils that
is dominated by the perfume industry. The oil particles are released
when the concrete substance is then mixed with alcohol. Once it is
separated out, the chemicals remain in the oil and the oil is used by the
perfume industry. These are perfume or aromatherapy grade oils.
E) Fractional distillation separates the volatile oil in different fractions or
portions at various boiling points. This is used in oil refineries for
distillation of petroleum products and is not suited for therapeutic grade
essential oils.

F) Carbon Dioxide Extraction or Super critical fluid extraction or carbon
dioxide extraction is also a fairly new solvent extraction process that
uses carbon dioxide at very high pressure. The carbon dioxide is injected
into the tank where the plant material is contained and acts as a solvent
to extract the oil.
G)The Phytonic process is a one of the newest methods of extracting
essential oils using non-CFCs (non-chlorofluorocarbons). It is also called
Florasol Extraction and the oils are referred to as phytols.
F) Carbon Dioxide Extraction or Super critical fluid extraction or carbon
dioxide extraction is also a fairly new solvent extraction process that
uses carbon dioxide at very high pressure. The carbon dioxide is injected
into the tank where the plant material is contained and acts as a solvent
to extract the oil.
G)The Phytonic process is a one of the newest methods of extracting
essential oils using non-CFCs (non-chlorofluorocarbons). It is also called
Florasol Extraction and the oils are referred to as phytols.

MICRO WAVE ASSISTED EXTRACTION
Micro wave works on high temperature produced by micro-waves
evaporate moisture in plant powder which dehydrates cellulos,ruptures
cell wall, and thus solubilizes phytoconstituents.
Microwave-assisted extraction (MAE) or simply microwave extraction
is a relatively new extraction technique that combines microwave and
traditional solvent extraction. Application of microwaves for heating the
solvents and plant tissues in extraction process, which increases the
kinetic of extraction, is called microwave-assisted extraction.
MAE has a number of advantages, e.g., shorter extraction time, less
solvent, higher extraction rate and lower cost,
MICRO WAVE ASSISTED EXTRACTION
Micro wave works on high temperature produced by micro-waves
evaporate moisture in plant powder which dehydrates cellulos,ruptures
cell wall, and thus solubilizes phytoconstituents.
Microwave-assisted extraction (MAE) or simply microwave extraction
is a relatively new extraction technique that combines microwave and
traditional solvent extraction. Application of microwaves for heating the
solvents and plant tissues in extraction process, which increases the
kinetic of extraction, is called microwave-assisted extraction.
MAE has a number of advantages, e.g., shorter extraction time, less
solvent, higher extraction rate and lower cost,

Traditional method of extraction of compounds from various matrices,
especially natural products.
The use of MAE in natural products extraction started in the late
1980s, and through the technological developments, it has now become
one of the popular and cost-effective extraction methods available today.
Several advanced MAE instrumentations and methodologies have
become available, e.g., pressurized microwave-assisted extraction
(PMAE) and solvent-free microwave-assisted extraction (SFMAE).
Traditional method of extraction of compounds from various matrices,
especially natural products.
The use of MAE in natural products extraction started in the late
1980s, and through the technological developments, it has now become
one of the popular and cost-effective extraction methods available today.
Several advanced MAE instrumentations and methodologies have
become available, e.g., pressurized microwave-assisted extraction
(PMAE) and solvent-free microwave-assisted extraction (SFMAE).

SONICATION
Sound waves of high frequency pulses of 20khz are generated in an ultrasonic
bath
Samples are kept in suitable vessel with solvent
Waves are generated from transmitters.
Thus increase cell wall permeability and cause solubilization of maximum
amount of active constituents
The frequency of a sound wave is a measure of how often the particles
of a substance vibrate
Sonicators either produce sound waves into a water bath, where
samples are placed, or can be probes that are put directly into the sample
to be sonicated.
SONICATION
Sound waves of high frequency pulses of 20khz are generated in an ultrasonic
bath
Samples are kept in suitable vessel with solvent
Waves are generated from transmitters.
Thus increase cell wall permeability and cause solubilization of maximum
amount of active constituents
The frequency of a sound wave is a measure of how often the particles
of a substance vibrate
Sonicators either produce sound waves into a water bath, where
samples are placed, or can be probes that are put directly into the sample
to be sonicated.

SUBLIMATION
Some substances on heating/cooling changes from solid to gas or vice
versa
Plant material is warmed and vapour allowed come in contact with
cold surface
The sublimating substance vaporize from plant material and condensed
as solid on cold surface .
SUBLIMATION
Some substances on heating/cooling changes from solid to gas or vice
versa
Plant material is warmed and vapour allowed come in contact with
cold surface
The sublimating substance vaporize from plant material and condensed
as solid on cold surface .

COUNTER CURRENT EXTRACTION
Liquid-liquid extraction-partition
The apparatus consists of series of tubes with one lower stationary
phase and upper immiscible liquids in moving mobile phase.
Mobile phase moves from one tube to other
Extract is placed in first tube and apparatus agitated, and layers are
allowed to separate
The components of mixture will distilled between the two layers
according to their partition coefficients
The upper moving phase is moved along to the second tube contains
lower phase.
COUNTER CURRENT EXTRACTION
Liquid-liquid extraction-partition
The apparatus consists of series of tubes with one lower stationary
phase and upper immiscible liquids in moving mobile phase.
Mobile phase moves from one tube to other
Extract is placed in first tube and apparatus agitated, and layers are
allowed to separate
The components of mixture will distilled between the two layers
according to their partition coefficients
The upper moving phase is moved along to the second tube contains
lower phase.

SUPERCRITICAL FLUID EXTRACTION SCF
A supercritical fluid SCF is any substance at
a temperature and pressure above its critical point, where distinct liquid and
gas phases do not exist.
It can effuse through solids like a gas, and dissolve materials like a liquid.
In addition, close to the critical point, small changes in pressure or
temperature result in large changes in density, Supercritical extraction and
fractionation of natural matter is one of the early and most studied
applications in the field of supercritical fluids.
supercritical Fluid Extraction (SFE) is the process of separating one
component (the extractant) from another (the matrix) using supercritical
fluids as the extracting solvent.
A supercritical fluid SCF is any substance at
a temperature and pressure above its critical point, where distinct liquid and
gas phases do not exist.
It can effuse through solids like a gas, and dissolve materials like a liquid.
In addition, close to the critical point, small changes in pressure or
temperature result in large changes in density, Supercritical extraction and
fractionation of natural matter is one of the early and most studied
applications in the field of supercritical fluids.
supercritical Fluid Extraction (SFE) is the process of separating one
component (the extractant) from another (the matrix) using supercritical
fluids as the extracting solvent.

Extraction is usually from a solid matrix, but can also be from liquids.
SFE can be used as a sample preparation step for analytical purposes,
Supercritical fluid chromatography (SFC) is a hybrid of gas and liquid
chromatography that combines some of the best features of each.
Supercritical fluid chromatography is of importance because it permits
the separation and determination of a group of compounds that are not
conveniently handled by either gas liquid or liquid chromatography.
SFC has been applied to a wide variety of including natural products,
drugs, foods, pesticides and herbicides, surfactants, polymers and
polymer additives.
Extraction is usually from a solid matrix, but can also be from liquids.
SFE can be used as a sample preparation step for analytical purposes,
Supercritical fluid chromatography (SFC) is a hybrid of gas and liquid
chromatography that combines some of the best features of each.
Supercritical fluid chromatography is of importance because it permits
the separation and determination of a group of compounds that are not
conveniently handled by either gas liquid or liquid chromatography.
SFC has been applied to a wide variety of including natural products,
drugs, foods, pesticides and herbicides, surfactants, polymers and
polymer additives.

METHODS OF PURIFICATIONMETHODS OF PURIFICATION
1.CHROMATOGRAPHY
2.DISTILLATION
3.EVAPARATION
4.CRYSTALISATION
5.SUBLIMATION
6.PRECEPTATION
1.CHROMATOGRAPHY
2.DISTILLATION
3.EVAPARATION
4.CRYSTALISATION
5.SUBLIMATION
6.PRECEPTATION
PHYSICALPHYSICAL CHEMICALCHEMICAL
1.HYDROLISIS ,
2.SALT FORMATION
3.ACETYLATION
1.HYDROLISIS ,
2.SALT FORMATION
3.ACETYLATION

SENNOSIDES FROM SENNA
Sennosides are obtained from dried leaflets of cassia angustifolia
belonging to the family Leguminosae
Sennosides are of A,B,C,D.Senna shows laxative property.
Extraction:
Senna leaves are dried and powdered to coarse
powdered senna is extracted with benzene in electric shaker 2 hrs
Filter the extract and dried with 70% methanol for 5hrs and again
extract marc with fresh methanol for 2 hrs
Filter and combine with extracts and concentrate up to 1/4 th of its

Add sufficient quantity of HCL to make pH 3.2 and keep a side for
2 hrs at 5°C
Filter and add sufficient quantity of alcoholic anhydrous calcium
chloride with continuous stirring
Add ammonia to bring pH 8 and kept a side for 2hrs filter and
collect ppt of calcium Sennosides
Dry the ppt in desiccators and calculate percentage yield

Chemical tests:
1.Borntragers test:
Take a little quantity of a solution of sample +water +CCL4 or ether
Separate organic layer and shake with dilute ammonia
Rose pink color of ammonia layer appears .
2.TLC/HPTLC:
a)Stationary phase silica gel G
b)Mobile phase :n-proponol: ethyl acetate :water: glacial acetic acid
(3:3:2:0:1 v/v)
c)Detection:366 nm,RF -0.35,0.25,0.61

3.HPLC:
a.Method : isocratic
b.Stationary phase:C18
c.Mobile phase :ethanol:water:acetic acid:tetrahydrofuron.
(60:38:2:2).
d. Detection:25μ nm.

EXTRACTION OF QUININE AND QUINIDINE
Quinine,quinidine,cichonine and cinchonoidine are the popular
quinoline alkaloids obtained from dried bark of cinchona officinalis
belonging to the family Rubiaceae.
All these have anti-malarial properties. Quinine also shows anti-
arrhythmatic agent.
Extraction:
Moist the weighed cinchona bark powder with alcoholic calcium
hydroxide or calcium oxide or potassium hydroxide (20%)
And sodium hydroxide (5%)keep the mixture as it is for few hours
so that alkali can convert cinchona alkaloids to free bases

Being non-polar in nature these free bases can be extracted with
benzene for 6 hrs in Soxhlet extractor.
After complete extraction filter the benzene extract and add 5%
sulphuric acid.
Shake the above contents mixture in separating funnel, separate
aq.layer from benzene layer and discharge benzene layer adjust pH 6.5
with sodium hydroxide.
Precipitate of quinine sulphate are formed filter and recrystalize with
hot water
Weigh and determine percentage yield and melting point
Dissolve this quinine sulphate dilute suphuric acid, add ammonia to
crystallize quinine as sharp needles separate these crystals from mother

Identification tests:
To the little quantity of sample and 0.25 ml of bromine water ,mix
well and add 1 m.l of chloroform, allow to stand few min,add
10%potassium ferrocyanide,shake well and add 3 m.l of 5N NaoH .
Quinidine :chloroform layer becomes red.
Quinine:chlorofom layer remains color less.
TLC/HPLC:
Stationary phase:silicagel-G
Mobile phase :diethylamine:ether:toulene(10:24:40)
Detection :spray with iodoplatinate reagent.

HPLC:
Method:Isocretic
Stationary phase:C18
Mobile phase: methanol 1 acetonitrile-0.1mol/L:ammonia
acetate(45:15:40:v/v) at a flow rate 1.0 ml/min
Detection:flurscence at Excitation-325 nm
Emission-375 nm

GLYCYRRHIZIN FROM GLYCYRRHIZA
Glycyrrhizine is a pentacyclic tri- terpenoid a-glycone obtained from
the roots and stolons of glycyrrhiza glabra belonging to the family
Leguminosae
Liquorice used as anti-ulcer.
Extraction :
Take accurately weighed quantity of coarse powder of glycyrrhiza
roots
Extract the powder with chloroform
Filter and discharge the filtrate
Extract the marc with 0.5 M H2SO4 for few hours

Filter and extract the filtrate with three portions of chloroform
Separate and combine the chloroform layers
Distill of the chloroform extract to yield a dry residue of
glycerrhetinic acid
Identification:
1.Libermann test:mix 2 ml of test solution with 2 ml acetic
anhydride boil and add 0.5 ml H2SO4.Blue color appears.
2.TLC:
a)Stationary phase: silica gel G
b)Mobile phase: ethyl acetate
c)Formic acid: glacial acetic acid (15:1:2) butanol-glacial acetic acid-
water (7:1:2)

LYCOPENE FROM TOMATO
Lycopene is a pigment principally responsible for the characteristic deep
red color of ripe tomato fruits. it is a natural anti- oxidant,lyccopene is a
carotonoid which also shows anti-cancer activity.
Lycopene fruits are the ripen fruits of lycopersicum esculentum belonging
to the family solanaceae.
Extraction :
50 gm of tomato paste was dehydrated by adding 65 ml methanol
After 24 hrs thick suspension was filtered
The dark red cake was shaken for another 15 min with 75 ml mixture of
equal volume of methanol and carbon tetrachloride and separated by
filtration

The ccl4 phase transferred to a reparatory funnel add volume of water and
shaked well after phase separation ccl4 phase evaporates
And residue was diluted with 2 ml of benzene using a dropper,1 ml of boiling
methanol was added in portion, then crystals as crude Lycopene were
appeared immediately and crystallization was completed by keeping the
liquid at room temperature and ice bath
The crystals were washed 10 times using benzene and boiling methanol
Long red Lycopene prisms were observed under microscope with some
colorless impurities

For more purification column chromatography on active acidic alumina
using toluene as eluent was done
The deep red zone was collected after complete evaporation of solvent
the residue was dissolves in 2 ml benzene
After re-crystallization using boiling methanol no colorless substance
observed
Crystalline Lycopene is not isomerized but has a tendency to auto-
oxidation, so it was kept in dark glass tube.

Chemical tests:
1.Lycopene dissolves in concentrated H2SO4—indigi blue color appears.
2.On addition of anti- mony chloride in chloroform to a solution of
Lycopene in chloroform on intense unstable blue color produces.
Identification:
UV-wave length—447.2 ,473.2,504.2 nm
IR- vibrational wave length
3100 cm-1
2918.92-2851.05 sp3
1670,1640-c=c(trans)

ISOLATION OF CURCUMIN FROM TURMERIC
Turmeric of zingiberaceae family wisely cultivated spice in India and others
Asian countries. Turmeric is rich in curcuminoids,curcuminnoids shows anti-
oxidant,anti-inflammatory,ant-carcinogenic effects, and Hypoglycemic
activity.
Extraction:
Fresh rhizomes were collected washed with deionised water
Sliced and dried in sun for one week and again dried at 50°c in hot air oven
for 6hrs
Dried rhizomes were cut in to small pieces powdered by electric mill
Take 20gm of sample in a thimble and placed in Soxhlet apparatus were set
up to various solvent from non-polar to polar

150 ml of solvent added and extracted 6 hrs hexane,B.P
69°C,chloroform 61°C,ethylacetate 77°C,methanol
65°C,acetone56°C,one with hexane 2 hrs
Hexane extract was discarded and the powder re extracts with
methanol 6 hrs
After completion of extraction dark brown extract was then cooled,
filtered ,concentrate, using rotatory evaporator finally by vaccum
suction to get a crude extract yield calculated.

Identification:
Curcumin orange yellow crystalline powder with M.P
183°C,insoluble in cold water, soluble in alcohol, glacial acetic
acid, dissolves in H2SO4 yields red color in 0.1N NaoH .
1.TLC:
a)Stationary phase:Silicagel-60F254
b)Mobile phase:chloroform:methanol (95:5)
Benzene:ethylacetate:18:2
UV:420 nm.

Extraction and identification of phyto-constituents

More Related Content

What's hot (20)

Viewers also liked (20)

Similar to Extraction and identification of phyto-constituents (20)

More from Dr K SUDHEER KUMAR KANDIBANDA (20)

Recently uploaded (20)

Extraction and identification of phyto-constituents