Extraction of Plant Material

Extraction is an age old process practiced by humankind in his day to day activities as in the case
of making tea and preparation of decoctions. With the advancement of science, the process of
extraction developed into a distinct area and contributes significantly to the development of
phytochemistry. It is only rarely the plant material as such can be used for analysis as in the case
of estimation of total ash and nitrogen. In certain cases the material to be worked out occurs
naturally in a solution such as latex, guttation fluid and nectar. In general, however, the first step
of phytochemical analysis is extraction, a method to separate the compounds that are being
studied from the mixture of solid bodies or liquids by a suitable solvent. In Latin the word
extraho means to draw out. A good extraction procedure should bring all the group of materials
we are looking for into solution and causes little or no change in the nature of compounds and
easy for further analysis. Though the extraction techniques were put as a separate chapter, it is
part of the separation process.
Rupturing of the Cells: Most of the compounds found inside the cells are not permeable
through the cell wall. Water soluble components of low molecular weights generally diffuse out
of the cell when the tissue is treated in such a way as to destroy its osmotic control, for example,
by heating to 60oC. Even when there is no osmotic barrier the diffusion from tissues is often slow
especially with large molecules as proteins or gums. So the tissues are disintegrated first and the
desired compounds are diffused from the cell sap to the solvent.
Disintegration of the tissues can be effected by;
Mechanical Methods: By the use of mortar and pistil, grinding mills and mixer grinder, the
plant tissues were powdered or grounded. The best way to rupture the cells of a plant tissue is to
freeze the tissue and pulvarise it using liquid nitrogen in a mortar and pestle. This method is
especially usefull for silicified or highly lignified plant materials. The freezing process burst the
cells, making extraction much easier. The plant tissue may be ground using a mortar and pistle
after adding some washed sand which facilitates the rupture of the cells. Rupturing the tissue
with the addition of a buffer is advised for the extraction of proteins and enzymes. Simply
powdering in a mill will also work. In the case of extraction of plant materials grown by cell
suspension culture methods, a sonicator is an effective technique for breaking the cell structure.
Enzymatic Degradation: The cell structure can be broken down by various enzyme
preparations. For example, cellulase breaks down cellulose of the plant cell wall.

Chemical Degradation: By using powerful chemical agents such as dimethyl formamide the
cell wall can be broken down so as to release all the contents. Detergents are used in some cases
to break molecular association between the substance that is to be extracted and the insoluble
part of the cell. Triton X-100, octyl glucoside and tween-20 are some of the non-ionic detergents
that possess uncharged hydrophilic head. Ionic detergents such as sodium dodecyl sulphate
(SDS) have a charged hydrophilic head.
Precautions to be taken during extraction processes: During the extraction process some
compounds may degrade by the influence of heat, other chemicals and reaction with enzymes
present in the cells and care should be given to minimize the same. On handling water soluble
compounds, specific buffer conditions need to be maintained as the structures are sensitive to pH
changes. In the case of proteins and enzymes specific buffer system has to be employed as a
change may degrade the protein or loss the enzyme activity. Polyphenols present in plant tissues
may be inactivated by complexing with insoluble polyvinyl polypyrrolidine (PVPP), soluble
polyvinyl pyrolidine (PVP) or by maintaining strong reducing environment to counteract the
effect of phenol oxidases.To remove divalent cations from the extracts, chelating agents such as
EDTA may be added. During the extraction of plant material producing intense foams, addition
of suitable antifoaming agents such as DC-544 (Dow Corning) or SAG-30 (Union Carbide) is
essential to control foaming.
Extraction with a Solvent
The ruptured cells are homogenized in the extracting solvent and the mixture is kept for some
time (half an hour to 24 h) to let the solvent penetrate all parts of the ruptured cells. The major
factors to be considered during the extraction process are the nature of the compound to be
extracted, selection of an ideal solvent and effect of temperature. An extraction method is
adopted by ensuring that the complete extraction of the desired compound takes place without
undergoing any decomposition, isomerisation or polymerisation. This demands a thorough
knowledge of the physicochemical properties of the compound of interest and the solvent used.
Stability of the compound in light, heat and in the particular solvent and polarity of the
compound are important determining factors. The extraction may be a batch process where at
every step the extracted solvent is removed and the process repeated, or a countinious process
where the extraction repeates cyclic, such as in the case of Soxhlet extraction and countercurrent
extraction.

Selection of an Ideal Solvent

An ideal solvent dissolves the desired compound, leaving the other constituents.
Polarity: Like dissolves like is the basic principle employed in the selection of an ideal solvent
for the extraction process. The solvents can be arranged in the order of polarity index (Table --).
Polarity index is a relative measure of the degree of interaction of the solvent with various polar
test solutes. For extracting non-polar compounds like fats, oils and lipids non-polar solvents are
used. The extraction of fixed oil, chlorophyll, steroids, terpenoids and aglycones can be effected
by the use of hexane. Extraction of highly polar compounds like glycosides, sugars, aminoacids,
proteins and polysaccharides can be done with polar solvents such as ethanol and water. In the
case of flavonoids, less polar ones such as isoflavones, flavanones, methylated flavones and
flavonols are extracted with low polar solvents such as chloroform, dichloromethane, diethyl
ether or ethyl acetate and the polar flavonoids and flavonoid glycosides are extracted with
alcohols or aqoues alcohol mixtures. Generally the extraction of polar compounds can be done
by using 50% alcohol or with 100% water. As it is difficult to treat the water extract using
common chromatographic techniques, the water or aqueous ethanolic extract is further
partitioned with chloroform, ethyl acetate or butanol for further studies.
Volatility: Also the solvent should be of low boiling point so that it can be easily removed,
without denaturing the compounds extracted at high temperatures.
Selective Extraction Techniques
Modification of the solvent by making it basic or acidic enhances specific extraction processes.
Acidified water preferentially extracts alkaloids, whereas water with basic nature is apt for
phenolic compounds. Thus 5% HCl is used for the extraction of alkaloids, amines and pyrazines,
5% NaOH or 5% KOH preferentially extracts phenolics and lactones and 1% NaHCO 3 is used
for the extraction of carboxylic acids. Methanol can be the best solvent for catechins and 70%
acetone for procyanidins. In the case of extraction of anthocyanins, a slight acidity of the solvent
keeps the anthocyanins in the most stable flavylium forms and thus 0.1% HCl in methanol is
used. 7% acetic acid or 3% trifluoroacetic acid are also used for the extraction of anthocyanins.
The use of mineral acid at high concentration or elevated temperature can lead to the hydrolysis
of acyl groups.
Very soluble: One part of the solute is soluble in less than one part of the solvent.
Soluble: One part of the solute is soluble in 10 or less than 30 part of the solvent.
Sparingly soluble: One part of the solute is soluble in 30 or less than 100 part of the solvent.

Insoluble: One part of the solute can not be dissolved completely in 10000 parts of the solvent.
Table x. Physical properties of common solvents used in phytochemistry
Solvent

Polarity

Refractive

n-Pentane
n-Hexane
Petroleum ether

Index
0.0
0.0
0.0

Index
1.358
1.375
--

36
69
40-60

(low boiling)
Petroleum ether

0.0

--

60-80

0.0
0.2
1.6
2.4
2.5
2.7
2.8
3.1

1.387
1.426
1.466
1.496
1.500
1.501
1.353
1.424

98
81
77
111
139
80
35
41

3.5

1.445

84

(high boiling)
Heptane
Cyclohexane
Carbon tetrachloride
Toluene
Xylene
Benzene
Diethyl ether
Dichloromethane
(Methylene chloride)
1,2-dichloroethane
(Ethylene chloride)
Isopropanol

B.P

Specific
gravity (20oC)
0.659

0.779
1.594
0.867
0.860
0.879
0.714
1.325

Solvent
Toxicity
Most of
the
solvents
that

widely
used

3.9

1.380

82

are
in

0.785

(2-Propanol)
n-Propanol
4
1.380
97
0.804
Tetrahydrofuran
4.0
1.407
65
0.887
n-Butanol
3.9
1.399
125
0.810
Chloroform
4.1
1.443
61
1.486
Ethyl acetate
4.4
1.370
77
0.901
Acetone
5.1
1.359
56
0.791
Methanol
5.1
1.329
65
0.792
Ethanol
5.2
1.361
78
Pyridine
5.3
1.510
0.982
Acetonitrile
5.8
1.344
82
0.782
Acetic acid
6.2
1.372
118
1,049
Dimethyl sulfoxide
7.2
1.477
189
1.101
Water
9.0
1.330
100
phytochemistry are toxic to some extent. It is better to have an idea of the toxicity of the solvents
along with other characteristics such as solubility and polarity. The solvents may act as
neurotoxins by interfering with the functions or structure of specific neural pathways and

systems. The blood brain barrier is a CNS protective system, but lipophilic and low molecular
weight susbastances may diffuse into CNS. Methanol affects the vision, hexane and toluene the
hearing and balance system, toluene the olfactory system. Aromatic amines and hydrazine are
potentially carcinogenic. Even a slight exposure may lead to tumors. Inhaling volatile alkane
solvents for long periods may eventually leads to damage to the lungs by dissolving non-polar
lung cell structures. Recommended limit (RL) for a solvent is the threshold for human exposure,
beyond which it may be harmfull.
The characteristics of some common solvents used in phytochemistry are elaborated here.
Hexane: It is the preferred solvent for extraction of low polar compounds such as lipids, fats,
fixed oils, low polar steroids and terpenoids. Hexane (C 6H8) BP 69oC is identical to petroleum
ether in solubility properties. Petroleum ether is available in the low boiling (BP: 40-60 oC),
which is used for cold extractions and the high boiling (BP: 60-80oC), which is used for hot
extractions and TLC development purposes. Highly polar compounds such as carbohydrates,
acids and proteins are insoluble in petroleum ether.
Benzene: Exposure to benzene for longer periods causes leukemia. Recommended limit (RL) for
benzene is 10 ppm. There should be minimum 75 ppm benzene in atmosphere if we can smell it.
Thus when one smells benzene, it is being inhaled in harmfull quantity. Benzene is replaced by
less volatile and less toxic toluene.
Dichloromethane: DCM has an additional toxic effect because it is metabolized to carbon
monoxide.
Chloroform: The solubility is similar to that of hexane but moderately polar compounds too
dissolve in chloroform. It is the preferered solvent for extraction of alkaloids especially after
changing the pH of the solvent. Chloroform is toxic and carcinogenic in animals and may also
cause reproductive hazards. It is hepatotoxic as it is metabolized to phosgene. Chloroform is less
toxic than CCl4.
Ethyl acetate: It has low toxicity compared to other organic solvents.
Methanol: Except the highly polar polysaccharides and proteins, most of the low polar as well
as medium polar and polar compounds are soluble in methanol. Methanol is metabolized to toxic
formaldehyde and acetic acid by alcohol dehydrogenase and aldehyde dehydrogenase. As little as
15 ml methanol can cause blindness and 70-100 ml is fatal to human beings. Ethanol is given for
reducing the risk of methanol poisoning as the oxidation rate of ethanol is 7-times that of

methanol and more over ethanol has 100 fold greater affinity for alcohol dehydrogenase
campared to methanol.
Ethanol: It is the preferred solvent for extraction of food grade isolates as methanol is toxic. In
the ethanol extract of a fresh plant sample, after decanting the first extract, a second extraction
with fresh ethanol gives surprisingly almost the same or more quantity of the extract as with the
first extraction. This is because, during the first extraction, the ethanol is diluted with water
present in the fresh plant tissue, yielding an aqueous ethanol solution that contains around 40%
water, whereas the less polar constituets are more readily soluble in the second extraction.
Water: It is the preferred solvent for the isolation of polar compounds such as sugars,
polysaccharides, glycosides, aminoacids, proteins and enzymes. Most of the traditional herbal
remedies are aqueous extracts. Chemical modification of water is essential in certain cases such
as for the extraction of alkaloids, where acidified water is used and basic water in the case of
extraction of certain phenolics. Addition of methanol to a water extract precipitate out most of
the proteins and polysaccharides.
Types of extraction processes
Based on the heat applied, extraction process can be classified into hot and cold extractions.
Based on the physical state of the material, the extraction process can be classified in to solid
liquid extraction and liquid liquid extraction.
Effect of Temperature on Extraction Process
Cold extraction: Here extraction process is carried out at room temperature. Percolation,
maceration and super fluid extraction are examples for cold extraction techniques. It is
recommended for thermo-labile compounds.
Hot extraction: The extent of extraction is higher in hot extraction as temperature is applied to
the system. Digestion, reflexion and steam distillation are examples for hot extractions. The
disadvantage of hot extraction is that at high temperatures the volatile components may escape or
polymerization may takes place and many of the alkaloids and proteins decompose during
heating.
Temperature of water bath
Hot water
Warm water
Cold water
Ice bath

: 98-100oC
: 70-80 oC
: 40-50 oC
: 2-10 oC
: below 2 oC

Sublimation: Caffeine easily sublimes and can be isolated from chopped tea leaves by heating in
a crucible, covered with a watch glass or funnel.
Extraction from liquids
When the starting material is in liquid form, partition method is applicable where the distribution
coefficient between the liquid form and solvent is appreciable. It is an example for liquid- liquid
extraction.
Extraction from solids
Maceration: It is the simplest mode of extraction where the powdered plant material is taken in
a stoppered container and soaked with the solvent for a specified period of time until the soluble
portions are dissolved in the solvent. It is an example for cold extraction process.
Percolation: The plant material is taken in a percolation tube (a cone shaped or cylindrical
vessel) plugged with cotton or fitted with a filter and a stopcock. Solvent is added into the plant
material after closing the stopper below and the plant material is allowed to macerate. The whole
system is kept for some time at room temperature and the solvent along with the extracted
material is collected by opening the stopper below. The process is repeated till a drop of the
solvent from the percolator when evaporated does not leave a residue.
Digestion: This is a form of maceration in which gentle heat (40-60 oC) is applied during the
process of extraction. It is used when moderately elevated temperature is not objectionable. The
process may be modified by mixing the material with the solvent using magnetic stirrer,
mechanical stirrer or by shaking occasionally by hand. After 8 to12 hours, the extract is filtered
and fresh solvent is added and the process repeated till all the desired solutes are extracted.
Infusion: In this extraction process, the plant material is macerated for a short period of time
with either cold or boiling water.
Decoction: Here the plant material is boiled in water, cooled and strained. This procedure is
suitable for extracting water soluble and heat stable constituents.
Extraction with boiling solvents (Refluxion): In this hot extraction process, the material is
treated with boiling solvent. The solvent vapor is recycled by a condenser fitted on top of the
container, preferentially a round bottomed flask.
Tincture: It is the extract of plant material in alcohol. Usually the plant material (fresh) and
ethyl alcohol are taken at the ratio of 1:5. Because of the alcohol content, the tinctures can be
stored at room temperatures without being decomposed.

Pressurized Liquid Extraction (PLE): The method is also known as accelerated solvent
extaction system (ASE) or enhanced solvent extraction system (ESE). The method uses elevated
pressure and temperature, where the increased temperature accelerates the extraction process by
increasing the diffusivity of the solvent, whereas the increased pressure keeps the organic solvent
in liquid state without boiling and also forces the solvent to penetrate the matrix pores. The
instrumentation options provide the possibility of working under an inert atmosphere and with
protection from light. More efficient extraction with less solvent at much less time is the
advantages of this process.
Soxhlet Extraction: Named after Franz Ritter von Soxhlet, a German agricultural chemist, it is
the best method for the continuous extraction of a solid by a hot solvent. Soxhlet apparatus is a
specialized glass refluxing unit mainly used for organic solvent extractions. The powdered solid
material is placed in a thimble made up of filter paper and is placed inside the soxhlet apparatus.
The apparatus is fitted to a round bottomed (RB) flask containing the solvent and to a reflex
condenser. The solvent in the RB flask is boiled gently, the vapor passes up through the side
tube, condensed by the condenser and falls into the thimble containing the material and slowly
fills the soxhlet. When the solvent reaches the top of the attached tube it siphons over into the
flask, thus removes the portion of the substance which it has extracted and the process repeats.
The capacity of soxhlet is quoted in terms of the siphoning volume as 100ml, 200 ml, 1 L etc.
Steam Distillation: It is the standard process employed for the isolation of essential oil from
crude plant material. Steam distillation is simple vaporization achieved by passing steam directly
through the material. Here the stem volatile essential oil is recovered by condensation, where oil
separates out from water.
Hydro Distillation: This is the widely used process for isolation of essential oils. The plant
material is soaked in water and boiled using a heating mantle. Due to the influence of hot water
the essential oil is freed from the oil glands in the plant tissues and passes along with the steam.
By using a typical glass apparatus known as Clevenger apparatus, the steam oil mixture is
condensed and oil is separated from water and the condensed water is recycled.
Expression: This is the process of forcibly separating liquids from solids by the application of
pressure. Essential oils from some citrus fruits are isolated by this method.
Enfluerage: This technique is employed for the extraction of delicate fragrances as that of some
flowers. The flower petals are spread over a layer of refined fat which picks up the odor of the

flowers and the saturated fat is treated with a solvent, usually alcohol in which the fragrant
components are soluble. The residual fat dissolved in alcohol may be removed by cooling the
alcohol extract to 20oC, when fat separates out. The volatile components are then recovered from
alcohol by concentrating the solution at reduced pressure in a rotavapor.
Supercritical Fluid Extraction (SFE): Critical point denotes the conditions above which
distinct liquid and gas phases do not exist, but a homogenous supercritical fluid state exists. The
temperature above which a substance can no longer exist as a liquid, no matter how much
pressure is applied is called the critical temperature and the pressure above which the substance
can no longer exist as a gas no matter how high the temperature is raised is called critical
pressure. Supercritical fluid is obtained by heating above the critical temperature and
compressing above the critical pressure. A super critical fluid has the properties of a liquid as
well as that of a gas. The lower viscocities and higher diffusion rates of supercritical fluids
compared to liquids enhances the extraction process. It penetrates the material like a gas under
pressure and can be handled as a liquid. Most commonly used supercritical fluid is CO 2. Other
gases such as ethylene, ethane, propylene, propane and nitrous oxide can also be used. At
supercritical fluid state, both the temperature and pressure equals or exceeds the critical point.
CO2 has a comparatively low critical temperature (31.1oC) and pressure (73.8bar). It is non
inflammable, chemically inert, odor free, easy for disposal and available at low coast with high
purity and also can be recycled. The extraction can be carried out at very low temperature with
CO2. The technique has been in use in industry for decaffeination of coffee and the removal of
nicotine from tobacco. With some modifications such as addition of a polar solvent like methanol
it is a more efficient method than solvent extraction for the extraction of taxol and baccatin from
the Yew tree. Other applications include the extraction of delicate flavour and perfume
chemicals. In this process the major advantage is the application of mild conditions, which avoid
the risk of thermal degradation compared to distillation and solvent extraction.
Ultrasonic Extraction: Here phytochemicals are liberated from the plant tissues by high
frequency sound, which damage the cell wall. Ultrasound assisted extraction can be used with
mixtures of immiscible solvents such as hexane with methanol/water. The process creates heat so
that heat labile compounds may decompose. In such cases the extaction container is placed in ice
bath to reduce the temperature. The method is not apt for isolation of large molecules like
proteins or DNA.

Microwave Assisted Extraction: Microwaves are electromagnetic radiations with the frequency
range 0.3 to 300 GHz and domestic microwaves generally operate at 2.45 GHz to avoid
intereference with radio communications. Microwave energy is applied to the sample suspended
in solvent, with short intervells of cooling time as this process creates much heat. The electric
field of the electromagnetic radition causes heating of substrates through dipolar rotation and
ionic conduction. With an increase in the dielectric constant of the solvent, the heating also
increases. The yield of the extract is comparable to Soxhlet extraction, but in much less time and
microwaves heat the whole sample simultaneously. Microvaves disrupt the weak hydrogen bonds
and in some cases, the matrix interacts with microwaves while the solvent with low dielectric
constant remains cold, thus facilitating the extraction of thermolabile compounds.Microwave
applications for the laboratory purposes should be provided with security measures such as
exhaust fans and solvent vapour detectors and the domestic microwave ovens are not advised for
laboratory purposes.
Solid Phase Extraction (SPE): This is a rapid, economical and sensitive technique that uses
different types of cartridges and disks, with a variety of sorbents, where the solute molecules are
preferentially attached over the stationary phase. Sample preparation and concentration can be
achieved in a single step. Nomal phase, reverse phase and ion exchange solid phase extraction
units are available. For example, with Sep-Pak C18 cartridges (reverse phase) it is possible to
remove polar components whereas the retained low polar ones can be eluted later.
Static head space sampling
Dynamic head space sampling: It is also very similar to the purge and trap technique except
that the incoming gas supply is introduced into the headspace rather than made to bubble through
the sample.
Solid Phase Microextraction (SPME): Developed in the early nineties, this sampling technique
does not require any solvent for extraction and is based on adsorbent fibers. SPME can be
combined to GC or HPLC. On-site sampling can be done with SPME and the properly stored
samples can be analysed days later. The technique is especially usefull for the study of volatiles.
Instead of using carrier gas to sweep or pulse the headspace vapor out of the sample vial into
some sort of trapping device, SPME essentially inserts a trap into the headspace vapor inside
the vial. This trap is normally implemented in the form of a retentive coating applied to a
narrow fused silica fiber which is located within the needle of a special syringe as shown in

Figure 33. The fiber is drawn back into the syringe needle which itself is withdrawn from the vial
and inserted into a heated GC inlet. The fiber is extended and absorbs heat from the injector liner
which desorbs the extracted analytes and carrier gas transfers them to the GC column for
analysis. The SPME apparatus is a very simple device. It looks like modified syringe (Fig. 2)
consisting of a fibre holder and a fibre assembly, the latter containing a 12 cm long retractable
SPME fibre. The SPME fibre itself is a thin fused-silica optical fibre, coated with a thin polymer
film (such as polydimethylsiloxane (PDMS)), conventionally used as a coating material in
chromatography. After sampling, the fibre is retracted into the metal needle (for mechanical
protection), and the next step is transfer of the analyte from the fibre into the chromatograph. Gas
chromatography (GC or GC/MS) is one of the preferentially used techniques. In this case,
thermal desorption of the analyte takes place in the hot GC injector. After inserting the needle
into the injector, the fibre is pushed outside the metal needle. The other common option is
analysis by HPLC (HPLC/MS). In this case the needle is placed into a modified Rheodyne or
Valco valve. The fibre is exposed and the analytes are eluted by the mobile phase.
Chromatography and detection (often by ms) take place in a conventional manner.
The common non polar liquid adsorbent phases are polydimethyl siloxane (PDMS), divenyl
benzene (DB), while polyacrylate (PA) and carbowax (CW) are polar adsorbants.
Head space-solidphase microextraction: HS-SPME is the most sought after technique for
capturing delicate volatile compounds emitted from plants. HS-SPME has assumed ever
increasing importance in the field of chemical ecology and fragrance industries. The technique
has advantages over the existing processes such as hydrodistillation like shorter sampling time,
field analysis, repeated and cost effective analysis.
Sequential and selective extraction: In sequential extraction, the extraction is carried out on the
same plant material successively in the order of polarity of the solvent. It is also known as
successive extraction. In selective extraction a particular solvent is used for the extraction and
once the extraction is over, fresh plant material is used for further extraction with other solvents.
Concentration of the Extract
The heating bath temperature is recommended to be around 20-30 oC higher than the boiling
point of the solvent. Usually the extract is concentrated on a water bath if the solvent is having
boiling point below 80oC, whereas a heating mantle, sand bath or hot plate is used for higher
boiling solvents.

Rotary Evaporator: If the compound is thermo labile, removal of the solvent is carried out on a
rotary evaporator (rotavapor), where by applying vacuum (reducing pressure) boiling point of the
solvent is reduced. Based on the equation, PV = nRT, a reduction of pressure lowers temperature.
The principle of rotavapor is the reverse to a pressure cooker, where by increasing the pressure,
the boiling point is increased. Boiling of a liquid takes place when the vapor pressure equals
atmospheric pressure and thus at reduced pressure only less vapor pressure is needed for boiling
and hence solvents boils at reduced temperature. At a vacuum of 72 mbar, water boils at 40 oC,
ethanol boils at 40oC at a vacuum of 240 mbar and butanol boils at 40oC at 25 mbar. The boiling
depends on heat of vaporization, molecular weight, density etc. The rotation improves heat
transfer, reduces the possibility of bumping of the solvents. During the rotation, the solvent
forms a layer on the inner surface of the flask, thus increasing the surface area which ultimately
increases the rate of evaporation. Rotavapor with diagonal condensers are used for simple
distillations whereas vertical condensers are employed for high boiling (>100 oC) solvents. Dry
ice and acetone are used as coolants in special applications involving low boiling solvents.
Methanol, ethanol or isopropanol are used as antifreeze agents when very low temperature is
needed.
Evaporator Centrifuge: It uses vacuum to evaporate solvents. The centrifugal force applied to
the samples prevents the bubling of the solvents. The evaporation of the solvent reduces the
temperature and the system has to be heated for compensating this loss of energy.
Freeze Drying (Lyophilization): To concentrate the water extract that contain thermo labile
entities such as proteins, antibiotics and enzymes the method of freeze drying is used. Here, in
principle the aqueous solution is frozen and the ice is sublimed off to leave a dry residue. The
process involves three steps. In the first step the material is cooled below its triple point and the
temperature is usually at the range 50 to 80 C. During the primary drying phase, where about
95% of the water in the material is sublimated, the pressure is lowered to a few millibars. The
sublimed water vapor is condensed on a cold surface (typically -50oC). The secondary drying
phase aims to remove any water molecules remaining attached to the frozen material and the
temperature is raised higher than in the primary drying phase (above 0C), to break the
attachments of water molecules and the pressure is still lowered in this stage to encourage
desorption (typically in the range of microbars). The energy for vaporization is taken from the
material and thus eventhough the material is kept exposed to room temperature, it remains frozen

till all the moisture escapes from the sample. When all the water was removed from the sample,
it reaches the surrounding atmosphere temperature. This is an indication of the completion of the
water removal. At the end of the operation, the final residual water content in the product is
extremely low, around 0.5%.
Preservation of the Extract
The factors that adversely affect the extract on storage are moisture, temperature, light, presence
of oxygen and mico organisms. If water contamination is suspected at any stage of an organic
solvent extraction process, a suitable drying agent such as anhydrous sodium sulphate, calcium
chloride, P2O5, CuSO4, K2CO3, Sodium, CaO, MgSO4, Molecular sieves (0.3mm, 0.4mm) may be
used for removing any traces of water. A few drops of toluene may be added to prevent fungal
growth in the extracts containing water. Darkening of the extract on standing occurs sometimes
because of the oxidation of phenolic substances by polyphenol oxidases present in the plant
tissue. Keeping the extract in N2 atmosphere prevents the decomposition to some extent and
addition of a reducing substance such as cysteine (1%, pH7) check the darkening to some extent.
To prevent decomposition of any compounds during storage, the extract may be stored in a
refrigerator or deep freezer or may be hermetically sealed (air tight and prevent moisture and
microorganisms)
Contemperory and Traditional Methods of Extractions
Extraction techniques have been used very effectively in traditionall medicinal practices and day
to day activities. Preparation of tea is the best example where hot water is used for the extraction
of the active constituents. The age old extraction process of herbal dyes also uses water as the
medium.Ayurvedic extractions in water, milk, oil and alcohol are other examples for traditionally
used extraction techniques. In traditional methods of extractions, mostly decoctions are prevelant
rather organic solvent extracts. Therefore the pharmacological screening of the traditional herbal
preparations using organic solvents may seldome bring the correct inference. Milk is an emulsion
of oil in water. The lipophilic, nonpolar constituents are extracted in the oil whereas water
extracts the polar constituents. Alcoholic extracts that contain residual alcohol is known as
tinctures. Alcohol dissolves both ionic and organic compounds. The ancient ayurvedic experts
were well versed in modifying the solvent for getting the exact dose of the active constituent. For
example, plumbagin is the active constituent in the rhizomes of Plumbago rosciae. At the

concentration present in the plant, it is toxic to human beings, but the modified extraction
process using cow urine or lime in water, plumbagin is extracted at the optimal level.
Scheme for general extraction
Plant powder
Sequential extraction with low polar solvent > medium polar solvent > high polar solvent
Scheme for polar extraction
Plant powder
Defatting by extraction with petroleum ether > extraction with 95% or 50% alcohol
Partition with chloroform (less polar compounds) > butanol (medium polar compounds)
Plant powder
Extraction with water > partition with butanol
Plant powder
Extraction with water.The aqoues extract after lyophilisation can be separated to
methanol (medium polar compounds) and water soluble (polar) fractions.