In that topic their is describe the different types of Extraction Methods, Parameters for Selecting appropriate Extraction methods, types of Extract, types of Separation techniques, types of distillation, chromatographic techniques.
2. 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
4. EXTRACTION PROCESS
• Definition:
• 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 Menstrum
and the inert insoluble material that remains after
extraction is called Marc
5. Strategies
1. Older strategies:
a. Focus on chemistry of compounds from natural sources, but not
on activity.
b. Straightforward isolation and identification of compounds from
natural sources followed by biological activity testing (mainly in
vivo).
c. Chemotaxonomic investigation.
d. Selection of organisms primarily based on ethnopharmacological
information, folkloric reputations, or traditional uses.
6.
7. 2. Modern strategies:
a. Bioassay-guided (mainly in vitro) isolation and identification of active ‘‘lead’’
compounds from natural sources.
culture, genetic
b. Production of natural products libraries.
c. Production of active compounds in cell or tissue
manipulation, natural combinatorial chemistry, and so on.
d. More focused on bioactivity.
e. Introduction of the concepts of dereplication, chemical fingerprinting, and
metabolomics.
f. Selection of organisms based on ethnopharmacological information, folkloric
reputations, or traditional uses, and also those randomlyselected.
8.
9. Extraction
• It can be defined as the process of obtaining the
constituents by separating it from the crude drug
by the use of solvent/s.
• Powdered material is extracted with suitable
solvent or mixture of solvents for extracting the
various phytoconstituents present in the crude
drug.
10. • Products obtained from plants are impure
liquids, semisolids or powders
• Such product are decoctions, infusions, fluid
extracts, tinctures, pilular (semisolid) extracts
and powdered extracts, called galenicals, named
after Galen, the second century Greek physician.
11. • Process of extraction is controlled by mass
transfer
• When a drug particle is immersed in a solvent,
the particle first surrounded by a boundary layer
of the solvent and the solvent starts penetrating
inside the particle and forms solution of
constituents within the cells.
12. • Constituents escapes from the cell wall
and gets dissolved in the boundary layer of
solvent.
• The process is continue till equilibrium is
set up between the solution of cell and the
free solution.
14. There is no general (universal) method for the
extraction of plant materials.
• The precise mode of extraction depends
on:
• The texture of the plant material.
• The water content of the plant material.
• The type of substances to be extracted or nature of
active constituents.
15. • The typical extraction process, especially for plant
materials incorporates the following steps:
• Drying and grinding of plant material or
homogenizing fresh plant parts (leaves, flowers, etc.) or
maceration of total plant parts with a solvent.
• Choice of solvents
• Polar extraction: water, ethanol, methanol (MeOH), and so on.
• Medium polarity extraction: ethyl acetate (EtOAc),
dichloromethane
(DCM), and so on.
• Nonpolar: n-hexane, pet-ether, chloroform (CHCl3), and so on.
16. The principal methods of extraction are:
• Maceration
• Percolation
• Infusion
• Decoction
• Digestion
• Continuous hot extraction techniqu (Soxhlet extraction process).
• Liquid-liquid extraction
• Solvent-solvent ppt.
• Distillation
17. • Choice of Solvent
As a general empirical rule
Non polar solvents (petroleum ether and hexane) will dissolve
non-polar compounds (fats and waxes).
While polar solvents (methanol, ethanol and water) dissolve
polar compound (alkaloid salts and sugars).
• Polar extraction: water, ethanol, methanol (MeOH), and so on.
• Medium polarity extraction: ethyl acetate (EtOAc),
dichloromethane (DCM), and so on.
• Nonpolar: n-hexane, pet-ether, chloroform (CHCl3), and so on.
18. • The affinity of the solute for the organic phase
may be greatly increased by using mixture of
solvents instead of single ones.
• (sometimes used mixtures of solvent to
increase the solubility).
19. • Example: solublization of an aliphatic carboxylic acid in
ethanol, acetone and a mixture of both.
In acetone R-C
O------------H-O-CH2-CH3
In ethanol -R-C
O-H
O
CH3
O-H---------O=C
CH3
20. • In a mixture of acetone and ethanol
R-C
OH
O HO-C2H5 (ethanol)
21. Solvent chosen for extraction is expected to possess
the following properties:-
• Should dissolve the phytoconstituents
• Non-inflammable
• Inert
• Non-toxic
• Easily removable
• Should be distilled or double distilled.
Solvent chosen depending upon the characteristic of the secondary
metabolites. Polarity, pH and thermo stability of the constituents are
also to be considered during extraction.
23. Maceration
• 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.
24. Infusion
Powdered drug is extracted with hot or cold water.
• Infusion:-
In this method, the powdered drug is soaked in hot
water at room temperature for the specified period
with or without stirring and filtered.
If necessary, mark again extracted with fresh water.
25. 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.
26. Decoction
• 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, heat-stable constituents.
• This process is typically used in preparation of
Ayurvedic extracts called “quath” or “kawath”.
27. • 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.
28. Percolation
• This is the procedure used most frequently
to extract active ingredients in the
preparation of tinctures and fluid extracts.
• A percolator (a narrow, cone-shaped vessel
open at both ends) is generally used.
29.
30. • The solid ingredients are moistened with
an appropriate amount of the Specified
menstruum and allowed to stand for
approximately 4 h in a well closed
container, after which the mass is packed
and the top of the percolator is closed.
31. • 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.
32. • 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
required
about three-quarters of the
volume of the finished product.
33. • 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.
34. Hot ContinuousExtraction
• In this method, the finely ground crude
drug is placed in a porous bag or “thimble”
made of strong filter paper, which is
placed in chamber E of the Soxhlet
apparatus.
• The extracting solvent in flask A is heated,
and its vapors condense in condenser D.
35. • The condensed extractant drips into the
thimble containing the crude drug, and
extracts it by contact.
• When the level of liquid in chamber E rises
to the top of siphon tube C, the liquid
contents of chamber E siphon into fl ask A.
36. • 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.
37. • This effects tremendous economy in terms of
time, energy and consequently financial inputs.
• At small scale, it is employed as a batch process
only, but it becomes much more economical
and viable when converted into a continuous
extraction procedure on medium or large scale.
38.
39.
40. Distillation
This method is most suitable for extraction of essential
oils(volatile oil).
• The plant material is heated to boiling with water in a
distillation unit. The vapors of the volatile oil component
are allowed to condense along with the vapors of the
steam. The oil separates from water upon cooling.
• This method is not suitable for volatile compounds which
deteriorate because of prolonged boiling at high
temperature.
41. • In hydro-distillation, steam is generated and
passed through the plant material suspended in
water, steam vaporizes the volatile components,
condensed and separated.
• In direct steam distillation, the material to be
extracted is spread on a mesh as thin layers and
the generated steam is passed through the
material.
42. Ultrasoundextraction
• Sound waves above 2000 Hz are used to
accelerate the extraction.
• Waves are generated in a magnetic transmitter
or piezoelectric transmitter.
• Increases cell wall permeability and cause
mechanical stressing of the cells.
43. Extraction by electricalenergy
• Electric energy accelerates the extraction and
improves the yield.
• Electromagnetic field is created in the drug
powder for extraction.
• This method is used for the extraction of
scopolamine from stramonium and valerianic
acid from valerian.
44. Counter currentextraction
• This is a liquid-liquid extraction process & is
based on the principle of partition
chromatography.
• It is developed by Craig in 1944.
• The apparatus consists of a series of tubes
connected and contain a lower, stationary phase
and an upper moving immiscible liquid.
45. • The mobile phase can be transferred from one
tube to another after shaking & allowing to
separate.
• The extract is placed in the first tube and the
apparatus is agitated and the layers are allowed
to separate.
46. • The components of a mixture will be distributed
between the two layers according to their
partition coefficients
• The upper moving phase is moved along to the
second tube containing the lower phase.
• More moving phase is brought into contact with
the lower phase of tube 1.
• The process is repeated and fraction is done.
49. • The distribution of each substance over given
number by the tubes can be increased by the
use of series of buffer solution of graded pH.
• This method is best suited for separation than
extraction.
• Extraction of alkaloids, amino acids, phenols,
antibiotics, anthraquinones, cardiac glycosides,
prostaglandins, vitamins, steroids etc.
50.
51.
52.
53.
54.
55. Supercritical fluidextraction
• The critical point of a pure substance is defined
as the highest temperature and pressure at
which the substance can exit in vapor-liquid
equilibrium.
• A temperature and pressure above this point a
single homogenous fluid is formed & said to be
super critical.
56. • Super critical fluid can dissolve wide variety of
organic compounds and their solvent power can
be varied near their critical points by small
pressure and temperature changes.
• Super critical fluids possess superior mass
transfer properties by virtue of their low
viscosity and high solute diffusivity along with
the ability to penetrate microporous materials.
57. • Super critical dioxide (SCO2) with its particular
attractive properties such as
• Non-toxic, non-flammable, non- corrosive, inert,
low critical temperature (304°K), moderate low
critical pressure (73 atm), easy availability, cost
effectiveness, environment acceptability is the
preferred solvent for many super critical fluid
extraction.
58. • Advantages:-
• No environmental pollution
• No solvent residue in the product.
• Permit the low temperature process and thermolabile, delicate,
expensive fragrances can be extracted.
• Non-corrosive in nature.
• Disadvantages:-
• Use of high temperature & high pressure may cause problems in
some cases.
59. • Application:-
• Pyrethrins from pyrethrum flower heads. (Increase up to 60%)
• Decaffeination of green coffee.
• Isolation of bitter principles from hops.
• Isolation of vindoline from Catharanthus roseus.
• Extraction of taxol from Taxus brevifolia & T.cuspidata.
• Extraction of pigments from annatto seeds.
• Volatile oil from rose petals, rosemary, coriander, illicium, juniper,
pimento etc.
60. Aqueous Alcoholic Extractionby
Fermentation
• Some medicinal preparations of Ayurveda (like
asava and arista) adopt the technique of
fermentation for extracting the active principles.
• The extraction procedure involves soaking the
crude drug, in the form of either a powder or a
decoction (kasaya), for a specifi ed 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.
61. • The alcohol thus generated also serves as a
preservative. If the fermentation is to be carried
out in an earthen vessel, water should first be
boiled in the vessel.
• In large-scale manufacture, wooden vats,
porcelain jars or metal vessels are used in place
of earthen vessels.
62. Solid phase microextraction
• Suitable for some drugs containing volatile oil
using methyl polysiloxane solid phase micro
extraction fibers.
• T.J. Betts extracted volatile oil from fresh cut
eucalyptus leaves (37 °C for 10 min.). Fibers were
then desorbed at 200°C by capillary gas
chromatography of the oil. Composition of oil
obtained is greatly differ from the oil obtained by
conventional steam distillation.
63. Advanced phytonics methodof
extraction
• This method is used for the extraction of volatile
oils.
• Some substances are gaseous at room
temperature & atm. Pressure. By changing the
pressure, they become liquids of low viscosity
and have good penetration power. 1,1,1,2
tetrafluroethane is a gas and can be liquefied.
This can be used for extraction fluid & is called
‘Phytosols’.(solvents which can solubalize
phytoconstituents from the drug.)
64. • Advantages:-
• Phytosols are better than steam distillation, solvent extraction
and SCFE & the temperature of extraction is not too high.
• Cost of production is low.
• Quality of essential oil is better & also the yield is very high.
65. Expression
• This method is adopted for the extraction of
fixed oils from the seeds and other part of plant.
• Mechanical pressure is applied to the crushed
material of the plant part either in cold or at
specified temperature to express the oil.
Hydraulic pressure can be used for extraction.
66. Enfluragemethod
• This method is used for extraction of delicate oils
from the petals.
• Solvent used for the extraction of the oils is a fat
which dissolves the oils from the material upon
prolonged contact. Fat is applied onto the glass plate
as a thin layer and petals are spread on this. Volatile
oils gets in to the fatty layer slowly and when once
the petals are exhausted, fresh petals are spread.
This continued till the fatty layer is saturated with
volatile oil. Dissolved oil is separated from the fatty
layer can be preserved for a longer time before
being separated.
67. • Application:-
This method has been successfully tried out for
the extraction of Rose oil from Rose petals.
68. MicrowaveTreatment
• Enhanced recovery of certain secondary
metabolites can be achieved if the plant material
is treated with microwave irradiation before or
during on extraction procedure.
• It is believed that heating of water in the
material causes generation of steam, which can
result in opening of the matrix and improved
porosity.
69. Pervaporation
• In this method, substances dissolved in a liquid
phase are removed by binding on to a
membrane.
• Hydrophilic membranes may be used to remove
polar materials, including water from the organic
solvents and, conversely hydrophobic
membranes can be used to remove organic
compounds from aqueous phase.
70. • Application:-
• This method has been used to remove aroma compounds from
fruit juices.
• This method is currently being developed, and its success will
depend on the generation of new membranes which show
selective binding for particular chemical groups, perhaps even for
specific enantiomers.
71. Sublimation
• In this process some substances, on heating or
cooling, changes from solid to gas or vice versa
without passing through a liquid state. This
property can be used to obtain the substance
from dried plant material or a dry crude extract.
72. • Plant material is warmed & the vapor allowed to
come into contact with a cold surface. The
subliming substance vaporizes from the plant
material & condensed as a solid on the cold
surface, from which it can be scrapped off.
• Application:-
• Caffeine of high purity can be obtained by this method from dry
tea leaves.
74. • Authentication of plant material should be done
before performing extraction. Any foreign matter
should be completely eliminated.
• Use the right plant part and, for quality control
purposes, record the age of plant and the time,
season and place of collection.
75. • Conditions used for drying the plant material
largely depend on the nature of its chemical
constituents. Hot or cold blowing air fl ow for
drying is generally preferred. If a crude drug with
high moisture content is to be used for
extraction, suitable weight corrections should be
incorporated.
76. • Grinding methods should be specifi ed and
techniques that generate heat should be avoided
as much as possible.
• Powdered plant material should be passed
through suitable sieves to get the required
particles of uniform size.
77. • Nature of constituents:
• If the therapeutic value lies in non-polar constituents, a non-polar
solvent may be used. For example, lupeol is the active constituent
of Crataeva nurvala and, for its extraction, hexane is generally
used. Likewise, for plants like Bacopa monnieri and Centella
asiatica, the active constituents are glycosides and hence a polar
solvent like aqueous methanol may be used.
78. • If the constituents are thermolabile, extraction methods like cold
maceration, percolation and CCE are preferred. For thermostable
constituents, Soxhlet extraction (if nonaqueous solvents are used)
and decoction (if water is the menstruum) are useful.
• Suitable precautions should be taken when dealing with
constituents that degrade while being kept in organic solvents,
e.g. fl avonoids and phenyl propanoids.
79. • In case of hot extraction, higher than required temperature
should be avoided. Some glycosides are likely to break upon
continuous exposure to higher temperature
80. • Standardization of time of extraction is important, as:
• Insuffi cient time means incomplete extraction.
• If the extraction time is longer, unwanted constituents may also be
extracted. For example, if tea is boiled for too long, tannins are
extracted which impart astringency to the fi nal preparation.
• The number of extractions required for complete extraction is as
important as the duration of each extraction.
81. • The quality of water or menstruum used should be specified and
controlled.
• Concentration and drying procedures should ensure the safety
and stability of the active constituents. Drying under reduced
pressure (e.g. using a Rotavapor) is widely used. Lyophilization,
although expensive, is increasingly employed.
82. • The design and material of fabrication of the extractor are also to
be taken into consideration.
• Analytical parameters of the fi nal extract, such as TLC and HPLC fi
ngerprints, should be documented to monitor the quality of
different batches of the extracts.
84. In order to extract medicinal ingredients
from plant material,
the following sequential steps are involved:
1. Size reduction
2. Extraction
3. Filtration
4. Concentration
5. Drying
86. • Numbers of different types of methods are used
for the extraction of herbal drugs, and the
extracts are used for different purposes ranging
from internal administration, external use, for
further purification of phytopharmaceuticals or
for it semisynthetic conversion to some
therapeutically more active compounds.
• The extracts are therefore prepared likewise to
achieve the objectives for which it is prepared.
87. • Extracts can be in the form of aqueous, hydroal-
coholic types in the form of infusion, decoction,
tinctures, etc., or they can be more concentrated
which may further be transformed into soft, dry
or liquid extracts.
88. AqueousExtracts
• These are the extracts which are medicinal
preparations intended to be used immediately
after preparation or to be preserved for use.
• The following methods are generally more in
utility for their preparation.
89. • Decoction:
• This is the ancient and more popular process of extracting water
soluble and heat stable constituents from crude drugs by boiling
in water for about 15 min.
• The boiled crude drug—water mixture is then cooled; filtered and
sufficient volume of cold water is passed through the drug to
produce the required volume.
90. • Infusion:
• An infusion is generally a dilute solution of the readily soluble
constituents of crude drugs.
• It is nothing but a type of periodic maceration of the drug with
either cold or boiling water.
• The infusion is filtered to remove the crude vegetable material
and then produced in a required volume by addition of water.
91. • Digestion:
• Digestion is also a type of maceration in which
moderate heating is preferred during extraction.
• Heating causes the digestion of drug material
and increases the solvent efficiency.
• It is preferred for the drugs in which the use of
moderately elevated temperature does not
cause the degradation of constituents.
92. • Tinctures:
• Tinctures are the alcoholic or hydroalcoholic solutions prepared
from crude drugs or from the pure organic or inorganic
substances.
• Tinctures of crude drugs may contain 10–20 g of drug per 100 ml
of tincture.
• The methods used for the preparation of tinctures are:
maceration and percolation. Iodine tincture is an example of
inorganic pharmaceuticals, belladonna tincture is prepared by
percolation while compound benzoin tincture, sweet orange peel
tincture are prepared by maceration.
93. • Liquid Extracts:
• The liquid extracts are also termed as fluid extracts in some
official books like USP.
• It is a liquid preparation of crude drugs which contain ethyl
alcohol as a solvent and preservative.
• It may contain active constituents to the extent of 1 g of drug per
ml.
• Pharmacopoeial liquid extracts are prepared by the percolation
or modified percolation techniques.
94. • Soft Extract:
• The extracts which are produced as semisolid or liquids of syrupy
consistency are termed as soft extracts.
• These extracts are used in the variety of dosage forms ranging
from ointments, suppositories or can be used in the preparation
of some other pharmaceuticals.
• Glycyrrhiza extract USP comes in the form of soft extract.
95. • Dry Extract:
• Dry extracts are also known as the pow- dered extracts or dry
powders.
• The total extracts obtained by using suitable process of
extraction, are filtered, con- centrated preferably under vacuum
and dried completely.
• The tray drying or spray drying is used for making dry extracts.
Just like soft extracts, these powdered extracts can be used for
the manufacture of some medicinal preparations.
• Powdered extracts are preferably used into a solid, dry dosage
forms like capsules, powders or tablets.
• The Belladonna extract, Hyoscyamus extract are the official dry
extracts.
96. Concentration
• The extract contains
huge solvent quantity
and small amount of
active molecule.
• Concentration:
• Evaporate the solvent and
enrich the active content
• Concentration under
film
vacuum, Thin
evaporator, Freeze
103. Floatation
•Is a separation method in which some
solids of a suspension mixture are
allowed to settle and the less dense
material is poured off.
104. Centrifugation
•Speeds up the separation of a solid
from a liquid through the centrifugal
force developed during the rotation
of the centrifuge.
•Ex. Complete blood count (CBC)
105.
106. Distillation
•This is the separation technique
where two miscible liquids are
separated.
•It is made possible due to the fact
that each liquid has its unique boiling
point.
108. SimpleDistillation
•This is a technique
used to separate a
mixture of a soluble
substance and a
solvent.
•E.g salt and water
109. Fractionaldistillation
•This is the technique used to separate
a mixture of two miscible liquids with
different boiling points.
•E.g. water and ethanol
•The boiling point for water is 100
while for ethanol it is 78.
110.
111. FractionalCrystallization
•Crystallization is the process in which
crystals are formed.
•Fractional crystallization is done by
lowering of temperature of a mixture
or solution so that the more insoluble
component crystallizes out first.
113. Introduction
• Chromatography basically involves the separation of
mixtures due to differences in the distribution coefficient
(equilibrium distribution) of sample components
between 2 different phases.
• One of these phases is a mobile phase and the other is a
stationary phase.
• 1903 Tswett - plant pigments separated on chalk columns
114. • The selection of a particular chromatographic
technique depends upon the nature of material
to be isolated and often several chromatographic
techniques may be used in sequence to achieve
complete purification.
123. LIQUID COLUMNCHROMATOGRAPHY
• A sample mixture is passed through a column packed
with solid particles which may or may not be coated with
another liquid.
• With the proper solvents, packing conditions, some
components in the sample will travel the column more
slowly than others resulting in the desired separation.
124. A + B + C
OOOOOOOOOOO
OOOOOOOOOOO
O O O O O O O O O O
OOOOOOOOOO O
OOOOOOOOOOO
O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O
OOOOOOOOOO O
OOOOOOOOOO O
O O O O O O O O O O
OOOOOOOOOOO
OOOOOOOOOO O
O O O O O O O O O O
OOOOOOOOOO O
OOOOOOOOOO O
O O O O O O O O O O
OOOOOOOOOO O
OOOOOOOOOO O
O O O O O O O O O O
OOOOOOOOOOO
OOOOOOOOOO O
O O O O O O O O O O
OOOOOOOOOOO
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O O O O
O O O O O O O O O O O
Sample
(A+B+C)
Column
Solid Particles
O O O O O O O O O O O (packingmaterial-
stationary phase)
Eluant (eluate)
OOOOOA O O O O
O O O O O O O O O O
OOOOOB O O O O
OOOOOOOOOO
OOOOOC O O O O
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
Solvent(mobile or
moving phase)
127. Principle
• The technique is simple, quick and allows a large
number of samples to be analyzed at one time.
• The technique can be used both for analytical
and preparative purposes.
128. Thin layerpreparation
• A slurry of stationary phase usually suspended in
water is applied to glass, plastic or foil plate
uniformly either manually or by using a spreader.
• For analytical uses, thickness of slurry is usually
0.25 mm and for preparative purposes, up to 5
mm.
• Where stationary phase is to be used for
adsorption chromatography, a binding agent
such as CaSO4 is incorporated into the slurry to
facilitate adhesion of the adsorbent to the plate.
129. • With exception to permeation chromatography,
after preparing the plates, they are heated to
leave the coating of stationary phase.
• In case of adsorbent drying is carried out in an
oven at 100-120oC. This also serves to activate
the adsorbent.
130. • Series of spots forms
• Compare samples in
mixture with known
substances.
• Measure Rf values.
• Coloured compounds &
colourless compounds.
131.
132. Sample application
• Sample in small quantities is applied by
micropipette and briefly dried in oven or with
the help of hair dryer where applicable.
• Spot size should be as small as possible and
therefore when a larger volume is to be loaded
sample is applied in small volumes, dried and
applied further.
• Sample is applied on plate at a position that it is
not dipping in the solvent.
133. Platedevelopment
• The solvent is filled in glass chamber to a
suitable height and filter paper is wrapped all
around the inner wall so as to saturate the
chamber with solvent vapours.
• The chamber is covered with air tight lid. It is
then left for several hours for equilibration with
solvent vapours.
• The plate loaded with sample is then placed in
the chamber. Chromatography is carried out at
moderately low and constant temperature.
134. Detection
• Spraying the plate with 50% H2SO4 or 25% H2SO4
in ethanol and heating will result in most
compounds becoming charred and giving brown
spots.
• Observation of plate in ultraviolet light will
reveal UV absorbing and fluorescent solutes.
• Subjecting the plate to iodine vapor will reveal
unsaturated compounds.
• Spraying of plate with specific colour reagents
will stain certain compounds.
135. Identification ofcompounds
• Although movement of compounds on TLC
plates can be found out by Rf values
• Identification of components separated by TLC is
more reliably done by comparing the
movements with reference compounds
chromatographed alongside the sample on the
plate.
136. Quantitation
• Quantitation may be achieved by use of
radiochromatogram using radiolabelled
compounds or generally by means of
densitometry.
• There are several other methods for quantitation
on plate such as measuring area of the spot and
comparing it with that of the reference.
• The developed spots may be scrapped off,
suspended in suitable solvent and assayed
colorimetrically after removing the gel.
138. • PC is applicable to water-soluble plant
constituents namely carbohydrates, amino acid,
nucleic acid base, organic acid and phenolic
compound
139. Principle
• Cellulose fibres of chromatography paper act as
the supporting matrix to the stationary phase
and separation occurs mainly by partition
chromatography, but adsorption also plays
significant role.
• This chromatography is done by either ascending
or descending of solvent on the paper.
140. Development
• There are three techniques employed for
developing paper chromatograms i.e. ascending,
descending and horizontal.
• In all the cases chamber should be air tight and
saturated with solvent vapours.
141. • Ascending chromatography is preferred for its
simplicity, in descending mode solvent flow is
faster.
• In horizontal method solvent flow is no way
affected by gravitation.
• Two dimensional chromatography on paper can
be performed in a manner similar to that of TLC
142. Componentdetection
• Identification of the spots can be made on the
basis of their Rf values which is constant for the
given solute under standard conditions.
144. Introduction
• Separation in Ion-exchange Chromatography is
based on the competition of different ionic
compounds of the sample for the active sites on
the ion-exchange resin (column-packing).
145. principle
• This technique is used for the separation of
charged solutes such as proteins and amino
acids.
• The separation is based on the differences in the
sign and magnitude of charge on the solutes
undergoing separation.
• Ion exchangers are either highly insoluble acids
or bases.
• Acids have positively charged exchangeable groups and are thus
called cation exchangers
• Bases have negatively charged exchangeable groups and are
called anion exchangers.
146. • When a solute mixture containing solutes having
different magnitude of positive charge is passed
through a bed of cation exchanger, positively
charged exchangeable groups are exchanged for
counter ion on the exchanger.
150. introduction
Gel-Permeation Chromatography is a mechanical sorting of molecules
based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.
151. Principle
• Separation of molecules on the basis of their
molecular size and shape uses molecular sieve
properties of a variety of porous materials.
• These polymeric organic compounds possess a
three dimensional network of pores which
confer gel properties upon them.
• Gels of various organic substances such as cross
linked dextran, agarose and polyacrylamide are
available.
152. • The pores are funnel shaped and therefore a
molecule larger than the size of the pore will
not be able to enter into the pore and will
move along with the solvent and come out of
the column first.
• Molecules of decreasing order of size will be
eluted in increasing order because smaller the
size deeper they will be able to enter into the
pores and thus their movement through the
column will be more restricted and they will
come out of the column with passage of larger
volume of eluent.
153. • The main application of gel filtration
chromatography is the purification of labile
biological macromolecules. Viruses, proteins,
enzymes, nucleic acids, hormones, antibodies,
polysaccharides all have been purified by this
technique.
156. introduction
• This separation process is based upon
distribution of solutes between two immiscible
phases.
• These phases may be mixtures of solvents,
buffers, salts and various complexing agents.
• The only difference in this technique from
partition chromatography is that in counter
current distribution there is no solid supporting
matrix.
157. • This is a liquid-liquid extraction process & is
based on the principle of partition
chromatography.
• It is developed by Craig in 1944.
• The apparatus consists of a series of tubes
connected and contain a lower, stationary phase
and an upper moving immiscible liquid.
158. • The mobile phase can be transferred from one
tube to another after shaking & allowing to
separate.
• The extract is placed in the first tube and the
apparatus is agitated and the layers are allowed
to separate.
159. • The components of a mixture will be distributed
between the two layers according to their
partition coefficients
• The upper moving phase is moved along to the
second tube containing the lower phase.
• More moving phase is brought into contact with
the lower phase of tube 1.
• The process is repeated and fraction is done.
160.
161.
162. • The distribution of each substance over given
number by the tubes can be increased by the
use of series of buffer solution of graded pH.
• This method is best suited for separation than
extraction.
• Extraction of alkaloids, amino acids, phenols,
antibiotics, anthraquinones, cardiac glycosides,
prostaglandins, vitamins, steroids etc.
164. Gas- LiquidChromatographyG.l.c.
Sample introduced by syringe.
Column separates components.
(Heated in oven)
Detector monitors compounds
emerging from outlet.
Recorder plots signals as
a chromatogram.
165. Introduction
• This technique is based on partitioning of
compounds between a liquid and gas phase.
• It is useful for separation of solutes of low
polarity.
• It is applicable to substances or their derivatives
which are volatilised under the temperatures
employed.
• GC is based on mechanisms of adsorption, mass
distribution or size exclusion.
166. • Stationary phase of liquid material such as
silicone grease is supported on an inert
granular solid.
• This material is packed into narrow glass or
steel coiled column which is of 2-4 mm inner
diameter and 2-3 meter long through which
an inert carrier gas (the mobile phase) such as
nitrogen, argon or helium is passed.
• Mobile phase is a carrier gas moving through
or passing the stationary phase contained in a
column.
167. • The column is maintained at an elevated
temperature which volatilizes the compounds
to be separated.
• The basis of separation is difference in the
partition coefficient of volatilized solutes
between the liquid and gas phases as the
compounds are carried through the column
by carrier gas.
• As the compounds leave the column they pass
through recorder. GLC may also be performed
using capillary columns of 0.03-1.0 mm
diameter and 100 m. in length
168. • GLC is mainly used for the separation of
volatile non-polar compounds.
• Very sensitive - small quantities of substances
detected, explosives, drugs etc.
• Separation of pure substances for collection.
• Can be connected to mass spectrometer for
direct identification of substances.
170. Principle
• Resolving power of a column increases with
column length and number of theoretical plates
per unit length.
• In all the chromatographic processes; the eluent
travels either under the influence of gravity or
low pressure pumping. This results in low flow
rates and gives greater time for band broadening
by diffusion.
171.
172. • Use of faster flow rates is not possible
because it creates a back pressure which can
damage matrix structure of the stationary
phase thereby reducing eluent flow and
impairing resolution.
• Now a days special very small particle size
stationary phases are available which can
withstand high pressures and pumping
systems which give high and reliable flow
rates.
173. • The choice of mobile phase depends upon the
type of chromatography to be performed.
• Isocratic separation can be done by a single
solvent or two or more solvents mixed in a
definite proportion.
• Gradient elution is also performed. All solvents
to be used in the HPLC should be highly pure,
since traces of impurity may affect the column
and interfere in the detection system.
• The solvents must also be degassed before use
as gassing tends to occur in most pumps.
174. • The detector system should be very sensitive
because a very small sample is analyzed. Most
commonly used detector is variable wavelength
UV-VIS spectrophotometer, a fluorimeter, a
refractive index monitor or an electrochemical
detector
175. • Almost all types of biological molecules have
been separated by HPLC.
• Reverse phase partition HPLC is particularly
useful for polar substances such as peptides,
vitamins, polyphenols and steroids.
• The technique is particularly useful in clinical
and pharmacological work.
• HPLC has been most successfully used for the
separation of proteins and oligopeptides.
Instrument used for separation of proteins has
given rise to the technique of fast protein
liquid chromatography (FPLC).
177. introduction
• The technique employs highly specific biological
interaction between solute to be separated and its
ligand for purpose of purification of the former.
• Enzymes are highly specific for their substrates,
inhibitors, activators or substrate analogues, which
specifically but non-covalently bind to the enzyme
for expression of their affect.
• This property has been utilized for purification of a
given enzyme from other enzymes and proteins.
• The technique requires that the material to be
isolated, is capable of selectively and reversibly
binding to the specific ligand which is covalently
attached to chromatographically inert matrix.
178. • Under appropriate experimental conditions,
when a complex mixture, containing specific
compound to be purified, is added to the
insolubilized ligand contained in a column, only
that compound would be bound to the matrix
containing ligand, and thus its movement be
retarded through the column.
• All other compounds can be washed away.
• The compound under study is then recovered by
displacement from the ligand by either passing
higher concentration of the substrate, changing
the pH or ionic strength of the eluent.
• The method requires a detailed knowledge of
structure and biological properties of the
compound to be purified, so that ideal separation
conditions could be carefully planned
179.
180. • A wide range of enzymes and other proteins
including receptor proteins and
immunoglobulins are purified by this
technique. The technique is limited in
application only on the availability of
immobilized specific ligand for the protein
under purification.
181. Purification
• Crystallisation
• Formation of crystals at the point of super saturation
in the solvent by concentration, slow evaporation,
cooling etc
• Fractional distillation:
• Fractionation based on boiling point
• Sublimation
• Chromatography separation :
• Column chromatography : separation based on the
solubility /partition coefficient
182. Industriallevel
• Structured approach
• R&D:
• Process development, Process Optimization, lab validation and
Scale up
• Production:
• Selection of suitable Equipment
• Unit operation
• Process validation and manufacturing
• Analysis:
• Analysis, Analytical technique, Pharmacopeia, & compliance
185. StructureElucidation/
Analytical technique
• Ultraviolet-Visible &Infrared Spectrometry
• Functional group identification and quantitative estimation
• Nuclear Magnetic Resonance Spectroscopy
(NMR)
• The number and nature of chemical entities in a molecule
• Mass Spectroscopy : Mass of the molecule
• TGA/ DTA: Thermal property
• Melting, Decomposition, hydration/ solvation, polymorph of
maolecule,
• XRD: crystal structure
