[Pharmacognosy
reference (first London Pharmacopoeia,
Formed from two Greek words:
Pharmakon- drug
Gnosis- knowledge
-To describe the study of medicinal plants and
their properties
-As an applied science that deals with the
biologic, biochemical and economic features of
natural drugs and their constituents
-It is a study of drugs that originate in the plant
and animal kingdom
Origins of Pcog
 Western medicine with origins in
Mesopotamia and Egypt
 the Unani (Islamic) and Ayurvedic
(Hindu) systems centred in western Asia
and
 the Indian subcontinent and those of
the Orient (China, Japan, Tibet, etc.).
How and when such medicinal plants were first
used is, in many cases, lost in pre-history,
indeed animals, other than man, appear to have
their own materia medica.
Following the oral transmission of medical
information came the use of writing
 (e.g. the Egyptian Papyrus Ebers c. 1600
bc)
 baked clay tablets (some 660 cuneiform
tablets c. 650 bc from Ashurbanipal’s
library at Nineveh, now in the British
Museum, refer to drugs well-known
today),
 parchments and manuscript herbals,
printed herbals (invention of printing
1440 ad),
 pharmacopoeias and other works of
1618; first British Pharmacopoeia,
1864), and most recently electronic
storage of data.
Scope and Practice of pcog
Contributions:
 alternative (complementary) therapies
 animal products which are traditionally
encompassed within the subject; these
include such items as beeswax, gelatin,
woolfat, vitamins, etc
 plant materials
 Other natural products such as the
antibiotics, hormones and others may
or may not be involved, depending on
the teaching practice of a particular
institution.
 Marine organisms, both plant and
animal, with potent pharmacological
actions are receiving increasing
attention in the search for new drugs.
Materials having no pharmacological action
which are of interest to pharmacognosists are
 [
 flavouring and suspending agents,
 colourants,
 disintegrants,
 stabilizers and filtering and support
media.
Other areas that have natural associations with
the subject are poisonous and hallucinogenic
plants, allergens, herbicides, insecticides and
molluscicides.
Vegetable drugs can be arranged for study
under the following headings.
1. Alphabetical.
-Either Latin or vernacular names may
be used.
 -This arrangement is employed for
dictionaries, pharmacopoeias, etc.
-Although suitable for quick reference it gives
no indication of interrelationships between
drugs.
2. Taxonomic.
-On the basis of an accepted system of
botanical Classification, the drugs are arranged
according to the plants from which they are
obtained, in classes, orders, families, genera
and species.
-It allows for a precise and ordered
arrangement and accommodates any drug
without ambiguity.
-As the basic botanical knowledge of pharmacy
students decreases over the years this system is
becoming less popular for teaching purposes.
3. Morphological.
-The drugs are divided into groups such as the
following:
-R. Pratt and H. W. Youngken Jr. were, in 1956,
the first to use this approach for an English
language textbook and now, with so many plant
materials being screened for specific
pharmacological activity, this type of listing is
found increasingly in the literature.
-However, it is important to appreciate that the
constituents of any one drug may fall into
different pharmacological groups.
5. Chemical or Biogenetic.
-The important constituents, e.g. alkaloids,
glycosides, volatile oils, etc., or their
biosynthetic pathways, form the basis of
classification of the drugs.
-This is a popular approach when the teaching
of pharmacognosy is phytochemically biased.
-Ambiguities arise when particular drugs
possess a number of active principles belonging
to different phytochemical groups, as illustrated
by liquorice, ginseng, valerian, etc.

a. organized drugs:
Plant Nomenclature
leaves, flowers, fruits, seeds, herbs and entire
Binomial System
organisms, woods, barks, rhizomes and roots
Scientific name
b. unorganized drugs
-first name denotes the genus, while the second
dried latices, extracts, gums, resins, oils, fats
(specific) name denotes the species.
and waxes
-All specific names may be written with small
These groupings have some advantages for the
initial letters although formerly capitals were
practical study of crude drugs; the identification
used where species were named after persons.
of powdered drugs is often based on micro-
morphological characters. -Thus, the species of Cinchona named after
Charles Ledger, who brought its seeds from
4. Pharmacological or Therapeutic.
Brazil in 1865, is now written Cinchona
-This classification involves the grouping of ledgeriana rather than Cinchona Ledgeriana
drugs according to the pharmacological action
of their most important constituent or their
therapeutic use.
Evaluation of Drugs?
Chemistry of natural Drug Products:?
Active constituents?
Inert Constituents?
Crude drugs
- undergo process of collection and drying
-are plants or animals, or their parts which after
collection are subjected only to drying or
making them into transverse or longitudinal
slices or peeling them in some case
Extractives- product of the extraction process
Extraction- removes only those substances that
can be dissolved in the liquid or liquid mixture
referred to as solvent (menstruum) like:
 ethyl alcohol with water
(hydroalcoholic),
 chloroform (when you want to extract
alkaloid),
 petroleum ether (extract fixed oil),
 purified water with Clevenger
apparatus (volatile oil)
Marc- Undissolved portion which remains until
the extraction process completed
Factors that influence the nature and amount of
active constituents present:
1. Environmental conditions
Temperature- a major factor controlling the
development and metabolism of plants.
Many tropical and subtropical plants will grow
in temperate regions during summer months,
but lack frost resistance to withstand the winter
Rainfall- The important effects of rainfall on
vegetation must be considered in relation to the
annual rainfall, its distribution throughout
the year, its effect on humidity and its effect
coupled with the water-holding properties of
the soil.
Variable results have been reported for the
production of volatile oils under different
conditions of rainfall and may in some
instances be coupled with the development of
glandular hairs.
Continuous rain can lead to a loss of water-
soluble substances from leaves and roots by
leaching; this is known to apply to some plants
producing alkaloids, glycosides and even volatile
oils.
This could account for low yields of some active
constituents in wet seasons from plants whose
general condition appears to be good.
Day-length and radiation characteristics
Plants vary much in both the amount and
intensity of the light which they require.
In the wild state the plant will be found where
its shade requirements are met, and under
cultivation similar shade must be provided.
In certain cases research has shown that light is
a factor which helps to determine the amount
of glycosides or alkaloids produced
Flavonoids and anthocyanins are also known to
be influenced by UV-B radiation.
Altitude
The coconut palm needs a maritime climate and
the sugar cane is a lowland plant.
Conversely, tea, cocoa, coffee, medicinal
rhubarb, tragacanth and cinchona require
elevation.

vegetative growth is more lush under irrigated
conditions at lower altitude, so the propagation
farms (for the vegetative multiplication of
plants) are, in Ecuador, situated at lower levels
than the final commercial farms.
 Cultivated and Wild Plants
Certain drugs are now obtained almost
exclusively from cultivated plants.
In other cases both wild and cultivated plants
are used.
Soils
Variations in particle size result in different soils
ranging from clay, via sand, to gravel.
Particle size is one factor influencing water-
holding capacity, and some plants (e.g. Althaea
officinalis) which produce mucilage as a water-
retaining material contain less mucilage when
grown on soil with a high moisture content.
The effect of nitrogen-containing nutrients on
alkaloid production has received considerable
study (solanaceous drugs including Nicotiana,
opium); generally, nitrogen fertilizers increase
the size of the plants and the amounts of
alkaloids produced but, as indicated elsewhere,
the method of expressing the results of such
experiments is important.
Propagation from seeds
To ensure success the seeds must be collected
when perfectly ripe.
If not planted immediately, they should
normally be stored in a cool and
dry place and must not be kiln-dried. Some
seeds such as cinnamon, coca and nutmegs
rapidly lose their power of germination if
allowed to dry or if stored for quite short
periods. Long storage of all seeds usually much
decreases the percentage which germinate.
Propagation by vegetative means
The following examples of vegetative
propagation may be mentioned.
1. By the development of
 bulbs (e.g. squill);
 corms (e.g. colchicum);
 tubers ( e.g. jalap and aconite); or
 rhizomes (e.g. ginger).
2. By division- a term usually applied to the
separation of a plant which has a number of
aerial stems or buds, into separate parts each
having roots and a growing point.
This method may be used for althaea, rhubarb,
gentian and male fern.
3. By runners or offsets (e.g. chamomile and the
mints).
4. By suckers or stolons (e.g. liquorice and
valerian).
5. By cuttings or portions of the plant severed
from the plant and capable of developing roots.
Success by this method has been extended to a
large number of plants by the use of rooting
hormones and by the employment of mist
propagation.
6. By layers- A layer is a branch or shoot which
is induced to develop roots before it is
completely severed from the parent plant.
This is done by partly interrupting the food
supply by means of a cut or ligature and
embedding the part.
Alternatively the slit portion of the branch is
enclosed in moist peat, surrounded by moss,
and the whole enclosed in polythene.
This method has been used successfully for the
propagation of cascara.
7. By grafting and budding.
Grafting is an operation in which two cut
surfaces, usually of different but closely related
plants, are placed so as to unite and grow
together.
The rooted plant is called the stock and the
portion cut off the scion or graft.
In Guatemala young Cinchona ledgeriana scions
are grafted on Cinchona succirubra root-stocks,
eventually giving a tree which produces bark
rich in the alkaloid quinidine.
Grafting of female scions of Myristica fragrans
on male stocks may be used to increase the
proportion of fruit-bearing trees in the
plantation.
The method has been used considerably in
phytochemical research to study sites of
synthesis of metabolites etc.
Budding consists of the introduction of a piece
of bark bearing a bud into a suitable cavity or T-
shaped slit made in the bark of the stock.
Budding is largely used for Citrus species,
selected strains of sweet orange, for example,
being budded on sour stocks.
8. By fermentation.
This process applies particularly to the
production of moulds and bacteria, and is
extensively used in the manufacture of
antibiotics, lysergic acid derivatives and some
vitamins.
9. By inoculation. Specific to ergot whereby the
spores of the fungus are artificially cultured and
injected into the rye heads by special machines,
10. By cell culture followed by differentiation;
Collection
Drugs may be collected from wild or cultivated
plants, and the task may
be undertaken by casual, unskilled native labour
(e.g. ipecacuanha) or
by skilled workers in a highly scientific manner
(e.g. digitalis, belladonna
and cinchona).
The season at which each drug is collected is
usually a matter of
considerable importance, as the amount, and
sometimes the nature, of
the active constituents is not constant
throughout the year.
Roots and rhizomes- should be collected after
vegetative processes
Bark- collected before vegetative processes
Leaves and flowering tops- when it is most
active
Flowers- prior to or just about the time of
pollination
Fruit- either before or after the ripening period
but unripe when fully ripe
Seeds- when fully matured
Drying
If enzymic action is to be encouraged, slow
drying at a moderate temperature
is necessary. Examples of this will be found
under ‘Orris
Drugs stored
Rhizome’, ‘Vanilla Pods’, ‘Cocoa Seeds’ and
in the usual containers—sacks, bales, wooden
‘Gentian Root’.
cases, cardboard boxes

and paper bags—reabsorb about 10–12% or

open-air drying more of moisture

- depends very largely on the

weather. In suitable climates open-air drying is Volatile

used for such drugs
oils should be stored in sealed, well-filled
as clove, colocynth, cardamom and cinnamon. containers in a cool, dark
Even in warm and dry
place.

Drying by artificial heat

is more rapid than open-air drying and

is often necessary in tropical countries (e.g.

West Africa, where the

humidity is very high, and Honduras for drying

cardamom fruits).

Rapid drying

helps flowers and leaves to retain their colour

and aromatic

drugs their aroma, but the temperature used in

each case must

be governed by the constituents and the

physical nature of the drug.

Storage

It has been

reported that the content of taxol in Taxus

baccata leaves and extracts

stored at room temperature for one year

decreased by 30–40% and

Phytochemistry
-is the study of phytochemicals in plants and
other natural sources
Phytochemicals
- are chemicals derived from plants? In
other words, the terms re often used to
described the large number of primary
and secondary metabolic compounds
found in plants
 Expression, aqueous extraction and
evaporation- had long been used for
the preparation of sugar from sugar-
cane
 extraction processes
-Alcohol is a general solvent for
many plant constituents (most fixed oils
excepted) and as such may give problems in the
subsequent elimination of pigments, resins, etc
 isolated sucrose- aside from sugar cane
there is sugar bits
 isolated acids- citric, gallic malic oxalic
tartaric and prussic
 isolated narcotine- first alkaloid isolated
like morphine, strychnine, emetine
 fats and fixed oils- fats (solid at ordinary
temp.) fixed oil (liquid at ordinary
temp.)
 Many constituents of plants-
particularly those associated with
herbal medicine, have medicinal
properties which manifest
themselves in more subtle and less
dramatic ways than the obviously
poisonous plants
 Plant metabolites
 Carbohydrates, fats and proteins- are of
dietetic importance
 Starches and gums- are used in
pharmacy but lack any marked
pharmacological action.
 Calcium oxalate, silica, lignin, and
coloring matters- may be of assistance
in the identification of drugs and the
detection of adulterations
Not all the chemical compounds
elaborated by plants are of equal
interest to the pharmacognosist. Until
relatively recently the so-called
‘active’ principles were frequently
alkaloids or specific glycosides
usually with pronounced
pharmacological properties; these
therefore received special attention,
and in large measure constituted the
principal plant drugs of the allopathic
system of medicine.
This has considerably widened the
scope of plant metabolites considered
worthy of more detailed investigation.
As a result of the recent interest in the
plant kingdom as a potential
source of new drugs, strategies for the
fractionation of plant extracts
based on biological activity rather than
on a particular class of compound,
have been developed. The chemical
examination follows after
the isolation of the active fraction.

Phytochemical investigation
-Authentication and extraction of the plant
material
-separation and isolation of the constituents
-characterization of isolated compounds
-investigation of the biosynthetic pathways to
particular compounds
-quantitative evaluation
-pharmacological assessment of the separated
components
Extraction of plant material
⁃ choice of extraction procedure but depends
on the nature of the plant material and
component or active constituent to be isolated
Flavonoids- methanol solvent
Alkanoids- chloroform
Volatile- purified water with Clevenger
apparatus and steam distillation is the method
of extraction
Fixed oil - petroleum ether
 Dried material
(I-powder then proceed to MoE)
fresh plants
(maceration or soaked sa solvent like
ethyl alcohol then proceed to method
of extraction)
 solvents
Alcohol is the most common
Water immiscible solvent like petroleum ether,
ether, chloroform
Alcohol is a general solvent for many plant
constituents (most fixed oils excepted) and as
such may give problems in the subsequent
elimination of pigments, resins, etc.
Water immiscible solvents are widely used—
light petroleum (essential and fixed oils,
steroids), ether and chloroform (alkaloids,
quinones)
 Extraction
The extraction of organic bases (e.g. alkaloids)
usually necessitates basification of the plant
material if a water-immiscible solvent is to be
used; for aromatic acids and phenols
acidification may be required.
Extraction itself may be performed by
repeated maceration with agitation
percolation
continuous extraction (in a Soxhlet extractor
Special methods for volatile oils, such as the
enfleurage process
Spouted bed extraction
in the production of annatto powder from the
seeds of Bixa orellana, the physical removal of
the pigment layer of the seed-coat can yield a
less impaired product than that produced by
solvent extraction.
Such methods can involve the use of a ball mill
or a spouted bed unit.
A development of the latter, the conical
spouted bed extractor, has been investigated
for annatto production.
Supercritical fluid extraction
The use of supercritical fluids for the extraction
of a range of materials including
 plant products of medicinal,
 flavouring and cosmetic interest has,
become of increasing economic and
research interest
Under these conditions the gas and liquid
phases both possess the same density and no
division exists between the two phases.
Further examples involving the extraction of
phytochemicals with supercritical carbon
dioxide follow:
1. Acylphloroglucinols: Oxygenated hyperforin
derivatives of Hypericum
2. Alkaloids:
Decaffeination of green coffee (industrial
application) Isolation of vindoline from
Catharanthus roseus
3. Diterpene:
Extraction of taxol from Taxus brevifolia
(extraction more selective than conventional
ethanol extraction), also from T. cuspidata
4. Fixed oils: Extraction of oil from evening
primrose
(subtle shift in triglyceride composition;
oxidation of γ-linolenic acid during extraction
reduced)
5. Pigments: Extraction of annatto seeds
6. Sapogenins: Smilax china (increased yield of
diosgenin)
7. Sesquiterpene lactones: In conjunction with
gas chromatography for the isolation of
parthenolide from feverfew.
Addition of methanol or methyl cyanide as CO2
modifiers gave higher yields but produced co-
extractives
The use of 10% methanol in CO2 for the
extraction of trilactones from ginkgo could be of
commercial significance
8. Volatile oils and resins:
Hops (commercial application);
frankincense and myrrh (efficient extraction);
juniper berries (significant difference in
composition compared with distilled oil, the
latter being significantly more enriched with
monoterpenoid hydrocarbons)
Solid phase microextraction
The method is suitable for some volatile oil-
containing drugs.
using methyl polysiloxane solid phase
microextraction fibres, has extracted the
volatile oil from the headspace above fresh cut
eucalyptus leaves (37° for 10 min.).
Separation and isolation of constituents
Sublimation
Sublimation may sometimes be possible on the
whole drug,
 as in the isolation of caffeine from tea
  for the purification of materials present
in a crude extract.
Modern equipment employs low pressures with
a strict control of temperature.
Distillation
Fractional distillation
-has been traditionally used for the separation
of the components of volatile mixtures;
-in phytochemistry it has been widely used for
the isolation of the components of volatile oils.
Steam distillation is much used to isolate
volatile oils and hydrocyanic acid from plant
material.
The TAS oven (see ‘Thin-layer chromatography’)
-involves steam distillation on a semi-micro
scale for the direct transfer of volatile materials
from a powdered drug to a thin-layer plate.
Fractional liberation
As an example,
a mixture of alkaloid salts in aqueous solution,
when treated with aliquots of alkali, will give
first the weakest base in the free state followed
by base liberation in ascending order of basicity.
If the mixture is shaken with an organic solvent
after each addition, then a fractionated series of
bases will be obtained.
A similar scheme can be used for organic acids
soluble in water-immiscible solvents;
in this case, starting with a mixture of the acid
salts, it is possible to fractionally liberate the
acids by addition of mineral acids.
Fractional crystallization
A method much used in traditional isolations
and still valuable for the resolution of often
otherwise intractable mixtures.
The method exploits the differences in solubility
of the components of a mixture in a particular
solvent.
Frequently, derivatives of the particular
components are employed (picrates of
alkaloids, osazones of sugars).
Adsorption chromatography
Of the various methods of separating and
isolating plant constituents, the
‘chromatographic is one of the most useful
techniques of general application.
The use of charcoal for the decolorization and
clarification of solutions is well known;
colored impurities are adsorbed by the charcoal
and a colourless solution results on filtration.
A light petroleum extract of green leaves is
allowed to percolate slowly through a column
of powdered calcium carbonate contained in a
vertical glass tube.
The pigmented contents of the solution are
adsorbed on the substance of the column and
undergo separation as percolation proceeds.
Substances are more readily adsorbed from
 non-polar solvents such as light
petroleum and benzene
 polar solvents
alcohol, water and pyridine
- are useful eluting media;
 -many substances are adsorbed at one
pH and eluted at another.
Various substances may be used as adsorbing
materials:
 alumina is the most common and
 other materials include
kaolin, magnesium oxide, calcium
carbonate, charcoal and sugars
Adsorption chromatography has proved
particularly valuable in the isolation and
purification of
 vitamins,
 hormones,
 many alkaloids,
 cardiac glycosides,
 anthraquinones, etc.
-It is commonly employed as a ‘clean-up’
technique for the removal of unwanted
materials from plant extracts prior to assay.
Partition chromatography
-The aqueous phase is usually the stationary
phase and
-is intimately mixed with a suitable ‘carrier’ such
as silica gel, purified kieselguhr or powdered
glass and
- packed in a column as in adsorption
chromatography
Partition chromatography on paper
-method of partition chromatography using
strips of filter paper as ‘carriers’ for the analysis
of amino acid mixtures.
-The technique was extended to all classes of
natural products, and although to a large
measure replaced by thinlayer chromatography
(TLC), it remains the method of choice for the
fractionation of some groups of substances
-Either the paper may be dipped in the solvent
mixture so that the solvent front travels up the
paper (ascending technique) or
- the trough of solvent may be supported at the
top of the chamber, in which case the solvent
travels down the paper (descending technique).
High-performance liquid chromatography
(HPLC)/ high-speed LC
HPLC is a liquid column chromatography system
which employs relatively narrow columns
(about 5 mm diameter for analytical work)
operating at ambient temperature or up to
about 200°C at pressures up to 200 atm (20
000 kPa
The apparatus is suitable for all types of liquid
chromatography columns:
 adsorption
 partition by the use of bonded liquid
phases,
 reversed phase,
 gel filtration
 ion exchange and
 affinity
HPLC can give much improved and more rapid
separations than can be obtained with the older
liquid chromatography methods and it is
therefore finding increasing use in numerous
areas
Supercritical fluid chromatography
-This technique, developed over recent years
for both the qualitative and quantitative
analysis of medicinal products
- utilizes supercritical fluids, particularly carbon
dioxide, as the mobile phase in liquid
chromatography
Counter-current extraction
-This is a liquid–liquid extraction process and
the principles involved are similar to those of
partition chromatography.
-The applications of counter-current extraction
covered
 many fields of plant chemistry,
including
alkaloids, amino acids, antibiotics,
antitumour compounds
 phenols including
anthraquinone derivatives, cardiac
glycosides, essential oils, fatty acids,
plant auxins, prostaglandins, steroids
and vitamins.
Other more recent developments involving the
counter-current principle are
 high-speed counter-current
chromatography (planet coil centrifugal
CCC)
 droplet counter-current
chromatography (DCCC) and
 centrifugal droplet CCC.
Thin-layer chromatography
TLC has certain advantages over paper
chromatography.
 Fractionations can be affected more
rapidly with smaller quantities of the
mixture;
 the separated spots are usually more
compact and more clearly demarcated
from one another;
 and the nature of the film is often such
that drastic reagents, such as
concentrated sulphuric acid, which
would destroy a paper chromatogram,
can be used for the location of
separated substances.
Adsorbent is the alumina
The solvents used for running the
chromatogram must be pure, and common
ones are
 methanol, ethanol and other alcohols,
 chloroform, ether, ethyl acetate, n-
hexane, cyclohexane, petroleum spirit
and
 mixtures of these
Kieselguhr and silica gel are also commonly
employed, and their properties as thin layers
can be modified by the inclusion of acids, bases
and buffer solutions
PreparativeTLC
To speed up separations and to make them on-
line for continuous recording, various
modifications of preparative TLC have been
developed.
These include centrifugally accelerated layer
chromatography and overpressure layer
chromatography
Gas–liquid chromatography
The use of a liquid stationary phase and a
mobile gaseous phase in chromatography for
the separation of the lower fatty acids.
1. The empty columns are made of glass or
metal and are either straight, often up to about
1.3 m in length, or coiled and up to 16 m in
length.
-The liquid stationary phase is held on an inert
material, commonly partially fused diatomite.
2. The operating temperature of the column is
critical.
Mixtures of low-boiling-point substances can be
fractionated at low temperatures;
some ethers, for example, can be dealt with at
room temperature.
Other materials require much higher
temperatures—volatile oils 150–300°C, steroids
250°C and pesticides 400°C.
3.The mobile phase is a gas which is inert in so
far as the other components of the
chromatogram are concerned.
The choice of gas is dependent on the detector
system and gases commonly used are
hydrogen, nitrogen, helium and argon.
The flow rate of the gas is important; too high a
flow rate will give incomplete separations and
too slow a rate will give high retention times
and diffuse peaks.
Typical flow rates for short columns are 10–50
ml min−1.
4.The detector system analyses the effluent gas
from the column.
It may be of the integral type, in which some
property—for example, titration value—of the
eluate is recorded or it may be of the
differential type, in which some property of the
effluent gas is compared with that of a
reference gas, often the mobile phase.
Some pharmacognostical examples of the
applications of gas chromatography include the
 examination of many volatile oils (see,
for example the BP assay of Clove Oil),
 camphor
 plant acids
 some alkaloids (opium, tobacco and
Conium and tropane derivatives),
 the resins of the Convolvulaceae and of
Cannabis, and
 steroidal compounds such as the
sapogenins and cardioactive glycosides
and aglycones.
The estimation of pesticide residues on crops is
of utmost importance, and here the sensitivity
of detector systems, such as the
electron capture detector, has made possible
the determination of the chlorinated pesticides
down to the parts-per-billion range.
Capillary-column gas chromatography
Such columns hold the stationary phase in a
number of ways.
(1), Wall-coated open tubular (WCOT) columns
have the inner wall of the tube coated with
stationary phase up to about 1 μm in thickness.
Greater thickness leads to column bleeding in
which the stationary phase moves down the
column and eventually leaks into the detector.
Thicker layers, and hence increased sample
capacity, can be achieved with silica columns
having specially bonded phases.
WCOT columns have the highest efficiency but a
low sample capacity.
(2), Support-coated open
tubular (SCOT) columns have the inner wall
lined with a thin layer of support material
coated with immobile phase.
This has the effect of increasing the available
area of immobile phase, affording the column a
greater load capacity.
The efficiency, while lower than that of the
WCOT columns is much higher than that for
packed columns.
(3) Micropacked
columns involve a coated support packed into
narrow-bore columns.
In all ways they represent a compromise, being
more efficient than the normal packed columns
but having the same problem in that column
length is restricted by the high back-pressure.
Examples include
 the separation of the two enantiomers
of linalool enabling the detection of
reconstituted bergamot oil in the
genuine oil and
 the detection of added reconstituted
lemon oil in the genuine cold-
pressedessential oil
Gel filtration chromatography, gel permeation
chromatography (molecular sieves)
-These techniques are used for the separation
of substances in solution according to their
molecular size.
 Hydrophilic gels such as those prepared
from starch, agar, agarose (a
component of agar), polyacrylamide,
polyvinylcarbitol and
 cross-linked dextrans
-have been used for the fractionation of
proteins, peptides, amino acids and
polysaccharides.
-The technique is important in DNA analysis for
the separation of those fragments that result
from the treatment of DNA with specific
restriction enzymes.
-Size-exclusion chromatography is used for the
determination of those smaller fatty acids
(oligomers) that need to be limited in fish oils,
such as Fish Oil, Rich in Omega-3-Acids BP/EP,
(oligomers, maximum 1.5%).
Electrochromatography
For the electrophoretic separation of mixtures,
a filter-paper strip is impregnated with a
solution of an electrolyte (usually a buffer
solution)
and supported in the centre; its two ends are
dipped into solutions in which electrodes are
immersed.
A spot of the material to be fractionated is
placed on the paper, the whole apparatus
sealed and
a potential difference of about 2–10 volts per
centimetre applied along the paper.
According to the nature of the charge on the
ions of the solute mixture, the solutes will move
towards either the anode or the cathode.
Many alkaloidal mixtures have been separated
by this method and also plant acids, the
component sugars of cardiac glycosides and
anthraquinone derivatives.
Capillary electrophoresis is a technique of
relatively recent introduction and can give
separation efficiencies of the order of 4 × 105
theoretical plates.
It provides a more rapid analysis than gel
electrophoresis and with detector systems such
as the laser-induced fluorescence detector
combines a high resolution with a 500-fold
increase in sensitivity over UV detection.
The method has been used for the analysis of
flavonoids.
Affinity chromatography
This method has been developed largely for the
resolution of protein mixtures, and it depends
on the specific, reversible binding of individual
proteins with a particular ligand such as an
enzyme substrate or inhibitor.
Affinity chromatography has been applied to
the purification of enzymes
 for potential clinical application,
 for the isolation of certain antibodies
and
 for the specific fractionation of
different types of cells (e.g.
erythrocytes and lymphocytes).
Characterization of Isolated Compounds
chemists are coming to rely more and more on
the use of physical techniques to establish
structures of new compounds and to identify
known compounds in plant sources.
 Ultraviolet,
 infrared,
 mass and
 nuclear magnetic resonance
spectroscopy
 together with X-ray crystallographic and
 optical rotatory dispersion methods
------have all played a significant role in
these developments.
have demonstrated the importance of such
methods for evaluating biosynthetic pathways
and for studying the fate of distant natural
product precursors in specific plants
Biogenetic Investigations
The components of such a mixture can be
isolated by centrifugation and the biological
activity of each fraction can be tested.
The penultimate stage in a biogenetic study is
the isolation of the enzymes involved in the
pathways under consideration and the in vitro
demonstration of their properties
Finally, it is becoming increasingly possible to
locate and clone the gene responsible for the
synthesis of a particular enzyme.
Tracer Techniques
Radioactive tracers
-In biological investigations
 the use of radioactive carbon and
hydrogen, and to a lesser extent and
 for more specific purposes sulphur,
phosphorus and
 the alkali and alkaline-earth metals,
-enables the metabolism of compounds to be
followed in the living organism.
For studies on proteins, alkaloids and amino
acids a labelled nitrogen atom may give more
specific information than a labelled carbon, but
the two available isotopes of nitrogen are both
stable, necessitating the use of a mass
spectrometer for their use as tracers
Autoradiography
-A technique used for the location of
radioactive isotopes in biological and other
material is autoradiography.
-the specimen is placed in contact with a
suitable emulsion (e.g. X-ray sensitive film) and
after exposure the latter is developed in the
usual manner.
-The resulting autoradiograph gives the
distribution pattern of the radioactive
substances in the specimen.
- by its ability to utilize a constituent of ‘brewer’s
solubles’ but not acetate
- led to the isolation of mevalonic acid, an important
intermediate of the isoprenoid compound pathway.
Ultraviolet-induced mutants of ergot auxotrophic
with respect to a number of amino acids have been
produced; cultures of these have been used to
inoculate rye and the resulting alkaloid contents of
the sclerotia have been investigated.

-The method can be applied to whole

morphological parts (e.g. leaves) or to
histological sections, for which the resulting
negative is viewed under a microscope

Isolated Organs, Tissues and Cells

The cultivation of isolated organs and tissues of

plants eliminates interference from other parts
of the plant which may produce secondary
changes in the metabolites.

It can be used for feeding experiments in

conjunction with labelled compounds and is
also useful for the determination of the site of
synthesis of particular compounds.

Grafts

Grafting techniques have considerable use in

biosynthetic studies, particularly for the
determination of the sites of primary and
secondary metabolism of some secondary plant
products.

Alkaloid formation by grafted plants has been

extensively studied in Nicotiana and the
tropane alkaloid-producing Solanaceae

Mutant Strains

A mutant of Lactobacillus acidophilus