Pharmacognosy

Chapter Two

General Methods in Studying

Constituents of Crude Drugs

1
 Introduction

 Studying the constituents of crude drugs is an essential aspect

of pharmacognosy.

 Various steps and methods are employed to analyze and

identify the chemical components present in crude drugs.

 Extraction is the cornerstone method of obtaining

constituents of crude drugs

2
 Extraction
Extraction
 The separation of medicinally active portions of plant or
animal tissues from the inactive or inert components by
using selective solvents in standard extraction procedures.
 Extraction can be
 Initial extraction
 Performed on small amount of crude drug to get
preliminary knowledge on the nature of exact &
class of metabolites present.
 Bulk extraction
 Is large scale extraction following initial extraction

3
 Extraction…

Extraction involves;

 Diffusion of the solvent into cells

 Dissolution of the metabolites in the solvent

 Diffusion of the solution out of the cells

 Continued till equilibrium is set up between the solution in

the cells and external environment.

4
 Extraction…
Extraction can be;
 Liquid—liquid
based on relative solubility of constituents in two
liquids that do not mix
Extraction from one liquid into another/ partitioning
 Solid—liquid
Separation of soluble components from a solid into a
solvent
5
 Extraction…
Methods of solvent extraction
Continuous extraction (percolation and Soxhlet extraction),
•Solvent continuously flows through the plant material
•Saturated solvents are replaced with fresh solvents
Discontinuous extraction (e.g. Maceration)
•The solvent is added and removed in batches.
•Extraction stops when equilibrium is reached b/n the
concentration of solute inside the plant material & the
solvent
•Needs decanting and replacement with new solvent.

6
 Extraction…
Terms of solvent extraction
 Menstruum :- the solvent used for extraction

 Crude extract: - plant constituents left after removal of the

dissolving media by evaporation.

 Residue or mark: - the undissolved constituents of the plant

left in the filter paper.

 Filtrate: - Solvent + dissolved plant constituent

7
 Extraction…
Choice of suitable solvents
An ideal solvent used for extraction should:
 Have good selective solubility for the targeted compounds
 Have high capacity of extraction
 Poor solubility of impurities and completely volatile
 Have a low price
 Be non reactive with the target/ other compounds in the
crude drug
 Be non toxic, non irritant and non flammable

8
 Extraction…
Choice of suitable solvents…
 Be readily available

 Be environmentally friendly
 Be non-explosive
 Not have any destructive effect to the constituent to
be extracted
 Penetrate the plant material and reach where the plant
constituent is found

9
 Extraction…
Choice of suitable solvents…
 Alcohol is used with water during extraction to
 Induce swelling of the plant particles
 Increase the porosity of the cell walls.
 Enhance diffusion of the solvent into the cell, and
 facilitates the diffusion of extracted substances from
inside of the cells to the surrounding solvent.

10
 Extraction…

Choice of suitable solvents…

 For extraction of barks, roots, woody parts and seeds the ideal
alcohol/water ratio is about 7:3 or 8:2.
 For leaves or aerial green parts the ratio 1:1 is usually
preferred in order to avoid extraction of chlorophyll.

11
 Steps Involved in the Extraction of Medicinal Plants
• The qualitative and quantitative studies of bioactive
compounds from plant materials mostly rely on the selection
of proper extraction method.
• In order to extract medicinal ingredients from plant material,
the following sequential steps are involved.

a. Drying and
grinding of crude d. Filtration
drug
b. Selection of e. Concentration
suitable solvents f. Drying
c. Extraction
12
Steps Involved in the Extraction of Medicinal Plants…

a. Drying and grinding of crude drugs

 Drying takes place at room temperature or in the oven at
no more than 30˚C.
 Compacted samples of fresh plant material with little air
circulation may experience fungal infestation and elevated
temperatures by fermentation if left to stand for several
days so needs attention

13
Steps Involved in the Extraction of Medicinal Plants…

a. Drying and grinding of crude drugs…

 Grinding assists the penetration of the solvents to the
cellular structure of the plant tissues.
 Equipment used for grinding include sophisticated mills,
blenders, simple axes, scissors, grinders or knives.
 When thermolabile or volatile phytocompounds are
extracted, the milling stage may be omitted to avoid losses
by heat generation during communition.
14
Steps Involved in the Extraction of Medicinal Plants…

b. Selection of suitable solvents

 Solvents should dissolve the secondary metabolites under
study, be easy to remove and be inert, nontoxic and not
easily flammable.
 Commonly employed solvents: water, ethanol, methanol,
chloroform, hexane etc.

15
Steps Involved in the Extraction of Medicinal Plants…

c. Extraction
• The most methods of extraction processes employed are;
Classical methods Modern methods
Ultrasound-assisted
Soxhlet extraction,
extraction
Maceration, Supercritical fluid
extraction
Digestion,
Microwave-assisted
Percolation and extraction
Pressurized liquid
Hydrodistillation
extraction

16
Steps Involved in the Extraction of Medicinal Plants…

d. Filtration
 The extract obtained is separated out from the marc.
e. Concentration
 The enriched extract (miscella) from extractors is concentrated
under vacuum to produce a thick concentrated extract.
f. Drying of extracts
 The concentrated extract is subjected to spray drying with a
high pressure pump at a controlled feed rate and temperature,
to get dry powder.

17
 Classical methods of extraction
techniques
• Bioactive compounds from plant materials can be
extracted by various classical extraction techniques
which are based on the extracting power of different
solvents in use.
• The existing classical techniques are: Soxhlet extraction,
maceration, digestion, percolation and hydrodistillation.

18
Maceration

 The whole or coarsely powdered crude drug is soaked in a

solvent (water, ethanol) and allowed to stand at room
temperature for a period of 3-7 days until the soluble matter
has fully dissolved with occasional stirring.
 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.

19
 Maceration…

 Employed for thermolabile phytoconstituents and repeated

maceration is better than single maceration as appreciable
amount of active constituent left in the marc.
 When the marc can’t be pressed, triple maceration is
employed.
 It is simple and inexpensive method, but time consuming and
may yield less extract

20
 Percolation

 A continuous flow of solvent is passed through a packed column

bed of plant material, allowing for the extraction of soluble
compounds.
 A narrow, cone-shaped vessel open at both ends is used and the
method is applied commonly in herbal tincture preparation
 This method is more efficient than maceration (provides products
of greater concentration), but requires more setup and solvent.

21
 Percolation…
Percolation involves;
i. Size reduction to fine/coarse powder
[Link]: powder moistened in menstruum and stand in well
closed container for about 4 hours
[Link]: moistened powder packed in the percolator
[Link]: additional solvent is poured on top of the plant
material and allowed to stand for 24 hrs.
v. Percolation: the lower tap opened and liquid allowed to drip

22
23
 Digestion

• This method involves heating a mixture of plant material and

solvent at a controlled temperature for an extended period of
time.
• It is used when moderately elevated temperature is not
objectionable.
Process: The mixture is usually placed in a water bath or similar
setup to maintain a specific temperature, allowing for the gradual
extraction of compounds.
24
 Decoction

 In this process, the crude drug is boiled in a specified

volume of water for a defined time; then cooled, filtered,
evaporated to dryness and concentrated.
• Suitable for extracting water-soluble, heat stable, non-
volatile constituents and hard plant parts (root, stems,
rhizomes and barks).

25
 Infusion
 Infusion involves steeping a plant material in hot water or
alcohol, to extract flavors, aromas, or others.
 The plant material is usually added to hot water and allowed to
steep for a specific period.
E.g. making herbal tea at home.
 Unlike decoction, infusion doesn’t involve boiling of the
powder and it is mainly used to extract polar constituents.

26
 Hot Continuous Extraction
(Soxhlet Extraction)
 The powdered plant material is loaded in the thimble (porous
cellulose) which allow passage of solvent) of the soxhlet
apparatus.
 The solvent is poured in the round bottom flask which is
exposed to direct heat by heating mantle or water bath.
 The vaporized solvent condensed at the condenser drips back to
the powder in the thimble continuously

27
 Soxhlet Extraction…

 The condensed solvent return into the round bottom flask

from the thimble through the siphon tube
 This siphoned solvent contains soluble plant
constituents.
 This process is continuous and is carried out until a
drop of solvent from the siphon tube does not leave
residue when evaporated.
 The solvent must be volatile.
28
29
 Soxhlet Extraction…

 Advantage of this method,

 Large amounts of drug can be extracted with a much
smaller quantity of solvent.
 Economical in terms of time, energy and consequently
financial inputs.

30
 Hydro-distillation
 Used for extraction of volatile oils from dried plant parts resistant to
decomposition by heat.
 Aromatic plant material is packed in a still with sufficient quantity of
water & subjected to boil.
 The hot water and steam generated free the essential oil from the oil
glands.
 The vapor mixture of water and oil is condensed by indirect cooling with
water.
 From the condenser, distillate flows into a separator, where oil separates
automatically from the distillate water.
31
 Hydro-distillation…

 Three types of hydrodistillation for isolating essential oils

from plant materials:

 Water distillation

 Water and steam distillation

 Direct steam distillation

32
 Modern Extraction Techniques
 To overcome limitations of conventional extraction methods,
new and promising extraction techniques were introduced.
 Ultrasound-assisted extraction (UAE)
 Supercritical fluid extraction (SFE)
 Microwave-assisted extraction (MAE)
 Pressurized liquid extraction (PLE)

33
 Ultrasound-assisted extraction (UAE) (Sonication)

 Utilizes sonic energy to assist solvent extraction of

phytoconstituents from plants.
 Ultrasound is a special type of sound wave beyond human
hearing, usually; it ranges from 20 kHz to 100 MHz.
 The possible mechanism is ultrasound intensification of mass
transfer and accelerated access of solvent to cell materials of
plant parts.
 Has inherent advantages over conventional techniques
 Shorter time, lesser solvent requirement and higher
extraction yield

34
 UAE (Sonication)…

 This mechanism involves two main types of physical

phenomena:
i. the diffusion across the cell wall and
ii. rinsing the contents of cell after breaking the walls.
 Disadvantage
 deleterious effect of US energy (if more than 20 kHz) on
the active constituents of medicinal plants through
formation of free radicals and consequently undesirable
changes in the drug molecules.

35
 Supercritical Fluid Extraction

 Using supercritical fluid as a solvent

 Critical point: the highest temperature and pressure at which
the substance can exist in vapor–liquid equilibrium
 Supercritical fluids: Existence of a substance above its
critical point
 Supercritical fluids are
 Homogenous fluids, highly compressible, and higher
diffusivity fluids
36
37
 Supercritical Fluid Extraction…

 CO2 is the widely used SCF for extraction above 31.1 0c and 73.8
bar
 In addition to its favorable physical properties, carbon dioxide is
 inexpensive, safe and abundant, sterile, bacteriostatic and
non-flammable.
• But while CO2 is the preferred fluid for SFE, it possesses several
polarity limitations and organic solvents are frequently added to
alleviate this problem.
• Instead of CO2, argon is being used because it is inexpensive and
more inert.
38
 Supercritical Fluid Extraction…

 Advantages:
 The extraction of constituents at low temperature, which
strictly avoids damage from heat and some organic
solvents.
 Environmentally friendly extraction procedure.
 SFE finds extensive application in the extraction of
pesticides, environmental samples, foods and fragrances,
essential oils, polymers and natural products.
39
 Microwave-assisted extraction (MAE)

 This is based on microwave energy (electromagnetic fields in

the frequency range from 300 MHz to 300 GHz)
 The principle of heating using microwave is based upon its
direct impacts on polar materials.
 Electromagnetic energy is converted to heat following ionic
conduction and dipole rotation mechanisms.
 MAE is recognized as a green technology because it reduces
the use of organic solvent

40
 Microwave-assisted extraction (MAE)…

 Several advantages of MAE have been reported;

 quicker heating for the extraction of bioactive substances
from plant materials
 reduced thermal gradients
 reduced equipment size
 increased extract yield.

41
 Pressurized liquid extraction (PLE)
 The concept of PLE is the application of high pressure to
remain solvent liquid beyond their normal boiling point.
 High pressure facilitates the extraction process.
 PLE technique requires small amounts of solvents because of
the combination of high pressure and temperature which
provides faster extraction.
 The higher extraction temperature can promote higher analyte
solubility by increasing both solubility and mass transfer rate
and, also decreases the viscosity and surface tension of
solvents improving extraction rate. 42
 Factors influencing phyto-chemical profile of
medicinal plants
a) Genetic variants leading to the variability in the chemical
constituents of the drug.
b) Geographical and nutritional factors: altitude, soil
composition, microbial load, climate, temperature etc.
c) Seasonal changes: rainfall, drought, water stress etc.
 alkaloid composition in the leaves of Adhatoda vasika is
low in Feb and March and highest in Aug and Sep.
d) Lack of quality standards: has resulted in mild to serious
adverse affects ranging from hepatotoxicity to death.
43
 Quiz (6%)
Define the following terms
1. Menstruum
2. Miscella
3. Extract
4. Marc
5. Termolabile
6. Write disadvantages of classical methods of
extraction

44
Isolation and purification of active
constituents
1. Classical Methods
2. Modern Methods

45
 Classical Methods of Isolation and Purification

1. Filtration
 Used to separate an insoluble solid from a liquid in a
suspension

46
 Classical Methods of Isolation and Purification…

2. Centrifugation
 Separation based on the difference in the density of the
mixtures.
 The suspension mixture is placed in centrifuge tube which is
plugged in to holders in a centrifuge.
 The centrifuge is turned on to rotate the tubes.

 The denser solid is collected as a lump at the bottom of the tube

with the clear liquid above.
47
 Classical Methods of Isolation and Purification…

Used when
 There is only a small amount of suspension,
 Much faster separation is required

48
 Classical Methods of Isolation and Purification…

3. Crystallization
 Crystals are solids that have a definite regular shape, smooth
flat faces and straight edges
 Crystallization (the process of forming crystals) occurs in two
ways:
– Crystallization by Cooling a Hot Concentrated Solution
– Crystallization by Evaporating a Cold Solution at Room
Temperature
49
 Classical Methods of Isolation and Purification…

I. Crystallization by Cooling a Hot Concentrated Solution

 To obtain crystals from an unsaturated aqueous solution
 The solution is gently heated to make it more concentrated.
The solubility of most solids increase with temperature.
 When a hot concentrated solution is cooled, the solution can’t
hold all of the dissolved solutes
– The “excess” solute separates out as crystals.

50
Classical Methods of Isolation and Purification…

51
 Classical Methods of Isolation and Purification…

II. Crystallization by Evaporating a Cold Solution at Room

Temperature
 As the solvent in a solution evaporates,

– the remaining solution becomes more and more

concentrated
– eventually the solution becomes saturated
• further evaporation causes crystallization to occur.
 However, it may take days or even weeks for crystals to form.
52
Classical Methods of Isolation and Purification…

53
 Classical Methods of Isolation and Purification…

4. Distillation
 To separate a solvent from a solution containing non-volatile
solutes.
 When a solution is boiled,

– only the solvent vaporizes

– the hot vapour formed condenses to liquid again on a cold
surface.
 The liquid collected is the distillate.
54
Classical Methods of Isolation and Purification…

55
 Classical Methods of Isolation and Purification…

5. Fractional Distillation
 To separate a mixture of two or more miscible liquids
 Separation is based on boiling point difference.
 A fractionating column (column packed with glass beads)

– Attached vertically between the flask and the condenser

– provide a large surface area for the repeated condensation
and vaporization of the mixture.

56
 Classical Methods of Isolation and Purification…

 The thermometer measures the temperature of the escaping

vapour
 When the temperature becomes steady, the vapour with the
lowest boiling point comes out from the column first.
 After all of the first liquid distilled off,
– The temperature reading rises again and becomes steady
leading to distillation of the second liquid.
 Fractions with different boiling points can be collected
separately.
57
Classical Methods of Isolation and Purification…

58
6. Sublimation
 Direct change of a solid to vapour
on heating
 When a mixture of two compounds
is subjected to heating, one
compound passes through these
phases while the other compound
remains unaffected by heating.

59
Modern Method of Isolation and Purification…

Chromatography

60
 Chromatography….
• Chromatography:
– Two Greek words
• Chroma- color
• Graphein- to write, hence chromatography means
to write in color
– First employed by Russian scientist Mikhail Tuswett
in 1903
• Used for separation of pigments such as
chlorophyll
 Chromatography…
Definition:
 Chromatography represents a group of methods for
separating and identifying molecular mixtures that
depend on the differential affinities of the solute
between two immiscible phases.
 It is a powerful analytical technique used to separate
and analyze components in a mixture of crude drugs.
 Chromatography…
• A technique of separating the components of a mixture based
on their relative partitioning characteristics between;
– Mobile phase (MP): the solvent moving through the column,
– Stationary phase(Sp): a substance fixed inside the column.
 Components move through the column based on the
difference in their interaction towards MP & Sp.
 Small differences in a compounds’ partition coefficient
result in differential retention on the stationary phase
leading to the separation of components of the mixture.
63
 Chromatography…
• Chromatography may be analytical or preparative

• Analytical chromatography

– For smaller amounts of material,

– For measuring the relative proportions of analytes in a mixture.

– Provide the qualitative profile or finger print of the compound.

• Preparative chromatography

– To purify sufficient quantities of a substance for further use.

– Provide the quantitative profile

– For isolation and purification of the components

64
 Classification

⦿ Shape of Stationary phase ⦿ Separation mechanism

◾ Column chromatography ◾ Adsorption
◾ Planar chromatography chromatography
 Paper chromatography ◾ Partition chromatography
 Thin layer chromatography ◾ Affinity chromatography
⦿ Physical state of mobile phase
◾ Ionexchange
◾ Gas chromatography chromatography
◾ Liquid chromatography ◾ Size-exclusion
◾ Supercritical fluid chromatography
chromatography
65
 Chromatography…
• Solute molecules move through the column only when
they are in the MP.
– The more the solute stays on the MP, the faster it
elutes.
– As the MP velocity increases the velocity of the
solute particles also increase and vice
versa.
• However, the longer the molecules of solute stay on
Sp the longer the retention time 66
 Chromatographic terms
• Analyte: substance to be separated during chromatography.
• Solute: the sample components in partition chromatography.
• Solvent: any substance capable of solubilizing another
substance/ the mobile phase in liquid chromatography.
• Stationary phase: substance fixed in place for the
chromatography procedure. Example the silica layer in TLC
• The mobile phase is the phase that moves in a definite direction.
It may be a liquid, a gas, or a supercritical fluid.
• Retention time: characteristic time it takes for a particular
analyte to pass through the system (from the column inlet to
the detector) under set conditions. 67
 Chromatographic terms…
• Chromatograph: equipment that enables a sophisticated separation.
• Chromatography: physical method of separation that
distributes components to separate between stationary and mobile
phases.
• Chromatogram: visual output of the chromatograph. peaks or patterns
on the chromatogram correspond to different components of the
separated mixture.
• Euate: is what is coming out of the column.
• Eluent: portion of the MP which carries the sample components with in
it. It is the solvent used as MP in LC & the carrier gas in GC.
• Eluotropic series: a list of solvents ranked according to their eluting
power.
 Chromatography…
Role of Chromatography in Pharmacognosy
1. Separation of Plant Constituents
 Chromatography helps in separating multiple
compounds in herbal extracts; alkaloids, flavonoids,
glycosides, terpenoids…
2. Identification of Bioactive Compounds
 Chromatography aids in identifying medicinally
active compounds in plants by comparing their
retention times or spectral data with known standards.
 Chromatography…
Roles…
3. Quality Control of Herbal Medicines
 Chromatographic techniques help ensure the presence
of active ingredients and detect adulterants.
4. Quantification of Phytochemicals
 Chromatographic methods help measure the
concentration of bioactive compounds in medicinal
plants, ensuring proper dosage in herbal formulations.
 Chromatography…

Principles of chromatography
 There are five basic principles of chromatography;
 Adsorption chromatography
Stationary phase: solid
Mobile phase: liquid/gas

E.g. Column chromatography, TLC, HPTLC,SC

 Chromatography…
Principles of chromatography…
 Partition chromatography
Stationary phase: liquid (polar & non-volatile)
Mobile phase: liquid or gas
E.g. HPLC, LLC, GC
 Affinity chromatography
Stationary phase: solid (porous beads or resin
with immobilized ligands)
Mobile phase: liquid
 Chromatography…
Principles of chromatography…
 Size exclusion chromatography
Stationary phase: solid (porous beads made of
dextran or silica
Mobile phase: liquid
 Ion exchange chromatography
Stationary phase: solid (charged beads of resin)
Mobile phase: liquid (aq. Buffer solutions with
various PH)
 Partition chromatography
 Chromatography of this principle separates components
based on their distribution between two liquid
immiscible phases (mobile phase and stationary phase).
 The components of the mixture get distributed into two
liquid phases due to differences in partition coefficients
during the flow of mobile phase in the chromatography
column.
 This method is commonly used in biochemical
applications, such as separating amino acids, sugars,
and other polar compounds.
 Partition chromatography…
Paper Chromatography
 Involves using a strip of paper as the
stationary phase.
 The sample is applied to the paper,
which is then placed in a solvent that
moves up by capillary action.
 Often used for educational purposes
and simple analyses, such as
separating plant pigments.
 Partition chromatography…
Liquid-Liquid Partition Chromatography:
Separates compounds based on their solubility in two
immiscible liquids.
The stationary phase is a liquid that coats a solid
support, while the mobile phase is another liquid.
Used for separating organic compounds from
mixtures.
 Partition chromatography…
High-Performance Liquid Chromatography (HPLC):

A sophisticated (advanced) form of liquid

chromatography that uses high pressure to push
solvents through a packed column allowing for better
separation and resolution.
 It can employ various stationary phases and mobile
phases to achieve separation based on partitioning.
High Performance Liquid Chromatography
(HPLC)
• Utilizes very small packing particles
and a relatively high pressure.
• Works by forcing the Mp through the
column at much higher rate
increasing the resolution rate.
• It can be
– Normal phase HPLC
– Reverse phase HPLC
39
 High Performance Liquid Chromatography
(HPLC)…
[Link] phase HPLC
• Polar Sp: SiO2, Al2O3
• Non polar Mp: Hexane, heptane,
cyclohexane…
• Polar compounds travel slower and eluted
slowly due to higher affinity to the Sp
2. Reverse phase HPLC
• Non polar Sp: n-octadecyl, n-octyl, phenyl
diol,
• Polar Mp: methanol or acetonitrile/water
• Polar compounds elute faster than non polar
compounds
 HPLC system

• Solvent Reservoir

• Degasser

• Solvent Delivery
System (Pump)

• Injector

• Column & oven

• Detectors
• Recorder (Data
Collection)
 Partition chromatography…
Gas Chromatography (GC)
 GC separates volatile compounds based on their
partitioning between a stationary phase (often a liquid
coated on a solid support) and a gas mobile phase.
 It primarily operates under the principles of partition
chromatography, where analytes are separated based
on their volatility and interaction with the stationary
phase.
 Gas chromatography (GC)
• Gas-liquid chromatography, (GLC) have.
– Mobile phase: inert gas (helium, nitrogen)
– Stationary phase: supported liquid (SiO2
coated with polymer)
GC:
 Involves a sample being vaporized and injected
onto the head of the chromatographic column.
 The sample is transported through the column
by the flow of inert, gaseous mobile phase.
 The column itself contains a liquid Sp which is
adsorbed onto the surface of an inert solid 35
 Instrumentation of GC

Flow meter

Injector Septum Detector GC

Pressure Flow Chart
regulator controller 
Recorder

Oven

Gas
supply Column
 Gas chromatography
(GC)…
• For GC analysis the sample must be volatile, or
be able to be converted to volatile derivative.
Application of GC

– Food Analysis

– Drug Analysis

– Environmental Analysis

– Forensic Analysis
 Adsorption Chromatography
 In adsorption chromatography, separation occurs based on
the differential adsorption of compounds onto a solid
stationary phase.
 The stationary phase typically consists a solid material (like
silica gel or alumina) that interacts with the sample
components.
 The compounds in the mixture are separated based on their
varying affinities for the stationary phase.
 Column chromatography
 Involves packing a column with a stationary phase
(solid or liquid) and allowing the mobile phase
(liquid or gas) to flow through it.
 As the mixture is introduced into the column,
different components move at different rates based on
their interactions with the stationary phase, leading to
separation.
Column chromatography…
 Column Chromatography…
Types:
 Flash Column Chromatography: A faster version of
column chromatography that uses increased pressure to
speed up the elution process.
 Gravity Column Chromatography: Relies on gravity
to move the mobile phase through the column.
 CC is used extensively for purifying compounds in
organic synthesis, isolating natural products, and
separating complex mixtures.
 Thin Layer Chromatography
(TLC)
• The Sp is a thin layer of adsorbent (usually silica gel
or alumina)
coated on a glass/ aluminum plate.
• Binder such as CaSo4 is spread on a glass plate
& allowed to dry.
• The plates are activated at 110oC for 30 minutes
and used.
• Compared to paper, it has the advantage of
faster runs, better
separations, and the choice between different
adsorbents.
89
 Thin Layer Chromatography
(TLC)…
• The mixture to be separated is applied as a
spot near the base of the plate, which is then
placed in a closed glass tank containing a
layer of developing solvent.
• The solvent moves up the plate by capillary
action
– carrying the less strongly adsorbed
components of the mixture with it,
• The more strongly adsorbed
compounds remain near the base of
the plate.
90
Thin Layer Chromatography
(TLC)…
Visualization
 If invisible, the spots
may be made visible by
Heating for a
specific period
Examining under U.V
light (if substances
are florescent).
Spraying the finished
chromatogram with a
suitable reagent
e.g. Dragendrof's reagent 91
 Thin Layer Chromatography (TLC)
…
• Each spot on TLC plate can be distinguished by
its retention factor (Rf ) value on the surface of
the stationary phase,
𝑅𝑓 value =Distance travelled by
compound Distance
travelled by solvent front

• Rf value is a constant for a compound

under constant chromatographic
conditions
– Used for identification of unknown
92
substance.
 Thin Layer Chromatography
(TLC)…
Advantages of TLC
- Simple, inexpensive
- Quick – results can be achieved from
between 30 minutes to a
few hours
- Good separation of spots (compounds)
- Very sensitive

- The chromatogram is resistant to the action

of chemicals used for the visualization of
compounds. 93
 Size Exclusion Chromatography(SEC)

• Is non-adsorption and non-destructive

method
• Applied to large molecules or
macromolecular complexes such as
proteins and industrial polymers.
• Unlike IEC, SEC does not involve
chemical interaction with sample

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 Mechanism of
separation
• Separate molecules in solution by their
hydrodynamic volumes
• Smallest molecules enter deep into the
pores and elute last.
• Medium sized components penetrate some
depth and elute separately according to
their size.
• Macromolecules, bigger than the pore size
will not be retained.
• Exclusion limit is maximum molecular
95
Figure 2: schematic representation of the operation of
SEC column
96
 Ion Exchange Chromatography (IEC)

• Separates ions and polar molecules based on their

charge and interaction with the stationary phase.

• Commonly used for analyzing anions, cations, and

small organic acids in environmental samples, food
products, and pharmaceuticals
 Affinity Chromatography
 This method utilizes specific interactions between an
immobilized ligand on the stationary phase and the
target molecule in the mobile phase.
 The adsorbent used is one of the biological substance
having a specific affinity for other substances.
 Frequently used for purifying proteins, enzymes,
antigens and antibodies.
 Affinity Chromatography
• These two substances are the biologically interacting pairs.
• Such adsorbent is attached to a porous stationary phase
and placed in a column.
• When a mixture containing the other component of the
adsorbent (interacting pair) is passed through the stationary
phase, selective separation occurs.
• Affinity chromatography is efficiently applied for
separation of enzymes, utilizing the technique of enzyme
substrate interaction.
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Thank You!!