EXTRACTION

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EXTRACTION
Definition:

Extraction is the process of isolation of soluble material from an insoluble

residue which may be liquid or solid, by treatment with solvent.

On the basis of physical nature of crude drug to be extracted, that is liquid or
solid, the extraction process may be liquid – liquid or solid – liquid
extraction.

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Is the separation of medicinally active portions of plant (and animal) tissues
using selective solvents through standard procedures.

 The products so obtained from plants are relatively complex mixtures of

metabolites, in liquid or semisolid state or in dry powder form (after
removing the solvent), & are intended for oral or external use.

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Menstrum: Solvent used for extraction (ex. water, alcohol, ether, acetone,
ethyl acetate)
Marc: The inert fibrous and other insoluble materials remaining after
extraction
Extractives: Concentrated preparations of vegetable or animal drugs
obtained by removal of the active constituents of the respective drug with
suitable menstrum, evaporation of all or nearly all solvent.
Tinctures: are alcoholic or hydro alcoholic solutions prepared from
vegetable material or from chemical substances. E.g. belladona tincture
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The Medicinal plants constitute an effective source of both traditional and

modern medicines, herbal medicine has been shown to have genuine utility
and about 80% of rural population depends on it as primary health care.
[WHO, (2005)]

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MEDICINAL PLANTS ARE THE RICHEST BIO-
RESOURCE

drugs of traditional systems of medicine

modern medicines
nutraceuticals
food supplements
folk medicines
pharmaceutical intermediates
chemical entities for synthetic drugs.
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Galenicals
These include classes of preparations viz.,
decoctions
infusions
fluid extracts
tinctures
pilular (semisolid) extracts
powdered extracts.

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STANDARDIZED EXTRACTION
The purpose of standardized extraction procedures for crude
drugs (medicinal & aromatic plant parts)

To attain the therapeutically desired portions

To eliminate unwanted material by treatment with a selective
solvent known as “menstrum”

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THE GENERAL TECHNIQUES OF MEDICINAL
PLANT EXTRACTION

Macération Aqueous-alcoholic extraction by

fermentation,
Infusion
Counter-current extraction,
Percolation
Microwave-assisted extraction,
Digestion
Ultrasound extraction (sonication),
Décoction
Supercritical fluid extraction,
Hot continuous extraction (Soxhlet)
Phytonic extraction (with
hydrofluorocarbon solvents).

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EXTRACTION TECHNIQUES FOR AROMATIC
PLANTS

Hydro distillation techniques (water distillation, steam distillation, water and

steam distillation)
Hydrolytic maceration followed by distillation, expression and enfleurage
(cold fat extraction)
Headspace trapping
Solid phase micro-extraction
Protoplast extraction
Microdistillation
Thermomicrodistillation and Molecular distillation.
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CHOICE OF SOLVENTS

Successful determination of biologically active compounds depends on the

type of solvent used in the extraction procedure.

The choice of solvent is influenced by what is intended with the extract.

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PROPERTIES OF A GOOD SOLVENT IN PLANT
EXTRACTIONS

low toxicity

ease of evaporation at low heat

promotion of rapid physiologic absorption of the extract

preservative action

inability to cause the extract to complex or dissociate.

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SOLVENTS USED FOR ACTIVE COMPONENT
EXTRACTION

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WATER

Water is universal solvent.

used to extract plant products with antimicrobial activity.
Traditional healers use primarily water & consistent antimicrobial activity is
obtained.
Plant extracts: organic solvents >>> water extract.
Water soluble flavonoids (mostly anthocyanins) have no antimicrobial
significance.
only water soluble phenolics are important as antioxidant compound.

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ACETONE

Dissolves many hydrophilic and lipophilic components.

a very useful extractant, especially for antimicrobial studies
(phenolic group extract).
extraction of tannins + phenolics:
aqueous acetone >>> aqueous methanol
Both acetone and methanol were found to extract saponins 
antimicrobial activity.

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ALCOHOL
The identified components from plants (antimicrobial) = aromatic or
saturated organic compounds most often obtained through initial ethanol
or methanol extraction.
Ethanol, found easier to penetrate the cellular membrane to extract the
intracellular ingredients(polyphenols) from the plant material.
Methanol is more polar than ethanol but due to its cytotoxic nature.
The higher concentrations of more bioactive flavonoid compounds were
detected with 70% ethanol
due to its higher polarity than pure ethanol.

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Chloroform
Used to obtain tannins and terpenoids.
Terpenoid lactones successive extractions of dried barks with chloroform.
Ether
Commonly used selectively for the extraction of coumarins and fatty acids.
Dichloromethanol
Specially used for the selective extraction of only terpenoids.

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Steps Involved in the Extraction of Medicinal Plants

1.Size reduction
2. Extraction
3. Filtration
4. Concentration
5. Drying

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1. Size Reduction
Objective:
To rupture plant organ, tissue & cell structures so that its medicinal
ingredients are exposed to the extraction solvent.
Size reduction maximizes the surface area, which in turn enhances the mass
transfer of active principle from plant material to the solvent.
The 30-40 mesh size is optimal.
Hammer mill or a disc pulverizer which has built in sieves controlled by
varying the speed of the rotor clearance b/w the hammers & the lining of the
grinder.

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2. Extraction
By using both modern extraction method Traditional extraction method
and traditional extraction method
Modern extraction method Macération
 Counter-current Percolation
 Microwave assisited Infusion
Ultrasound extraction (sonication), Décoction
Supercritical fluid extraction,
Hot continuos extraction (Soxhlet)

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Parameters influencing the quality of an extract

Plant part used as starting material

Solvent used for extraction
Extraction procedure

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Effect of extracted plant phytochemicals depends on

The nature of the plant material

Its origin
Degree of processing
Moisture content
Particle size

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Selection of Plant
Plant based natural constituents can be derived from any part of
the plant like bark, leaves, flowers, roots, fruits, seeds, etc.
Plants are usually air dried to a constant weight before extraction.
 oven drying: every part were cut into pieces dried in an oven @
60°C for 9 hrs. & pulverized.
Other method for drying the plants is the oven drying at about
40°C for 72 h.

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3. FILTRATION

The extract so obtained is separated out from the marc (exhausted

plant material) by allowing it to trickle into a holding tank through
the built-in false bottom of the extractor, which is covered with a
filter cloth.
The marc is retained at the false bottom, and the extract is
received in the holding tank.
From the holding tank, the extract is pumped into a sparkler filter
to remove fine or colloidal particles from the extract.

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4. CONCENTRATION
The enriched extract from percolators or extractors, known as miscella, is fed
into a wiped film evaporator where it is concentrated under vacuum to produce
a thick concentrated extract.
The concentrated extract is further fed into a vacuum chamber dryer to produce
a solid mass free from solvent.
The solvent recovered from the wiped film evaporator and vacuum chamber
dryer is recycled back to the percolator or extractor for the next batch of plant
material.
The solid mass thus obtained is pulverized and used directly for the desired
pharmaceutical formulations or further processed for isolation of its
phytoconstituents.
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5. DRYING
The filtered extract is subjected to spray drying with a high pressure pump at a
controlled feed rate and temperature to get dry powder.

The desired particle size of the product is obtained by controlling the inside
temperature of the chamber and by varying the pressure of the pump.

The dry powder is mixed with suitable diluents or excipients and blended in a
double cone mixer to obtain a homogeneous powder that can be straight away
used (for example, for filling in capsules or making tablets).
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VARIATION IN EXTRACTION
METHODS
Length of the extraction period
Solvent used
pH of the solvent
Temperature
Particle size of the plant tissues
The solvent-to-sample ratio.

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PARAMETERS FOR SELECTING AN
APPROPRIATE EXTRACTION METHOD
Authentication of plant material by botanist.
Use the right plant part + the age of plant + the time, season & place
of collection.
The nature of its chemical constituents.
Grinding methods & powdering techniques.
Nature of constituents (polar/nonpolar).
The quality of water / menstruum.
The design & material of fabrication of the extractor.
Analytical parameters of the final extract,(TLC/HPLC).

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Methods of extracton

Solvent extraction Distillation Expression

(Mechanical extraction)
Mostly for non-volatile For volatile plant
plant constuituents and constuituents For both volatile and
sometimes for voilatile ones non-volatile plant
constituents

Volatile solvent Non-volatile

extraction solvent
extraction
> pet. ether
> hexane > petrolatum
> methanol > silicon polymers
> butter
> chloroform
> acetone etc. > fixed oils

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1. MACERATION
The whole / coarsely powdered crude drug is placed in a stoppered container
with the solvent.
Allow to stand @ room temperature for a period of at least 3 days with
frequent agitation until the soluble matter gets dissolved.
The mixture then is strained, the marc (the damp solid material) is pressed,
The combined liquids are clarified by filtration or decantation after standing.
This method is best suitable for
use in case of the thermolabile drugs.

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2. INFUSIONS
Fresh infusions are prepared by macerating the crude drug for a
short period of time with cold or boiling water.
These are dilute solutions of the readily soluble constituents of
crude drugs.

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3. 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.
Image=microwave
Digestion system

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4. DECOCTION
In this process, the crude drug is boiled in a specified volume of water (1;4) for a
defined time.
Volume is reduced to 1/4th the original
It is then cooled and strained / filtered.
This procedure is suitable for extracting water-soluble, heat-stable constituents.
Typically used in preparation of Ayurvedic extracts = “quath” / “kawath”

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5. PERCOLATION

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Percolation is a continuous flow of the solvent through the bed of crude drug
material to get the extract.

The percolator is generally V- shaped vessel open at both the ends.

Percolation process is dependent on the flow of the solvent through

powdered drug and it yields a product of greater concentration than the
maceration process.

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Used most frequently to extract active ingredients in the preparation of tinctures and
fluid extracts.

The solid ingredients are moistened with an appropriate amount of the specified
menstruum,

Allowed to stand for approximately 4 hours in a well closed container, After stand
time, the mass is packed & the top of the percolator is closed.

the mixture is allowed to macerate in the closed percolator for 24 h.

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Additional menstruum is added as required, until the percolate measures
about three-quarters of the required volume of the finished product.
The marc is then pressed and the expressed liquid is added to the percolate.
Sufficient menstruum is added to produce the required volume.
The mixed liquid is clarified by filtration or by standing followed by
decanting.

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6. HOT CONTINUOUS EXTRACTION (SOXHLET
EXTRACTION)

Soxhlet extraction is the process of continuous extraction in which the same

solvent can be circulated through the extractor several times.

The process involves extraction followed by evaporation of the solvent.

The vapours of the solvent are taken to a condenser and the condensed liquid
is returned to the drug for continuous extraction.

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The finely ground crude drug is placed in a porous bag or “thimble” made of
strong filter paper, which is placed in chamber of the Soxhlet apparatus.

The extracting solvent in flask is heated, and its vapors condense in

condenser.

The condensed extract drips into the thimble containing the crude drug &
extracts it by contact.

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SOXHLET APPARATUS

When the level of liquid in chamber rises to the top of siphon tube, the liquid
contents of chamber siphon into flask
This process is continuous and is carried out until a drop of solvent from the
siphon tube does not leave residue when evaporated.

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Advantage:

Less solvent is needed for yielding more concentrated products.

The extraction can be continued until complete exhaustion of the drug.

Disadvantage:

It is restricted to pure boiling solvents or to azeotropes.

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AQUEOUS ALCOHOLIC EXTRACTION BY
FERMENTATION

Some medicinal preparations of Ayurveda (asava & arista) adopt

the technique of fermentation for extracting the active principles.
The extraction procedure involves soaking the crude drug,
[powder / a decoction (kasaya)], for a specified period of time
Undergoes fermentation & generates alcohol in situ.
This facilitates the extraction of the active constituents contained
in the plant material.
The alcohol thus generated also serves as a preservative.
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Some examples of Ayurvedic preparations:
karpurasava
kanakasava
dasmularista.
If the fermentation is to be carried out in an earthen vessel, it should not be
new: 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.

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12/01/2023 AB 48
COUNTER-CURRENT
EXTRACTION
Wet raw material is pulverized using toothed disc disintegrators to produce a
fine slurry.
Material to be extracted is moved in one direction generally in the form of a
fine slurry within a cylindrical extractor where it comes in contact with
extraction solvent.
The further the starting material moves, the more concentrated the extract
becomes.
Complete extraction is thus possible when the quantities of solvent &
material. Their flow rates should be optimized.
sufficiently concentrated extract comes out at one end of the extractor while
the marc, practically free of visible solvent falls out from the other end.
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ULTRASOUND EXTRACTION
(SONICATION)
The procedure involves the use of ultrasound with frequencies ranging from
20 kHz to 2000 kHz.
This increases the permeability of
cell walls & produces cavitation.
Eg: extraction of rauwolfia root.

Deleterious effect: Ultrasound energy (>20 kHz) on the active constituents of

medicinal plants through formation of free radicals and consequently
undesirable changes in the drug molecules.
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SUPERCRITICAL FLUID EXTRACTION

Certain gases behave like free flowing liquids or super critical fluids at the
critical point of temperature and pressure.
Such super critical fluids have very high penetration power and extraction
efficiency.
Gases such as CO2 are held as supercritical fluid at the critical point of 73.83
bar pressure and 31.060C.
At this point CO2 behave like liquified gas or free flowing liquid and assists
the extraction of the Phyto- chemical constituents from the crude drug.

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Advantages of CO2 in super critical fluid extraction are that it is sterile and
bacteriostatic, and it is non- combustible and non- explosive.
CO2 is harmless to the environment and no waste products are generated
during this process.
The component recovery rates generally increase with increasing
pressure/temperature.
The highest recovery rates in case of argon:@ 500 atm & 150° C.

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PHYTONICS PROCESS
A new solvent based on hydrofluorocarbon-134a, a new technology to optimize the
extraction of plant materials.
Advanced Phytonics Limited (Manchester, UK) has developed patented technology
termed “phytonics process”.
The products are fragrant components of essential oils & Biological/phytopharmacological
extracts.
The core of the solvent is 1,1,2,2-tetrafluoroethane, (HFC-134a).
HFC-134a developed as a replacement for chlorofluorocarbons. (Boiling Point -25° C).
the solvents can be customized: by using modified solvents with HFC-134a.
The process can be made highly selective in extracting a specific class of
phytoconstituents.
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ISOLATION AND
PURIFICATION
The physical methods used for purification are chromatographic
technique and methods such as fractional crystallisation, fractional
distillation, fractional liberation and sublimation.
Fractional crystallisation:
It is depends upon the inherent character of the compound which
forms crystals at the point of super saturation in the solvent in
which it is soluble.
Compounds such as sugars, glycosides, alkaloids, steroids,
triterpenoids, flavonoids etc. shows crystalline nature.
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Processes such as concentration, slow evaporation and refrigeration are used
for crystallising the product.
Fractional distillation:
For distillation compound should have volatile nature.
It is used for separation of essential oil components.
This process widely used for separation of hydrocarbons from oxygenated
volatile oil components.
Components such as citral, citronellal and eucalyptols.

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Fractional Liberation

In this process, groups of compounds having tendency of precipitation comes

out of the solution.

Alkaloids, is modified by converting it to its salt form or free base.

This process is used for separation of cinchona alkaloid quinine, isolation of

morphine and many other alkaloids.

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Sublimation:

In this process the compound, if subjected to heat, changes from solid to
gaseous state directly without passing through the liquid stage.

The process is traditionally used for the separation of camphor from the chips
of wood of Cinnamomum camphor to obtain solid sublimate of camphor.

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CHROMATOGRAPHY

Chromatography is usually introduced as a technique for

separating and/or identifying the components in a mixture.

The basic principle is that components in a mixture have

different tendencies to adsorb onto a surface or dissolve in a
solvent.

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Chromatography is a physical method of separation in which the components to
be separated are distributed between two phases (KD/P = Distribution/partition
constant)
one of which is stationary (stationary phase) while the other (the mobile phase)
moves through it in a definite direction.
The chromatographic process occurs due to differences in the distribution
constant of the individual sample components.
 The sample under go repeated equilibration
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BASIC CHROMATOGRAPHIC TERMINOLOGY

Chromatograph: Instrument employed for a chromatography.

Stationary phase: Phase that stays in place inside the column
Mobile phase: Solvent moving through the column, either a liquid in LC or
gas in GC.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through the column.
Chromatogram: Graph showing detector response as a function of a time.
Flow rate: How much mobile phase passed / minute (ml/min).
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TYPE OF CHROMATOGRAPHY

1. Paper chromatography

2. Thin layer chromatography (TLC)

3. Gas chromatography (GC)

4. liquid chromatography (LC)

5. High performance liquid chromatography (HPLC)

6. Ion exchange chromatography

7. Gel filtration chromatography.

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All chromatographic methods require one static part (the stationary phase)
and one moving part (the mobile phase).
The techniques rely on one of the following phenomena: adsorption;
partition; ion exchange; or molecular exclusion.
Adsorption versus Absorption:
 In absorption one substance penetrate in to the bulk of another substance.
 A sponge and water
 Adsorption is a surface phenomenon where interaction takes place only on
the surface of one substance.

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Mobile phase - flows through column, carries analyte.
Gas = Gas Chromatography (GC)
Liquid = Liquid Chromatography (LC), Thin Layer Chromatography (TLC)
Supercritical fluid = Supercritical Fluid Chromatography (SFC)
Stationary phase - stays in a place, does not move.
GC, LC placed inside of the column
TLC – layer of a sorbent on the plate
The SEPARATION is based on the partitioning between the mobile and
stationary phase.
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CLASSIFICATION OF CHROMATOGRAPHY

2. Based on the Configuration of the instrument (the way the stationary phase is
dispersed)
a. Planar Chromatography: - the SP is immobilized in the plane. Ex.. TLC, PC
b. Columnar Chromatography: - the SP is immobilized in the column
Ex. GC, CC, HPLC
3. Based on the nature of the Mobile phase: -
A. Liquid Chromatography
B. Gas Chromatography

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4. Based on the nature of the Stationary Phase
A. Normal phase Chromatography: - stationary phase is polar,
mobile phase is non polar
B. Reverse phase Chromatography: - stationary phase is non-
polar, mobile phase is polar

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5.Based on column type
1. Closed column chromatography
• stationary phase is packed inside a column
• mobile phase + solute flows through the column -> separation
2. Open column chromatography
 Paper chromatography- sheet of paper is used to support the stationary
phase
 Thin-layer chromatography- adsorbent is spread evenly over the surface of a
flat sheet of glass
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Adsorption-Chromatography
It has a solid stationary phase and a liquid or gaseous mobile
phase
Each solute has its own equilibrium between adsorption onto the
surface of the solid and solubility in the solvent
The least soluble or best adsorbed ones travel more slowly.
The result is a separation into bands containing different solutes.

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One of the oldest types of strength
chromatography around
It involves
a stationary solid phase
a liquid or gaseous mobile phase
Not used as widely as partition
chromatography
 used mainly on TLC & CC packed with
silica gel/alumina
Separation is based on adsorption
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PARTITION
In partition chromatography the stationary phase is a non-volatile liquid which is
held as a thin layer (or film) on the surface of an inert solid.

The mixture to be separated is carried by a gas or a liquid as the mobile phase.

The solutes distribute themselves between the moving and the stationary phases,
with the more soluble component in the mobile phase reaching the end of the
chromatography column first

Paper chromatography is an example of partition chromatography.

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Based on a thin film formed on the
surface of a solid support by a liquid
stationary phase.
Solute equilibrates between the mobile
phase and the stationary liquid.
Partition - based on the relative
solubility of analyte in mobile and
stationary phases
Reversed phase chromatography (GC,
HPLC)
Paper chromatography

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SELECTION OF THE SOLID SUPPORT

The support material should:

 adsorb & retain the liquid stationary phase.
 be mechanically stable.
 be easy to pack.
 not retard the solvent flow
 expose as large surface as possible to the mobile phase
Examples of solid supports:
Silica gel, diatomaceous earth (as kieselguhr, celite etc.) & cellulose.

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1. PURPOSE OF CHROMATOGRAPHY

Analytical :
 For identification and analysis purpose
 Chemical profile (composition) of a sample
 To check the purity of the compound
 for screening purpose
 A very thin layer of stationary phase coated on supporting
materials
 Deals with analytes in micrograms quantities
eg. TLC; HPLC; GC
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Preparative :
 vital for Isolation of compounds
 purify and collect one or more components of a sample
Relatively thicker layer of stationary phase coated on
supporting materials
 Deals with analytes in milligrams or large quantities
eg. PTLC; Prep-HPLC; Prep-GC

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PAPER CHROMATOGRAPHY

A chromatographic analytical separation technique for complex mixtures

involving the progressive adsorption of the dissolved component onto a
special grade of paper.
SP: Water adsorbed on cellulose
The SP can be modified in to reversed phase or ion exchange
MP: A binary mixture of incompletely miscible components
MoS: Partition

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 PRINCIPLE

 It is an example of a partition chromatography

 Liquid-Liquid chromatography
 Cellulose fibers strips serve as inert solid support.
 The factor governing separation of mixtures of solutes on filter paper
is the partition between two immiscible phases.
 One is usually water adsorbed on cellulose fibers in the paper
(stationary phase).
 The second is the organic solvent flows past the sample on the paper
(stationary phase).

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What are some limitations of paper
chromatography?

Paper chromatography is a very simple and cost effective method for separation of
components.
But it has some major disadvantages:-
 paper chromatography gives better sample resolution, but it is a very slow technique.
 Paper chromatographic techniques can not be used in separation of volatile substances
such as hydrocarbons and volatile fatty acids.
 The lower limit for the detection of most compounds is 1-5 mg.
 In paper chromatography utmost care has to be taken while hanging the paper in the
chromatography chamber. If the paper is tilted, the solvent front will be uneven due to
which calculating the correct Rf value will be almost impossible. A similar problem
arises when the paper is cut unevenly.
 Paper chromatography also requires some skill in sample spotting.
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Theretention factor, or Rf, is defined as the distance traveled by the
compound divided by the distance traveled by the solvent

The Rf value of a compound in a particular solvent system is constant

under identical conditions of the experiment, e.g. temperature, pH, etc.
For example, if a compound travels 2.1 cm and the solvent front travels
2.8 cm, the Rf is 0.75:

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SPOT DETECTION

- Color spot  observed by naked eye

- Non – color spot  color reagent will give specific colors for different
compound.
Example :
 Ninhydrin – a.amino
 Iodine–alkaloid

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Thin layer chromatography (TLC)

Overview of TLC
TLC—one form of solid-liquid chromatography
Adsorbent—solid phase; it won’t dissolve in the associated
liquid phase
Ex: Silica Gel (Si02) & Alumina (Al2O3)
Eluting—liquid phase
Solvatedsubstances are spotted onto a TLC plate coated with
the adsorbent and allowed to elute up the plate by capillary
action
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 Capillary action occurs because molecules of a pure substance is sticky,
thanks to the forces of cohesion (similar molecules of a pure substance
like to stay close together) and adhesion (molecules of a given pure
substance are attracted and stick to other substances).
 Adhesion of a liquid to the walls of a vessel will cause an upward force
on the liquid at the edges and result in a meniscus which turns upward.
The surface tension acts to hold the surface intact.
 Capillary action occurs when the adhesion to the walls is stronger than
the cohesive forces between the liquid molecules. The height to which
capillary action will take the liquid in a uniform circular tube (picture
to left) is limited by surface tension and, of course, gravity.
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Four Purposes of Performing TLC

Isolation components in a mixture (preparative TLC)

Assess purity (how many spots are present)
Identify a substance (analytical TLC)
Assess compound polarity

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TLC Development Setup

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This technique manipulates POLARITY

More polar substances bind strongly to the adsorbent and elute
SLOWER
Less polar substances bind weakly to the adsorbent and elute
FASTER

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Overview of TLC—The R f Value

A given compound will always travel a fixed distance relative to the distance
the solvent travels
This ratio is called the Rf value and is calculated in the following manner: .
distance traveled by substance .
distance traveled by solvent front

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TLC Visualization Methods

 Ultraviolet Light—some organic compounds illuminate or fluoresce under

short-wave UV light
 Iodine Vapor—forms brown/ yellow complexes with organic compounds
 Fluorescent Indicators—compounds fluoresce when placed under UV light
 Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to
light

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Plate Preparation
Adsorbent + Calcium sulfate (gypsum) + Water = thick slurry
Spread, pouring, dipping and spraying done on glass, aluminum foil, or
plastic.
analytical TLC 0.1–0.25 mm
preparative TLC around 1–2 mm
30 minutes at 110 °C. “Activation”
Stored over desiccant.

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Advantages Disadvantages
 Convenient and simple.  Lack of automation
 Simple detection of separated bands  HPTLC (automated TLC)
 Fast screening of raw materials  the problems of reproducibility which
 Inexpensive sometimes occur
 Consumes smaller amounts of  Lack of accuracy in quantitation
solvents than HPLC.
 Simultaneous application and
determination of samples on a single
TLC plate

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Column chromatography
It is used for the separation and isolation of different constituents of
extracts.
Column chromatographic grade adsorbents of choice are used are
used for packing the column and various organic solvents are used
as eluting solvents.
A glass column is used in this technique.
Silica gel is used as a packing column.
Sample applied on the top of the column, in such a way that narrow
band is formed, for further elution.
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The column is subsequently eluted with various proportions of solvents in the
order of increasing polarity.
The fractions collected from the bottom are distilled to recover the solvent .

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HPLC (High Performance Liquid Chromatography)

It utilises a column of packing material of particle of small size and regular

shape.
As the column finely packed, pressure up to 5000 lb in-2 can be employed to
achieve acceptable flow rate.
This technique has advantages of quantitatively analysing the components of
the mixture at a high level of resolution and sensitivity.
The disadvantages of this method include the cost of pump / detector /
recorder / column and the requirement of injecting only highly pure, particle
free solution on the column to avoid blockage and column.

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