TYBSC Analytical Chemistry

THIN LAYER
CHROMATOGRAPHY
Dr.Bhagure G.R.

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 Contents
2.2.1 Principle of Thin Layer Chromatography (TLC),
Retardation factor Rf.
2.2.2 Techniques in Thin Layer Chromatography
A) Stationary phase coating materials
B) Preparation of Plates
C) Mobile phase : Selection of Solvents
D)Methods of Plate Development Ascending development,
Descending Development, Two dimensional Development,
Continuous Development, and Multiple Development.
E) Detection methods for locating separated components:
Qualitative analysis and Quantitative analysis.
2.2.3 Applications of Thin layer chromatography
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 THIN LAYER
CHROMATOGRAPHY
Principle
Materials used to perform TLC
Experimental technique
Application
Advantages
Disadvantages

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 Principle:
 The sample is dissolved in in a volatile solvent

 Sample is applied with the help of capillary on to the base line

drawn on the solid adsorbent.
 The plate is dipped in to the solvent working as mobile phase.

 As the mobile phase rises up the TLC plate by capillary action,

the components dissolve in the solvent and move up the TLC
plate.
 In adsorption separation will occur on the basis of differences
in adsorption (preferential adsorption). Weekly adsorbed
component will separate first and strongly later on.
 In partition , if the component is soluble in the mobile phase it
will move with the solvent and vise versa .
 The movement of the solute is measured relative to that of
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solvent is expressed as the retardation factor.
 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
The strength of interactions between the adsorbent
and eluting components vary approximately in this
order:
Salt formation > coordination > H-bonding >

dipole-dipole > van der Waals

More Less
Polar Polar

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Polarity decreases
 OH
Silica Gel OH

OH
Silica
OH Gel

OH

OH
OH
Mobile
phase
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 Less
Polar
More
Polar
Adsorbs weakly
and separate
very fast

Adsorbs stronger
and separate
very slowly

Sample to be applied
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on this area
 The Rf 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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 THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front

Rf (B) = 3.0 cm = 0.60

5.0 cm
Distance solvent
migrated = 5.0 cm
4.0 cm
Distance A
migrated = 3.0 cm Rf (C) = 0.8 cm = 0.16
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm
The Rf is defined as the distance the center of the spot moved divided
by9 the distance the solvent front moved (both measured from the
origin)
 Materials used in TLC

Glass Plate
Adsorbents
Oven for activation of plate
Developing chamber
Mobile Phase
A device for applying the adsorbent layer
Storage facility for the prepared plate

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 Materials used in TLC

Hooper
A device for applying
Glass the adsorbent layer
Mobile
phase
Plate

Developing chamber

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 Stationery phase
Stationery phase Description Application

Silica gel G Silica gel with average Used in wide range

particle size 15µm pharmacopoeial test
containing ca 13%
calcium sulfate binding
agent

Silica gel G254 Silica gel G with Same application with Silica
fluorescence added gel G where visualization is to
be carried out under UV light.

Alumina

(Al2O3)
Cellulose Cellulose powder of less Identification of tetracycline's
12 than 30µm particle size.
 MOBILE PHASE
 TLC Solvents or Solvent Systems.
 A single solvent or mixture of two solvents can work
as mobile phase in TLC .Ex. petroleum ether, carbon
tetrachloride, chloroform, ethyl acetate, hexane can
used as mobile phase.
 The ability of mobile phase to move up is depend on
the polarity itself
 Volatile organic solvents is preferably used as
mobile phase.

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 MOBILE PHASE
SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0
Water

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 Experimental Procedure

Thin layer
Chromatograp
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hy
 TLC Plate Preparation


Methods used
to apply
adsorbent

Dipping
Spreading Spraying plate in
slurry

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  TLC Plate Preparation
TLC plates are usually commercially available, with
standard particle size ranges to improve
reproducibility.
They are prepared by mixing the adsorbent, such as
silica gel , with a small amount of inert binder like
calcium sulphate (gypsum) and water. This
mixture is spread as thick slurry on an uncreative
carrier sheet, usually glass, thick aluminum foil, or
plastic.
The thickness of the adsorbent layer is typically
around 0.1 – 0.25 mm for analytical purposes
Around 0.5 – 2.0 mm for preparative TLC.
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 Materials used in TLC

Spreading the slurry

Glass by Hooper Mobile
phase
Plate

Developing chamber

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 Activation of plate

 Plate is kept for drying in oven at

100OC.
Glass  This step is called as activation of
Plate plate.
 By doing this surface area of the
adsorbent increases.

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 Drawing a Line and circle to apply
the sample

Circle to apply
Glass sample Mobile
phase
Plate

Developing chamber

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 Experimental Procedure
TLC Chamber Preparation
Cut the filter paper so that it fits in the jar, touching the bottom
and reaching a height of about 1cm from the top of the jar
To ensure that the filter paper will work, put it in the jar, and
then place an unused TLC plate in the jar. If the above criteria
are met and the plate doesn’t make any contact with the filter
paper, the setup should work
Remove the TLC plate, and then completely saturate the filter
paper with the development solvent using a pasteur pipet.
Fill the jar with development solvent to a depth no greater than
0.5cm
Put the lid on the jar to preserve the saturated conditions
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 Application of sample
Spotting the TLC Plate
Dip the open end of a capillary tube into the solvent
containing the compound to be eluted
Touch the end of the capillary tube lightly and very briefly to
the coated surface of the TLC plate
Your spots should be made on the line drawn across the plate
in the correct lanes and shouldn’t have a diameter much larger
than the capillary tube
After spotting the plate, place it in the saturated chamber and
close the lid
 Substances should be eluted until the solvent front reaches a
height of about 0.5cm from the top of the TLC plate

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 Materials used in TLC

Circle to apply
Glass sample Mobile
phase
Plate

Developing chamber

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 Development of Chromatogram

Chromatoplate is kept in a tank at an angle 45o

The bottom the tank is nearly covered up to 1 mm by solvent.
 Three sides of tanks are lined with solvent saturate paper.
The top of the tank is covered tightly.
 Solvent moves up and separation takes place in ascending way with in

few minutes.

Plate is removed and dried.

 The separated components are located by either physical or chemical
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method.
 Methods of Plate Development
Ascending development
 Descending
Development
 Two dimensional
Development
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 Application of sample and development of
Chromatogram by Ascending way

High
Polar

Less
Pola
r

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Development of chromatogram by
descending way

Mobile Phase
 Two dimensional chromatography
Sample
A B A B
X X
AB AC
is is
Solvent immersed immersed
in mobile in mobile
phase phase

C
C
Solvent front
 TLC Visualization Methods

Physical
Methods:

Ultraviolet Light—some organic

compounds illuminate or fluoresce
under short-wave UV light:
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 Chemical methods

Iodine Vapor—forms Fluorescent Indicators Silver Nitrate Spray (for Sulfuric Acid Spray +
brown/ yellow —compounds fluoresce Alkyl Halides)—dark Heat—permanent
complexes with organic when placed under UV spots form upon charred spots are
compounds light exposure to light produced

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 Applications of TLC
Qualitative analysis: -
If the separated components are colored then
identification is very easy. All the visualizing
agents used in paper chromatography (Detecting
agents or indicators) can be used in TLC.
From Rf value qualitative analysis can be
performed.

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 The size of the spot increases with
the amount. Square root of the spot
area is find out from that amount of
solute can be found out.

Potentiodensitometry of
the plate is carried out.
Quantitative
analysis Flurometry or
emission
Spectroscopy is also
used.
Separated spot is removed by knife
edge .Its dissolved in proper solvent
and its amount is finding out by
volumetric analysis.

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 TLC can is applicable in the
field of medicinal preparations,
pharmaceutical preparations,
natural product extract and
related compounds.
Other
Applications
Assaying the radiochemical
of purity of radiopharmaceuticals.
TLC
Determination of the pigments in
plants.
In forensic science Laboratory
detection of pesticides and
insectides in food, poison etc.

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 Other Applications of TLC:-
Analyzing the dye composition of fibers in forensic study
or Identifying compounds present in a given substance
For Monitoring organic reactions.
In clinical study to carry out qualitative and quantitative
analysis of biological and metabolic samples to detect
disease.
Semi quantitative analysis can also performed by
extracting the spot in suitable solvent and it’s determined
by volumetric analysis or any other instrumental
technique.

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 Advantages of TLC over Paper Chromatography:

Separation is sharper in TLC than paper

Chromatography.
TLC is much more rapid
The use of inorganic layer eliminates
background organic effects in spectroscopic
analysis.
More reactive reagents like Sulphuric acid can
be used.
The separated spots are more distinct hence
detection methods are more sensitive.
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THANK YOU

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