Thin layer Chromatography

1. Dr.Gurumeet C
wadhawa
THIN LAYER
CHROMATOGRAPHY
TYBSC Analytical
Chemistry
1

2. THIN LAYER
CHROMATOGRAPHY
Principle
Materials used to perform
TLC
Experimental technique
Application
Advantages
Disadvantages
2

3. Principle:
 As the mobile phase rises up the TLC plate by capillary action, the
components dissolve in the solvent and move up the TLC plate.
 Individual components move up at different rates, depending on
intermolecular forces between the component and the silica gel
stationary phase and the component and the mobile phase.
 The stationary phase is SiO2 and is very “polar”.
 More polar analyte's interact more strongly with the stationary phase
in move very slowly up the TLC plate.
 By comparison, the mobile phase is relatively nonpolar and is capable
 of interacting with analyte's by stronger London forces, as well as by
dipole-dipole and H-bonding.
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4.  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
4
Polarity decreases
More
Polar
Less
Polar

5. 5
OH
OH
OH
OH
OH
OH
OH
Mobile
phase
Silica Gel
Silica
Gel

6. More
Polar
Less
Pola
r
Adsorbs
stronger and
separate very
slowly
Adsorbs
weakly and
separate very
fast
Sample to be applied
on this area
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7. 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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8. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The Rf is defined as the distance the center of the spot moved divided
by the distance the solvent front moved (both measured from the origin)
A B C
U
x x
x x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
Rf (A) =
Rf (B) =
Rf (C) =
Rf (U1) =
Rf (U2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
Rf (D) = = 0.80
4.0 cm
5.0 cm
4.0 cm
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9. 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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10. Materials used in TLC
Glass
Plate
Hooper
A device for
applying the
adsorbent layer
Developing chamber
Mobile
phase
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11. Stationery phase
Stationery phase Description Application
Silica gel G Silica gel with average
particle size 15µm
containing ca 13%
calcium sulfate binding
agent
Used in wide range
pharmacopoeial test
Silica gel G254 Silica gel G with
fluorescence added
Same application with Silica
gel G where visualization is to
be carried out under UV light.
Alumina
(Al2O3)
Cellulose Cellulose powder of less
than 30µm particle size.
Identification of tetracycline's
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12. 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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13. 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
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14. Thin layer
Chromatogra
phy
Experimental Procedure
14

15. TLC Plate
Preparation
 Methods
used to apply
adsorbent
Spreadin
g
Sprayin
g
Dipping
plate in
slurry
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16. 16
 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.

17. Materials used in TLC
Glass
Plate
Spreading the
slurry by Hooper
Developing chamber
Mobile
phase
17

18. Materials used in TLC
Glass
Plate
Spreading the
slurry by Hooper
Developing chamber
Mobile
phase
18

19. Activation of plate
Glass
Plate
Plate is kept for drying in oven at
100OC.
This step is called as activation
of plate.
By doing this surface area of the
adsorbent increases.
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20. Drawing a Line and circle to
apply the sample
Glass
Plate
Circle to apply
sample
Developing chamber
Mobile
phase
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21. 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
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22. 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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23. Materials used in TLC
Glass
Plate
Circle to apply
sample
Developing chamber
Mobile
phase
23

24. Development of Chromatogram
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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 method.

25. Application of sample and development of
Chromatogram by Ascending way
High
Pola
r
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Les
s
Pola
r

26. 26
Physical
Methods:
Ultraviolet Light—some organic
compounds illuminate or fluoresce
under short-wave UV light:
TLC Visualization Methods

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Chemical methods
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
Sulfuric Acid Spray +
Heat—permanent
charred spots are
produced

28. 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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29. 29
Quantitative
analysis
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.
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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Other
Applications
of
TLC
TLC can is applicable in the
field of medicinal
preparations, pharmaceutical
preparations, natural product
extract and related
compounds.
Assaying the radiochemical
purity of radiopharmaceuticals.
Determination of the pigments
in plants.
In forensic science Laboratory
detection of pesticides and
insectides in food, poison etc.

31. Other Applications of TLC:-
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 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.

32. 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