Principle:: Intermolecular Forces Between The Component and The Silica Gel
Principle:: Intermolecular Forces Between The Component and The Silica Gel
Principle:: Intermolecular Forces Between The Component and The Silica Gel
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 SiO 2 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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• Thin Layer Chromatography (TLC) is an
important technique used for identification
and separation of mixture of chemical
compounds into its individual components.
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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
The strength of interactions between the adsorbent
and eluting components vary approximately in this
order:
Salt formation > coordination > H-bonding >
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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 .
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THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
2.0 cm
Rf (A) = = 0.40
5.0 cm
Solvent Front
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
by8 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 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
than 30µm particle size.
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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
Hexane 0.1
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0
Experimental Procedure
Thin layer
Chromatograp
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hy
TLC Plate Preparation
Methods
used to apply
adsorbent
Sprayin Dipping
Spreading plate in
g 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
Developing chamber
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Materials used in TLC
Developing chamber
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Activation of plate
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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
High
Polar
Less
Pola
r
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TLC Visualization Methods
Physical
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: