Thin Layer Chromatography

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“A method of separation or identification of a mixture of
components into individual components by using finely
divided adsorbent solid / (liquid) spread over a glass
plate and liquid as a mobile phase.”
 TLC is one of the simplest, fastest, easiest and least
expensive of several chromatographic techniques
used in qualitative and quantitative analysis to
separate organic compounds and to test the purity of
compounds.

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TLC is a form of liquid chromatography consisting of:
 A mobile phase (developing solvent)
 A stationary phase (a plate or strip coated with a form of silica gel)
 Analysis is performed on a flat surface under atmospheric pressure and
room temperature
The two most common classes of TLC are:
“Normal phase is the terminology used when the stationary phase is
polar; for example silica gel, and the mobile phase is an organic
solvent or a mixture of organic solvents which is less polar than the
stationary phase or non-polar.”
“Reversed-phase chromatographyis the terminology used when the
stationary phase is a silica bonded with an organic substrate such as a
long chain aliphatic acid like C-18 and the mobile phase is a mixture of
water and organic solvent which is more polar than the stationary
phase.”

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• Thin layer chromatography was first
introduced by IZMAILOV & SHRAIBER.
• They both described basic principle and
used it for separation of plant extracts.
1938
• Consden, Gorden & Martin started
using filter papers for separation of
amino acid.1944
• Kirchner who used impregnated glass
plate coated with alumina, identified
terpenes.1950
• Ergon stahl introduced a standard
equipment for preparing uniform thin
layers of known thickness.1958
• Michael Tswett is credited as being the father of liquid
chromatography. Tswett developed his ideas in the early 1900’s.

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Similar to other chromatographic methods, TLC is also based
on the principle of separation.
 The separation depends on the relative affinity of
compounds towards the stationary and the mobile phase.
 The compounds under the influence of the mobile phase
(driven by capillary action) travel over the surface of the
stationary phase. During this movement, the compounds
with higher affinity to stationary phase travel slowly while
the others travel faster. Thus, separation of components in
the mixture is achieved.
 Once separation occurs, the individual components are
visualized as spots at a respective level of travel on the plate.
Their nature or character are identified by means of suitable
detection techniques.

Principle Of TLC
Adsorption Partition
Ion-
Exchange
Reversed
Phase Partition
• Chromatography
on adsorbents such
as silica gel and
alumina has widely
been employed for
the fractionation of
mixture of non polar
material into class of
compounds that
migrate at different
rate by virtue of their
respective polarities.
• Eg:- Hydrocarbons
are easily separated
from Acids, alcohols
& aldehydes.
• Separation
of mixture is
effected by
virtue of
difference in
the solubilities
of its
components in
developing
solvent and
the stationary
liquid phase
• In ion
exchange
system the rate
of migration of a
compound can
be found by the
total charge of
ionised group
per molecule.
• The stationary
phase is non
polar and the
mobile phase is
polar.
Reversed phase
partition has
widely use for
fractionation of
hydrocarbons
and other non
polar petroleum
products

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Coating Materials
Preparation of thin Layer In plates
Adsorbent For TLC
Sample Application
Development Tank
Solvent System
Plate Development
Detection Of Component
Evaluation Of Chromatogram

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 A large no. of coating materials are used which are
commercially produced as a thin adsorbents.

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Pouring
• Disadvantage is
that uniformity
in thickness
can not be
ensured.
Dipping
• Disadvantage is
that a larger
quantity of slurry
is required even
for preparing
fewer plates
Spraying
• The disadvantage
is that the layer
thickness cannot
be maintained
uniformly all over
the plate.
Spreading
• .
• Commercial spreaders
are of two types (a)
Moving spreader, (b)
Moving plate type. It
gives layer thickness
from 0.2 to 2.0 mm.
The adsorbent of
finely divided and
homogeneous
particle size is
made into slurry
and is poured on a
plate and allowed
to flow over it so
that it is evenly
covered.
This technique is
used for small
plates by dipping
the two plates at
a time in to slurry
& are separated
after removing
from slurry & later
dried.
Resembles that of using
a perfume spray on a
cloth. Slurry is diluted
further for the operation
of sprayer. The
suspension of adsorbent
or slurry is sprayed on a
glass plate using a
sprayer
All the above
methods fail to give
thin and uniform
layers. Modern
methods utilize the
spreading devices
for preparation of
uniform thin layers
on glass plates.

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• In the beginning of TLC method, only few coating materials were
used as adsorbents such as silica gel, alumina etc.
Factors to be considered while choosing the adsorbents:-
1. Characteristics of compound to be separated.
2. Solubility of compounds.
3. Nature of substance to be separated i.e. acidic, basic, amphoteric,
4. To see whether compound is liable to react chemically with adsorbent (or solvent),
or not.
General properties:-
• Two general properties that decide its application are :
1.Particle size
2.Homogeneity
• Particle size of 1-25mm is generally preferred.
• Adsorbents do not generally adhere to glass plates & hence binders like gypsum,
starch are added.
• Gypsum (calcium sulphate) in 10-15% w/w is widely used as binder.

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•Organic adsorbants:
•Cellulose & its acetylates.
•Charcoal & activated carbon.
•Inorganic adsorbants:
•Silica gel.
•Alumina.
•Magnesia.
•Magnesium silicate.
•Calcium silicate

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• Usually to get good spots, the concentration of the sample or standard solution has to
minimum, 2-5ml of a 1% solution of either standard or test sample is spotted using a
capillary tube or micropipette.
• The spots should be kept at least 1-2cm above the base of the plate and the spotting area
should not be immersed in the mobile phase in the development tank.
• The developing container for TLC can be a specially
designed chamber, a jar with a lid, or a beaker with a watch
glass on the top.
• Pour solvent into the chamber to a depth of just less than 0.5
cm. To aid in the saturation of the TLC chamber with solvent
vapors, you can line part of the inside of the beaker with filter
paper.
• In TLC the plate is placed in a development chamber at an
angle of 45.

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The choice of the mobile phase is depends
upon the following factors:-
1. Nature of the substances to be separated
2. Nature of the stationary phase used
3. Mode of chromatography (Normal phase or
reverse phase)
4. Separation to be achieved – Analytical or
preparative
• The organic solvent mixture of low polarity
is used.
• Highly polar solvents are avoided to
minimize adsorption of any components of
the solvent mixture.
• Use of water as a solvent is avoided as it
may loosen the adhesion of a layer on a
glass plate.
• Solvents with an increasing
degree of polarity are used in
liquid-solid or adsorption
chromatography.
• The solvents listed in eluotropic
series are selected

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•Allow the plate to develop until the solvent is
about half a centimeter below the top of the
plate.
•Remove the plate from the beaker and
immediately mark the solvent front with a
pencil. Allow the plate to dry.
•The prepared TLC plate is placed in the
closed container saturated with developing
solvent.
•Cover the beaker with the watch glass, and
leave it undisturbed on your bench top.
• The solvent will rise up the TLC plate by
capillary action. Make sure the solvent does
not cover the spot.

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After the development of chromatogram, the spots
should be visualised. Detecting coloured spots can
be done visually, But for detecting colourless spots,
any one of the following techniques can be used.
a. Non specific method: Where the
number of spots can be detected but
not exact nature of compound
Example
i. Iodine Chamber Method: Where
brown or amber spots are
observed when the TLC is kept tank
with few iodine crystals at the
bottom
ii. UV Chamber for flourescent
compounds: When compounds are
viewed under UV chamber at 245
nm or at 365 nm flourescent
compounds can be detected.
b. Specific methods: Specific spray
reagents or detecting agents
visualizing agents are used to find
out the nature of compounds for
identification purposes. For example
i. Ferric
chloride
ii. Ninhydrin in
acetone
iii. Dragendroff’s
reagent
iv. 3,5-Dinitro
benzoic acid
v. 2,4-
Dinitrophenyl
hydrazine-
For phenolic
compounds & tannins
For amino acids.
For alkaloids.
For cardiac
glycosides.
For aldehydes &
ketones.

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METHODS
QUALITATIVE QUANTITATIVE

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•This is done by the help of measurement of migration parameters.
Migration
Parameters
Rƒ Rx

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 Rf = Distance traveled by sample
Distance traveled by solvent.
 If Rƒ value of a solution is zero,
the solute remains in the
stationary phase and thus it is
immobile.
 If Rƒ value = 1 then the solute has
no affinity for the stationary
phase and travels with the
solvent front.
 Retention Factor can never be
greater than one.
Factors affecting Rf value:
 It depends on following factors:
 Nature adsorbant, mobile phase,
Thickness of layer, Temperature,
Dipping zone, Chromatographic
techniques

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Visual assessment
of chromatogram
Measurement of
spot area
spectrophotometry
Densitometry
Densitometer is an instrument which
means quantitatively the density of
the spots. When the optical density
of the spots for the STD & test
solution are determined, the quantity
of the substance can be calculated.

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ADVANTAGES OF TLC DISADVANTAGES OF TLC
 It is simple process with short
development time.
 It helps in visualization of separated
compound spots easily.
 The method helps to identify the
individual compounds.
 It helps in isolation of most of the
compounds.
 The separation process is much faster
and the selectivity for compounds is
higher even small differences in
chemistry is enough for clear
separation.
 The purity standards of the given
sample can be assessed easily.
 It is a cheaper chromatographic
technique.
 TLC plates do not have long
stationary phases.
Therefore, the length of
separation is limited
compared to other
chromatographic techniques.
 Also, the detection limit is a
lot higher. If you would need
a lower detection limit, one
would have to use other
chromatographic techniques.
 TLC operates as an open
system, so factors such as
humidity and temperature can
be consequences to the
results of your chromatogram.

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1. Over-large spot: Spotting sizes of the
sample should be not be larger than
1-2 mm in diameter.
 The component spots will never be
larger than or smaller than your
sample origin spot.
 Over-large spot overlapping
of other component spots with similar
Rf values on your TLC plate.
 If overlapping occurs, it would prove
difficult to resolve the different
components.


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3. Streaking:
 If the sample spot is too
concentrated, the substance
will travel up the stationary
phase as a streak rather than a
single separated spot.
4. Spotting:
 The sample should be above the
solvent level. If the solvent level
covers the sample, the sample
spot will be washed off into
the solvent before it travels up the
TLC plate
Rarely, water is used as a solvent because it produces an uneven curve front
which is mainly accounted for by its surface tension.
2. Uneven Advance of Solvent
phase: This uneven advance can be
caused by a few factors listed below:
No flat bottom. When placing the
TLC plate into the chamber, place the
bottom of the plate on the edge of the
chamber (normally glass
container)e.g. beaker.
Not enough solvent. There should be
enough solvent (depends on size of
chamber) to travel up the length of the
TLC plate.
Plate is not cut evenly. It is
recommended that a ruler is used so
that the plate is cut evenly.

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 To check purity of given samples.
 Identification of compounds like acids, alcohols,
proteins, alkaloids, amines, antibiotics etc
 To evaluate reaction process by assessment of
intermediates, reaction course etc.
 To purify samples i.e. for purification process.
 To keep a check on the performance of other
separation processes.

Thin Layer Chromatography

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