The document presents a detailed overview of thin layer chromatography (TLC), outlining its principles, procedures, and applications in the analysis of various compounds. TLC is characterized by the separation of compounds based on their affinity to stationary and mobile phases, using adsorbents on a glass plate. Applications include the separation and identification of drugs, carbohydrates, and other organic substances, with various detection methods described.

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Thin Layer Chromatography
PRESENTED BY,
Devdatt Jani
M-Pharm 1st year
Dept. of Pharmaceutics

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Contents
 Introduction
 Principle
 Types
 Experimental details
 Applications
 References

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Introduction :
 TLC is simple & rapid method carried out using
thin layer of adsorbents on plates.
 Principle :
 TLC has been included under both adsorption
& partition chromatography.
 Separation may result due to adsorption or
partition or both phenomenon depending upon
nature of adsorbents & solvent system used for
development.

4. PRINCIPLE :
The main principle of TLC is adsorption One or
more compounds are spotted on a thin
layer of adsorbent coated on a
chromatographic plate. The mobile phase
solvent flows through because of capillary
action (against gravitational force). The
component with less affinity towards the
stationary phase travels faster.
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5. PRINCIPLE :
 The component with more affinity towards
the stationary phase travels slower. Thus
the components are separated on a thin
layer chromatographic plate based on the
affinity of components towards stationary
phase.
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OUTLINE OF GENERAL PROCEDURE OF
TLC :-
Make slurry from loose powder of an
adsorbents or finely ground material .
glass plate coated with it .
The unknown substance & reference
material are dissolved in water or organic
solvent is
Drying as thin layer

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applied in a row of spots , 1-2 cm from
the edge of the with the help of capillary ,
micropipette or micro syringe .
Chromatoplate is placed in jar or beaker
containing the solvent for development
Jar or beaker is covered with an air tight
lid . Solvents ascends through the layer by
capillary action

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Sample is resolved into fractions .
Plate or sheet is carefully withdrawn after
the solvents front has migrated about
75% length of the plate .
Dry the plate .
spray with reagent for detection of
components or more commonly
expressed to iodine vapours .

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Solute position are indicated by brown
spots.
origin
New
position

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PRACTICAL REQUIREMENTS
:-
1. Glass plates .
2. Preparation & activation
of TLC plates .
3. Application of sample .
4. Development tank .
5. Mobile phase .
6. Development technique
7. Stationary phases .
8. Detecting or visualizing
agents .

11. Methods for application of
adsorbent.
 Pouring
 Dipping
 Spraying
 Spreading.
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Pouring: 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.
Dipping : This technique is used for small
plates by dipping the two plates at a time, back
to back in a slurry of adsorbent in chloroform or
other volatile solvents. Exact thickness of layer
is not known and evenness of layer may not be
good.

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Spraying : Slurry is diluted further for the
operation of sprayer. But this technique is not
used now a days as it is difficult to get uniform
layer.
Spreading : 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. Commercial
spreaders are of two types (a) Moving spreader,
(b) Moving plate type.
It gives layer thickness from 0.2 to 2.0 mm.

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Precoated plates of different adsorbents
either on glass or polymeric sheets are
available in uniform and optimal layer
thickness for intended purpose and are
abrasive resistant. They can be sprayed
with almost all spraying agents including
corrosive agents. But these are very
expensive. Also called as ready to use TLC
plates.

16. ACTIVATION OF PLATES
 After spreading plates are allowed to
dry in air and further dried and
activated by heating at about 100
0
c
for 30 mins.
 By removing the liquids associated
with layer completely, the adsorbent
layer is activated.
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17. SOLVENT SYSTEM
 The choice of the mobile phase is depends
upon the following factors:-
1. Nature of the substance 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.
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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 elutropic
series are selected.

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 n-Hexane
 Cyclohexene
 Toluene
 Benzene
 Diethylether
 Chloroform
 Dichloromethane
 1,2 dichloroethane
 Acetone
 Ethyl acetate
 Acetonitrile
 Propanol
 Methanol
 Acetic acid
 Water.
Increasing
polarity

20. APPLICATION OF SAMPLE
 Sample solution in a non polar solvent is applied.
 The concentration of a sample or standard
solution has to be minimum 2-5 ul of a 1% solution
of either standard or test sample is spotted using a
capillary tube or micropipette.
 The area of application should be kept as small as
possible for sharper and greater resolution.
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21. DEVELOPMENT OF
CHROMATOGRAMS
 Generally ascending method is used
to greater extent but various other
methods are also used. They are
 Ascending Development : The
plates after spotting of the sample
are placed in chamber containing
solvent at bottom. Flow of solvent is
from bottom to top. (as in fig.)
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 Descending : Flow of the solvent from
reservoir to the plate is by means of a
filter paper strip. Solvent moves from
top to bottom of the plate (as in fig.)
ASCENDING DESCENDING

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 Two dimensional development :
 It is used if the component of the mixture are
not completely separated by development in a
single direction.
 In these sample spot is applied at corner of
plate.
 First development is carried out by ascending
method in one solvent. The plate is taken out,
solvent allowed to evaporate.
 second development is carried out in another
solvent by changing the edge of plate at 90
0
(as
in fig.)

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Two dimensional
development.

25. REVERSE PHASE TLC
 Here stationary phase is non polar in nature and mobile
phase is polar in nature.
 These chromatoplates are prepared by immersing the
adsorbent layer very slowly in 5-10% of paraffin, silicone
oil, undecane in petroleum ether or diethyl ether. After
removing the plate and evaporating the solvent , the plate
is ready for chromatography. Paraffin and silicon oil
provides the permanent impregnation whereas undecane
can be removed after development after heating the plate
at 120
0.
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APPLICATIONS OF TLC :-
The application of TLC are wider & there
is no limitation to the compounds that can be
analysed by TLC . Anyhow different types of
application are listed below .
1. Separation of mixtures of drugs of chemical or
biological origin , plant extracts , etc .
2. Separation of carbohydrates , vitamins ,
antibiotics , proteins , alkaloids , glycosides ,
etc .

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3.Identification of drugs .
DRUG STATIONAR
Y PHASE
MOBILE
PHASE
DETECTIN
G AGENTS
Aminocapro
ic acid
Silica gel G Alcohol:H2O
:NH3
(25:3:
4)
Ninhydrin in
alcohol &
pyridine .
Amoxycillin
trihydrate
Silica gel G
H.F. -254
Buffer pH 6
: acetone
(4:1)
NaoH +
Starch +
glacial acetic
acid +
iodine in
potassium
iodate

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4.Identification of related compounds in
drugs :-
DRUGS NAME OF
RELATED
COMPOUND
STATIONARY
PHASE
MOBILE
PHASE
DETECTING
AGENTS
Allopurinol 3-amino
pyrazole -
4-carbonamide
hemisulphate
Cellulse powder
with flurescent
additive
n-butanol :
ammonia
UV 254 nm
Bethanidine
sulphate
Methylamine
benzylamine
trimethyl
guanidine
sulphate
Silica gel G Ethyl
acetate :
glacial
acetic acid :
water :
alcohol(25:
12:8:5)
1.Ninhydrin in
isopropanol
2. Potassium
iodobismuthate

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5.To detect the presence of foreign substance in
drugs :
DRUG NAME OF
THE
FOREIGN
SUBSTANC
E
STATION
ARY
PHASE
MOBILE
PHASE
DETECTING
AGENT
Butylated
hydroxy
anisole
2 & 3- t-
butyl-1-4-
methoxy
phenol
Silica gel
G
Chloroform Phospho
molybdic acid
+ NH3
vapour
Carbimazo
le
methimazole Silica gel Chloroform
: acetone
(4:1)
Potasssium
iodobismuthat
e

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6.To detect decomposition products in drugs
:
DRUG NAME OF
THE
DECOMPO
SION
PRODUCT
STATIONA
RY PHASE
MOBILE
PHASE
DETECTIN
G AGENTS
Chlordiaze
poxide
7-chloro-1-3-
dihydro-5-
phenyl-1,4-
benzodiazepi
ne-2-one-4-
oxide
Silica gel Alcohol :
ethyl
acetate (1:
24)
N-1-(NED)
Diazepam - Silica gel
G.F-254
Hexane
:ethyl
acetate
(1:1)
UV 254nm

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References
 “Instrumental Methods Of Chemical Analysis”, Gurdeep R.
Chatwal & Sham K.Anand....Himalaya publishing house.
 “Textbook of Pharmaceutical Analysis”, S. Ravi Shankar…RX
publications.
 “Pharmaceutical Analysis”, volume 2…Instrumental methods
by Dr.A V.Kasture. Dr. K R.Mahadik,
Dr.S G.Wadodkar,Dr.H N. More.
 www.google.com

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