Thin layer Chromatography and it's Techniques

Detail study about thin layer Chromatography

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Thin Layer Chromatography
PRESENTED BY,
Devdatt Jani
M-Pharm 1
st
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 adsorbentson 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 :
ThemainprincipleofTLCisadsorptionOneor
morecompoundsarespottedonathin
layerofadsorbentcoatedona
chromatographicplate.Themobilephase
solventflowsthroughbecauseofcapillary
action(againstgravitationalforce).The
componentwithlessaffinitytowardsthe
stationaryphasetravelsfaster.
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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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appliedinarowofspots,1-2cmfrom
theedgeofthewiththehelpofcapillary,
micropipetteormicrosyringe.
Chromatoplateisplacedinjarorbeaker
containingthesolventfordevelopment
Jarorbeakeriscoveredwithanairtight
lid. Solvents ascends through the layer by
capillary action

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Sampleisresolvedintofractions.
Plateorsheetiscarefullywithdrawnafter
the solventsfronthasmigratedabout
75%lengthoftheplate.
Drytheplate.
spraywithreagentfordetectionof
components or more commonly
expressedtoiodinevapours.

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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 platesof 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
Herestationaryphaseisnonpolarinnatureandmobile
phaseispolarinnature.
Thesechromatoplatesarepreparedbyimmersingthe
adsorbentlayerveryslowlyin5-10%ofparaffin,silicone
oil,undecaneinpetroleumetherordiethylether.After
removingtheplateandevaporatingthesolvent,theplate
isreadyforchromatography.Paraffinandsiliconoil
providesthepermanentimpregnationwhereasundecane
canberemovedafterdevelopmentafterheatingtheplate
at120
0.
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APPLICATIONS OFTLC:-
TheapplicationofTLCarewider&there
isnolimitationtothecompoundsthatcanbe
analysedbyTLC.Anyhowdifferenttypesof
applicationarelistedbelow.
1.Separationofmixturesofdrugsofchemicalor
biologicalorigin,plantextracts,etc.
2. Separationofcarbohydrates,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 GAlcohol:H
2O
:NH
3
(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
Allopurinol3-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
ChloroformPhospho
molybdic acid
+ NH
3
vapour
Carbimazo
le
methimazoleSilica 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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