1. This document provides an overview of thin layer chromatography (TLC) including adsorbents, preparation techniques, mobile phase selection, and reverse phase TLC. 2. Common adsorbents for TLC plates include silica gel, alumina, and cellulose. Factors like particle size and chemical properties are considered when selecting an adsorbent. 3. TLC plates are prepared by coating glass or plastic plates with a thin, uniform layer of adsorbent using various techniques like pouring, dipping, spraying, or spreading. 4. Mobile phase selection depends on factors like the compounds being separated and the adsorbent used. Mobile phases range from non-

1. 1
Adsorbents for TLC,
preparation techniques,
mobile phase selection,
reverse phase TLC
NIVEDITHA G
1st year M.Pharm
Pharmaceutics
NCP

2. 2
Contents
• Introduction to TLC
• Technique
• Adsorbents for TLC
• Preparation techniques
• Mobile phase selection
• Reverse phase TLC
• References

3. 3
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. 4
Techniques
 In TLC the separation is carried on a glass
or a plastic plate which is coated with a
thin uniform layer of finely divided inert
adsorbents such as silica gel or alumina.
 The plates are activated, in solution of the
sample in a volatile solvent is applied by
using capillary tube or micropipette to a
spot keeping 1-2cm from bottom of TLC
plate.

5. 5
The position of the sample spot is
indicated by marking the origin line
on the plate with the lead pencil.
When spot has dried, the plate is
placed vertically in a suitable tank
with its lower edge immersed in
selected mobile phase.
Solvent rises by capillary action,
resolving the sample mixture into
discrete spots.

6. 6
At the end of the run the solvent is
allowed to evaporate from the plate
& the separated spots are located &
identified by various physical &
chemical method.

7. 7
Adsorbents for TLC
In the beginning of TLC method, only
few coating materials were used as
adsorbents such as silica gel,
alumina, kieselguhr, etc.
However, now a days , there is
variety of adsorbents which can be
selectively utilized.

8. 8
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.

9. 9
 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.

10. 10
Types of adsorbents
1. Inorganic adsorbents
2. Organic adsorbents

11. 11
• Silica gel
• Alumina
• Kieselguhr
• Magnesia
• Magnesium silicate
• Calcium silicate
Inorganic adsorbents

12. 12
• Cellulose & its acetylates
• Charcoal & activated carbon
Organic adsorbents

13. 13
Inorganic adsorbents
Silica gel:
• It is prepared by the hydrolysis of
sodium silicate to polysilicic acid
which on further condensation &
polymerization yields silica gel.
• Binder (10%w/w) is added to confer
greater mechanical strength to the
layer & enhance adhesion to the
plate.

14. 14
• Starch is another binder which has limited
use since it does not allow the use of
corrosive locating agents like 10% v/v
alcoholic sulphuric acid, 5% potassium
dichromate.
• Silica gel commonly used in TLC has mean
particle size of 15mm with a particle size
range of 5-40mm.
• Adsorption properties of the silica gel can
be modified for reverse phase by
incorporating non polar functional group
such as octadecyl siliyl (ODS).

15. 15
• As such, surface of silica is acidic due
to presence of many silanol hydroxyl
groups therefore best suited for
analysis of acidic & polar compounds
like amino acids, fatty acids, lipids,
essential oil, terpenoids, sugar, etc.

16. 16
Alumina (Al2O3):
• It has basic surface & is used next to
silica gel for separation of weakly
polar group.
• It often contains sodium carbonate &
bicarbonate whose presence affects
its adsorptive properties.
• Alumina can be produced with its
surface either acidic, basic, or
neutral.

17. 17
• Neutral alumina is used with organic
eluents. It is suitable for use with
substances that are either liable or
bound to strong alkalies.
• Acidic alumina is used for separation
of neutral or acidic materials.
• Basic alumina is used to separate
alkaloids, aromatic & unsaturated
hydrocarbons.

18. 18
Kieselguhr (Diatomceous earth):
It has neutral pH. It is available with
or without binder. It has less capacity
of resolution than alumina and silica
gel.
 Magnesia (MgO) :
Often replaces alumina. It is too
finely divided to allow filtration and
can be mixed with filter aid. It is
obtained by dehydration of the
hydroxide.

19. 19
 Magnesium Silicate, Calcium Silicate :
These are utilised for separation of sugars
and its acetates, phenylosazones. In these
adsorptive power increases with
decreasing water content.
 Others :
Aluminum silicate, bentonites, barium
sulphate, calcium carbonate, calcium
hydroxide, calcium sulphate, dicalcium
phosphate, fuller’s earth,
zinc carbonate.

20. 20
ORGANIC ADSORBENTS
Cellulose and its acetylates:
• These adsorbents are fibrous and can be
used with relative advantages over
paper as the flow is more even and
there is les diffusion of the dissolved
substances. The flow is also faster.
Modified cellulose powders are used to
obtain ion-exchange separations in TLC
and can be used with or without binder.

21. 21
• Cellulose contains adsorbed water which
bring separation by partition
mechanism. And commonly used for
separating hydrophilic substances like
amino acids and sugars.
 Charcoal and activated Carbon :
• Charcoal has specific property of
adsorbing strongly aromatic substances.
Adsorptive property of activated carbon
can be modified by depositing on it a
film of a non electrolyte or a fatty acid.

22. 22
Others :
Dextran gels, ion-exchange resins,
polyamides, polyethylene powder,
sucrose are used for variety of
separations.

23. 23
PREPARATION OF
CHROMATOLATES
• Glass plates or flexible plates are
commonly used for adsorbent. Size used
depends on type of separation to be
carried out, the type of chromatographic
tank and spreading apparatus available.
• The standard sizes are 20 x 5 cm, 20 x 10
cm or 20 x 20 cm .
• The surface should be flat without
irregularities.
• The standard film thickness is 250um

24. 24
Methods for application
of adsorbent.
Pouring
Dipping
Spraying
Spreading.

25. 25
• 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.

26. 26
• 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.

27. 27

28. 28
• 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.

29. 29
ACTIVATION OF PLATES
• After spreading plates are allowed to
dry in air and further dried and
activated by heating at about 1000
c
for 30 mins.
• By removing the liquids associated
with layer completely, the adsorbent
layer is activated.

30. 30
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.

31. 31
• 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.

32. 32
• n-Hexane
• Cyclohexene
• Toluene
• Benzene
• Diethylether
• Chloroform
• Dichloromethane
• 1,2 dichloroethane
• Acetone
• Ethyl acetate
• Acetonitrile
• Propanol
• Methanol
• Acetic acid
• Water.
Increasing
polarity

33. 33
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.

34. 34
DEVELOPMENT
CHAMBERS
• TLC plates are placed vertically in a
rectangular chromatography tank
(as in fig)

35. 35
• They are classified according to the
separation technique used.
(a) Tanks for ascending development
(b) Tanks for descending development
(c) Tanks for horizontal development
(d) Tanks for thin layer electrophoresis
• For first three methods glass or S.S is
most suitable.
• Degree of saturation will affect Rf value

36. 36
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.)

37. 37
• 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.)

38. 38
 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 900
(as in
fig.)

39. 39
Two dimensional
development.

40. 40
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 1200.

41. 41
REFERENCES
1. ‘Instrumental method of chemical
analysis’ by B.K. Sharma.
2. ‘Instrumental method of chemical
analysis’ by Chatwal & Anand.
3. Text book of pharmaceutical analysis by
Dr. Ravi sankar.
4. Pharmaceutical Analysis Volume – ii
Instrumental Methods by Dr. A. V
Kasture, Dr. S. G Wadodkar, Dr. K. R.
Mahadik, Dr. H. N. More.

42. 42