Thin layer-chromatography

1. THIN LAYER
CHROMATOGRAPHY
Mahpara Gondal
Rashid Latif College Of Pharmacy

2. Learning Objective
1. State the definition of TLC
2. Explain the phases used in TLC
3. List the materials & methods used in
TLC
4. List the application of TLC
5. List the advanteges & disadavantages
of TLC

3. Thin Layer Chromatography
 What is TLC?...
One of analysis method that is used to
identify the unknown compounds and to
determine the purity of mixture.
 This method is simple, rapid and cheap
 Widely used in pharmaceutical & food
stuff industry.

4. Introduction
 TLC is a method of analysis in which the
stationary phase (a finely divided solid) is
spread as a thin layer on a rigid supporting
plate and a mobile phase, a liquid is allowed to
migrate across the surface of plate.
Supporting material made by glass, coated by solid matter act as stationary phase

5. TLC can be used to:
 Verify a substance identity
 Monitor the progress of a reaction
 Determine the number of components in a
mixture.
 Determine appropriate conditions for column
chromatography
 Analyze the fractions obtained from column
chromatography

6. Theoretical Aspect
 The equilibrium between the free and
absorbed states depends on three
factors:
 Polarity and size of the analyte molecule
 Polarity of the stationary phase
 Polarity of the solvent

7. -A plate of TLC can be made from aluminium
or glass which is coated by a solid matter as
a stationary phase which is typically alumina
or silica gel. The covalent networks of these
absorbents create very polar materials.
- The coated material has 0.1-0.3mm in
thickness
-some of them has been added by
fluorescent indicator that will make it
florescence during the UV light exposure.

8. STATIONARY PHASE
 Silica is commonly used as stationary
phase
 The separation of sample mixture will be
depend on the polarity of sample.
 Some modified silica is also used in
certain purposes.

9. Stationery phase Description Application
Silica gel G Silica gel with average
particle size 15µm
containing ca 13%
calcium sulfate binding
agent
Used in wide range
pharmacopoeial test
Silica gel G254
Silica gel G with
fluorescence added
Same application with
Silica gel G where
visualization is to be
carried out under UV
light.
Cellulose Cellulose powder of less
than 30µm particle
size.
Identification of
tetracyclines

10. Adsorbents
 Two important properties of adsorbent are its
particle size and its homogeneity.
 A particle size of 10-25um is usually
recommended.
 A coarse grained will not produce a satisfactory
thin layer, so for greatly enhanced resolution
should use fine-grained adsorbent.

12. Silica Gel
 Most commonly used
adsorbent in tlc.
 Prepared by
hydrolysis of sodium
silicate to polysilicic
acid which on further
condensation and
polymerization yeil
silica gel material.

13. Silica gel matrix structure

14. Cont…
 With regard to specific requirments and high purity, synthesis can
be change.
 Suffix “G” is used universally to denote silica gel with gypsum
binder, namely calcium sulphate hemi hydrate (CaSO4 (0.5H2O)).
 Resolution depends on particle size and particle size distribution.
 TLC mean particle size 12um,
range 5-25um,
pore diameter 6nm
Commercial plates particle size 10um.

15. Kieselguhr (celite)
 Kieselguhr (celite) are
diatomaceous earths
composed of the silica rich
fossilised skeletal remains of
microscopic sea organisms
called diatoms.
 The material has high
porosity and large surface
area , exhibits very little
absorptive properties, so
that’s why mostly use as a
support for stationary phase.
 Celite when mixed with clay
forms fire brick which can be
crushed and sieve graded.
 The firebrick has good flow
and packing properties with
an increased adsorptive
capacity.

16. Alumina
Alumina (Aluminium oxide) can be synthesized to the same
degree of purity and specification as silica gel by a series of
non-uniform dehydration processes of various crystalline
modifications of aluminium hydroxide.
For optimum results it is necessary to activate the alumina, by
heating at a specified temperature (125C- 150C) for a defined
time.
Basic aluminium oxide when used with organic eluants will
adsorb aromatic and unsaturated hydrocarbons , carotenoids
steroids, alkaloids and other natural products.

17. Structure of alumina

18. CELLULOSE
Cellulose can be more
accurate and precise when
use in tlc plates.
Cellulose use for separating
hydrophilic substances as
compared to silica gel or
alumina which are use for
hydrophilic substances.

19. Structure of cellulose molecule

20. Chemically modified cellulose
DEAE act as strong anion exchanger and carries positive
charges at neutral and acidic pH.
Used to separate delicate molecules like proteins and nucleic
acids.

23. MOBILE PHASE
 The ability of mobile phase to move up is
depend on the polarity itself.
 Volatile organic solvents is preferably used as
as mobile phase.
 Mixture of two or more solvent of different
polarity give better separation than the single
solvent.

24. MOBILE PHASE
SOLVENT POLARITY INDEX
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0

25. MATERIALS
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp

26. METHOD

27. 1. Coating Material
 Silica gel, alumina, or similar material
coated on glass , metal or a plastic film
as a thin layer ( approx 0.25mm).
 In addition gypsum as a binder and
fluorescent powder for simplify the
visualization of spot later on.

28.  Applicators are commercially
available which are used for
coating the plate .
 Commercialy obtained with
5cm x 20cm in size.
 Prepare your size when
neccesary.
2. TLC Plate Preparation

29. Methods for preparing layers
 Pouring
 Dipping
 Spraying
 Spreading
 Precoated plate

30. 3. Activation of adsorbent
 The plates are dried
in air and then dried
in oven at 110-140C
for 30 minutes.
 On heating, the
plates are said to be
activated.

31. 4. Capillary spotters
 Place capillary tube in
burner flame, hold it
there until it softens and
start to stags.

32. 5.Spotting’ TLC plates
 Make sure that your sample is
liquified already.
 If not then dissolve a few
milligrams of material in a volatile
solvent create a dilute solution.
 stick it using capillary pipe &
spott onto the line you have made
 Smearing, smudging, and spots
that overlap will result making
identification of separated
components difficult

33. 6.Developing Container
Preparation
Solvent is transferred
into the container with
0.5-1cm in dept from the
bottom
A piece of filter paper is
put around the inner side
of the bottle.

34. 7.‘Develop the plate’
 after spotting, put the plate inside
the chamber in the ascendant
position
 Make sure that the dept of solvent
doesn’t touch the spots
 Let it develop up to the 1cm from
the top of plate
 After that, pull out the plate from
the chamber and let the solvent be
vaporized

35. 8. Visualization
- The color samples are easy to be seen and no need to
use UV lamp to detect them.
- Mostly organic compounds are colorless then for the
identification of such kind of compounds need UV
lamp.

36. UV lamp

37. Cont…
 Another visualizing
technique is an iodine
chamber. Iodine
sublimes and will absorb
to organic molecules in
the vapor phase.
 The organic spots on the
plate turn brown and can
be easily identified.

38. DETECTION OF SPOT
1) Iodination-put the plate in which the spots face to
the iodine crystall and see what is the spot color
changing
2) Ninhydrin:
-specific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when it
is sprayed to the amino acid spot.
3) KMnO4
used to identify a reducing agent such as glucose,
fructose, vitamin C and others.
4) Alkaline tetrazolium blue
specificaly used for corticosteroid identification

39. The use of Rf as separation
parameter
- The distance taken through by the solvent to move up will be
assigned as solvent front
- The distance taken through by the sample to move up will be
assign as sample front
- Rf value is obtained by dividing the sample front toward
solvent front
Rf = sample front
solvent front
-

40. Thin-Layer Chromatography:
Determination of Rf Values
solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
More polar!
Less polar!

41. Quantitative determination of known sample
- Done by scratching the spot using
spatula, and extract the compound using
the suitable solvent
- The liquid extract can be determined its
content using other method such as
spectroscopy.

42. Prob;ems commonly occur in TLC and how to solve
a. The spot shape is too broad
- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- non proportionality in stationary phase
surface will inhibit the movement of solvent
c. streaking formation
- caused by too concentrated sample

43. TLC Compared to Paper Chromatography
1. Precise and effective
2. More stable toward various organic
solvents

44. Advantages
 Cheap
 Simple
 The developing can be monitored visually
 Able to use various chemical as a detector

45. Identification unknown drugs using standard
Reference

46. Thanks for Attention