1. THIN LAYER CHROMATOGRAPHY
ADSORBENTS FOR TLC, PREPARATION
TECHNIQUES, MOBILE PHASE SELECTION,
REVERSED PHASE TLC
-: Presented By :-
Amruta S. Sambarekar
1st Year M.Pharm
Dept. of Pharmaceutics
M M C P, BELGAUM
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2. THIN LAYER CHROMATOGRAPHY
• TLC is a technique in which the components of
the extract are distributed between two
phases, stationary phase & mobile phase.
• The principle of separation is adsorption.
3. • REQUIREMENTS OF TLC:
Stationary phase and papers used.
Preparation and activation of TLC plates.
Application of the sample.
4. • COMPARISION OF THIN LAYER CHROMATOGRAPHY AND PAPER
THIN LAYER CHROMATOGRAPHY PAPER CHROMATOGRAPHY
The principle of separation is adsorption. The principle of separation is partition
More amount of substance is required. Less amount of substance is required.
Less time consuming i.e. 15—45 minutes More time consuming i.e. 2—3 hours
TLC plates can be heated in an oven for a Paper cannot be heated in an oven for a long
long time time.
Separation is more sharp Separation is less sharp.
Thin layer have physical strength. Ascending Due to lack of physical strength in paper,
techniques are preferred for this type descending techniques are used.
In TLC, chromatoplates are prepared by Here paper is generally used. Silica gel or
coating thin layers of silica gel or alumina on alumina cannot be used because they cannot
glass plates. be made into sheets.
in TLC, corrosive reagents may be coated on Here, corrosive reagents destroy the paper.
Sensitivity of detection of the fraction on Less sensitivity of detection. Spots easily
plate is high. The spots are less diffused diffused
TLC can be evaluated under UV. Paper chromatography cannot be evaluated
5. • COMPARISION OF TLC & HPTLC:
Parameter TLC HPTLC
Chromatographic plate used Hand made /pre- Pre-coated
Adsorbent layer thickness 250 μm 100-200μm
Particle size range 5-20μ 4-8μ
Pre-washing of the plate Not followed Must
Application of sample Manual/Semi Semi
Shape Spot Spot/Band
Spot size 2-4mm 0.5-1mm
Sample volume 1-10μL 0.2-5μL
Application of larger volume Spotting which Can be applied as
leads to over bands
6. No. of samples/plate (20X20) 15-20 40-50
Optimum development 10-15cm 5-7cm
Development time Depends on 40% Less than
mobile phase TLC
Reproducibility of results Difficult Reproducible
7. ADSORBENTS FOR TLC
• In the beginning of TLC method, only few
coating materials were used as adsorbents
such as silica gel, alumina etc.
• However, now a days , there is variety of
adsorbents which can be selectively utilized.
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8. Factors to be considered while choosing the
1. Characteristics of compound to be
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.
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9. Two general properties that decide its application
1. Particle size
Particle size of 1-25 m 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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11. INORGANIC ADSORBENTS
• Silica gel
• Magnesium silicate
• Calcium silicate
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12. ORGANIC ADSORBENTS
• Cellulose & its acetylates
• Charcoal & activated carbon
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13. PREPARATION OF CHROMATOPLATES
• 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
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14. Methods for application of adsorbent.
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15. • 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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16. • 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
It gives layer thickness from 0.2 to 2.0 mm.
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17. ACTIVATION OF PLATES
• After spreading plates are allowed to dry in air
and further dried and activated by heating at
about 100 c for 30 mins.
• By removing the liquids associated with layer
completely, the adsorbent layer is activated.
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18. 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
4. Separation to be achieved- Analytical or
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19. • 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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21. APPLICATION OF SAMPLE
• Sample solution in a non polar solvent is applied.
• The concentration of a sample or standard solution
has to be minimum 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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22. Sample Application (spotting)
Process “finishing line”
A. Draw “guide lines”
lightly with pencil
B. Dissolve solid
sample in CH2Cl2
C. Use TLC capillary
to transfer and spot
dissolved sample “starting line”
T-stillbene benzoic acid
23. DEVELOPMENT CHAMBERS
• TLC plates are placed vertically in a
rectangular chromatography tank (as in fig)
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24. • They are classified according to the separation
(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
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25. 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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26. • 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.)
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27. Two dimensional development :
• It is used if the component of the mixture are not
completely separated by development in a single
• 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 0 another solvent
by changing the edge of plate at 90 (as in fig.)
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29. REVERSED 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
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30. HIGH PERFORMANCE THIN LAYER
Multiple development technique &quantitative
method for TLC/HPTLC
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• HPTLC or High-Performance Thin Layer
Chromatography is an analytical technique
based on thin layer chromatography, but with
enhancements intended to increase the
resolution of the compounds to be separated
and to allow quantitative analysis of the
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32. Pre-conditioning (chamber saturation)
• Pre-conditioning has pronounced influence
on the separation profile
• What happen plate are introduced in non
1. Lager quantity of solvent are required for
2. Increase in Rf values
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33. Reason for chamber saturation
• If chamber is saturated prior to
development solvent vapour get uniformly
distributed in chamber
• As plate are introduced in such chamber it
soon get pre-loaded with solvent
• Less solvent required to travel particular
• Lower the Rf values
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34. Development &drying
• Techniques used for devolvement in HPTLC/TLC
3. Two dimensional
4. Horizontal (continuous)
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• After development the plate are removed
form the chamber & mobile phase
removed completely as quickly as possible]
• Drying preferably be performed in fume
cup board to avoid contamination of lab
• During drying plate should always laid
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36. • Usually hand dryer are used ( cold or hot).
• The following considerations are taken care of
during the drying process:-
i. Essential oil component may evaporate.
ii. Compound sensitive to oxygen may get
iii. Particle of dust from lab. may deposit on
It is precisely for this reason the drying of
chromatogram should preferably be done in
dessicator with protection from heat and light.
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37. Detection and visualization
• One of the most characteristic feature of
the HPTLC is possibility to utilize post
chromatographic off-line derivatization.
• The zones can be located by various
physical, chemical, biological, physiological
• There is no difficulty in detecting the
colored substance or colorless substance
absorbing the short wave in UV region (
254nm ). Contd…
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• Or with intrinsic fluorescence such as
riboflavin or quinine sulphate.
• The substance which do not have above
properties have to be transferred into
detectable substance by means of
chromogenic and flurogenic reagent.
• Iodine is the universal detecting agent.
• Derivatization is essentially required for
detection when individual compounds does
not respond to UV or does not intrinsic
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• Other detection methods based on wetting
and solubility phenomena.
• As aluminium oxide, kieselguhr or silica gel
are hydrophilic adsorbent, on dipping or
spraying the chromatogram with water
lipophilic such as steroid hydrocarbon
appears as a white spot against semi
• Instead of water one can employ
hydrophilic or lipophilic dye solution for
spraying or dipping.
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• In case of hydrophilic dyes such as methylene
blue the background is stained blue, whereas
non-wetted zone appear pale.
• While in case of lipophilic dyes non-wetted
zone appears as a deeply colored against as a
• Fluorescent chemicals are used for the
detection of lipophilic substances by wetting or
non-wetting technique. E.g. Rhodomin B.
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• Several corrosive reagents are also used for
detection of organic compounds.
• These compounds produce color or fluroscent
zone on heating. E.g. 10% v/v alcoholic
sulphuric acid, 5% potassium dichromate.
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42. Visualization of TLC Results
A. Allow solvent to evaporate
from surface of TLC plate.
B. View results under UV light.
look for grayish spots on the UV
fluorescent green background
C. Mark spots with a pencil
while viewing under UV.
43. Quantitative method in TLC and HPTLC
• Techniques for quantitative analysis
A. Analysis of fraction on plate:-
i. Visual comparison with standard.
ii. Precise measurement of spot area or spot
iii. Photo densitometry.
iv. Direct spectrometry.
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B. Determination of fraction after elution from
i. Scooped area containing adsorbed compound
with vacuum cleaner and then analyzed by
various analytical methods like gravimetry,
C. Radioactive substance can be assayed by
i. Photo densitometry of x-ray film.
ii. Direct radio-scanning of chromatoplates.
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45. QUALITATIVE ANALYSIS:
Rf value is calculated for identifying the
spots i.e in qualitative analysis. Rf value
ranges from 0 – 1. but the ideal values are
from 0.3 to 0.8 . when Rf value of a sample
and reference standard.
APPLICATIONS OF TLC:
Separation of mixtures of drugs of
chemical or biological origin,Plant extracts
Separation of carbohydrates, vitamins,
antibiotics, glycosides, etc.
Identification of related compounds in the
To detect the presence of foreign
substances in drugs.
46. IMPORTANT QUESTIONS
• What do you mean by reversed phase TLC?
• Explain various techniques used for the
visualisation of spot in TLC? (5)
• Difference between TLC and HPTLC?(5)
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1. ‘Instrumental method of chemical analysis’ by B.K.
2. ‘Instrumental method of chemical analysis’ by
3. Text book of pharmaceutical analysis by Dr. Ravi
4. Pharmaceutical Analysis Volume – ii Instrumental
Methods by Dr. A. V Kasture, Dr. S. G Wadodkar, Dr.
K. R. Mahadik, Dr. H. N. More.
5. Internet source.
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