THIN LAYER CHROMATOGRAPHY IN BRIEF

1. Presented by:
Khaleel Basha S.G
M.Pharmacy
ACHARYA NAGARJUNA UNIVERSITY
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2.  Introduction to Chromatography
 Thin layer chromatography (TLC)
 Definition
 History
 Principle Involved
 Experimental Techniques Of TLC
 Chromatographic Parameters
 Factors Affecting Resolution
 Applications
 Conclusion
 References
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3. o Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of
which is stationary(stationary phase) while the other (The mobile phase)
moves in a definite direction.
o Types of Chromatographic techniques:
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Technique Stationary Mobile
Phase
Column/Adsorption
Chromatography
Solid Liquid
Partition Chromatography Liquid Liquid
Paper Chromatography Liquid Liquid
Thin Layer Chromatography (TLC) Solid Liquid
Gas - Liquid Chromatography
(GLC)
Liquid Gas
Gas - Solid Chromatography (GSC) Solid Gas
Ion Exchange Chromatography Solid Liquid

4. o Thin Layer Chromatography (TLC) can be defined as a method
separation or identification of mixture of components into
individual components by using finely divided adsorbent solid
spread over a glass plate and liquid as a mobile phase.
o TLC is often named by other names such as drop, strip, spread
layer, surface chromatography and open column chromatography.
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5. o Simple Equipment
o Short Development Time
o Wide Choice Of Stationary Phase
o Early Recovery of Separated Components
o Easy Visualization of Separated components
o Sensitivity
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6. o The history of TLC dates back to 1938 when IZMAILOV
and SHRAIBER separated plants extracts using 2mm
thick and firm layer of alumina set on glass plate.
o In 1944 CONSDEN , GORDEN & MARTIN used filter
papers for separating Amino acids.
o In 1950, KIRCHNER identified terpenes on filter paper
and later glass fiber paper coated with Alumina.
o In 1958, STAHL developed standard equipment for
analysing by TLC.
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7. Principle of
separation is
“ADSORPTION”
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8. o Based on principle of separation
o The separation depends on the relative affinity of compounds
towards the stationary phase.
o The components with more affinity towards stationary phase
travels slower.
o The components with less affinity towards stationary phase
travels faster.
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9. TLC system components :
 TLC plates :
 Should be stable and chemically inert
 Stationary phase on the plate is of uniform thickness,
consists of fine particles.
 TLC chamber :
• Should maintain uniform Environment inside
• Prevent evaporation of solvent
 Development solvent
• Mobile phase
 Filter paper
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10. 1. Coating material ( Adsorbent ) :
• Large no. Of coating material are commercially produced as
thin layer adsorbent.
• Popularly Used are :
• Silica gel
• Alumina
• Kieselghur
• Cellulose powder
• Calcium phosphate
• Magnesium trisilicate
• Ferric oxide hydrate
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11.  Generally adsorbent do not adhere satisfactorily to the
glass plate to overcome this binders like gypsum, starch,
hydrated silicon oxide. Etc.. Are added to the adsorbents.
Gypsum is most widely used.
 In silica gel G And alumina G the G indicates gypsum.
 Characteristics To be checked while choosing an
adsorbent :
• Solubility
• To as certain whether it is basic, acidic or amphoteric.
• Chemical reactions with adsorbent or chemical changes
due to the action of adsorbent or binders.
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12. 2. Preparation of thin layer In plates :
The various methods of preparing layers are as follows :
 Pouring - measured amount of slurry is put on a given
size of plate which is kept on a level surface.
• This method is simple but not used to a great extent.
 Dipping - plates are prepared by dipping at a time back to
back in chloroform or chloroform-methanol slurries of
adsorbent. Not much used.
 Spraying - small portion of slurry is separated on the
glass plate.
• Difficult to obtain uniform layers on single plate and
there may be variation from plate to plate.
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13.  Spreading : - slurry placed in an applicator. This is either
moved over the stationary plate or it is held stationary and the
plate is pushed or pulled through.
 It gives layer thickness from 0.2 to 2.0 mm.
 This method is most important now-a-days to obtain thin and
uniform layers.
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14.  Precoated plates :- available on glass or plastic sheets.
• Quite Expensive
• Avoid purchase of expensive coating equipment.
• Thickness of precoated plates usually varies from 0.1 to 2.0
mm.
• Contain no gypsum.
• It may be safely activated at 110-120° C
• Ex: Ready-for-use TLC plates with cellulose-F
Ready-for-use TLC plates with Silica Gel-F-254
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15. 5. Development tank :
• Ascending chromatography is most common technique in
TLC.
• Plate placed in the chamber at 45• angle.
• Bottom of the chamber covered up-to 1mm by solvent.
• Top of the chamber covered tightly with the lid for saturation
With solvent vapors to avoid "edge effect ".
• Descending TLC is also possible.
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17. 6. Solvent systems :
• Trail and error Method is used to found best eluent if one
doesn't know the nature of components to be separated.
• If chemical nature is known then suitable solvent is selected
using STAHL'S triangle.
• Mixture of two or more solvents of different polarity give
better separation than chemically homogeneous solvents.
• Solvents used must be as pure as possible.
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18. Elutropic series of selected one or two component solvent
systems with increasing polarity :
• High boiling paraffin's
• Cyclohexane
• Benzene
• Cyclohexane: ethyl acetate( 95:5 )
• Chloroform : acetone (9:1)
• Benzene : methanol ( 95:5 )
• Ethyl acetate
• Ethyl acetate : methanol ( 99:1 )
• Acetone
• Methanol
• Water.
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19. 7. Plate development :
• A drop of sample placed near one edge of the plate ( 20×5) or
20×20cm) position marked with pencil.
• After sample solvent has evaporated, the plate is placed in
closed chamber saturated with vapors of development
chamber.
• After the development has transversed one half to two third
the length of the plate the latter is removed and dried.
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20. 8. Detection of Components :
• Colored substances can be seen visually.
• Colorless compounds can be detect either under UV or by
treating with a visualizing reagent.
• Specific detecting reagents :
 Aniline-phthalate reagent for carbohydrates
 Ninhydrin reagent for Amino acids
 Dragendroff’s reagent for alkaloids.
 Ferric chloride for phenols
 Iodine for unsaturated and aromatic compounds
 Chromic acid or sulphuric acid .
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TLC Profile Of Extracts
Visible light b. short Wavelength (274 nm) c. Long Wavelength (375 nm)

22. 9. Evaluation of chromatogram
Evaluation may be either qualitative or quantitative :
Qualitative : Rf values for TLC show inferior
reproducibility.
 Depends upon factors such as :
 Quality of the layer material
 Activation of grade of the layer
 Quality of solvent
 Equilibration of chamber
 Presence of impurities
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23. Quantitative : Can be carried out in two ways
1. Direct methods
2. Indirect methods
1. Direct methods :
Quantitative determination is undertaken directly on the
layer.
Methods involves :
I. Visual assessment of chromatograms
II. Determination by measurement of spot areas
III. Quantitative TLC incorporating densitometry
IV. Direct spectrophotometry on thin layer chromatograms
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24. 2. Indirect Methods
The substances removed from the adsorbent and then determined
after elution. These methods also know as elution techniques.
 Colorimetry
 Flourimetry
 Radiometry
 Flame photometry
 Gravimetry
 UV spectrophotometry
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25. The position of migrated spots on the chromatogram are indicated
by different terms such as Rf, Rx, Rm, evaluated.
Rf: The R is related to the migration of the solute front relative to
the solvent front as :
Rf = Distance travelled by solute from the original line
Distance travelled by solvent from the original line
Rf defines the movement of the substance relative to the solvent
front in a given chromatographic system.
 Rf values are less than 1.
 Higher the Rf value the substance is very non-polar, low Rf
value refers to a substance that is very polar.
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Rx : In some cases, the solvent front runs off the end of the filter paper, the
movement of a substance in such cases is expressed as Rx but not Rf.
Distance travelled by the substance from the origin line / Distance
travelled by the standard substance x from the
original line

27.  Retention factor : absolute movement of the chemical
depends on how far solvent travels.
 Thin layer chromatography plate :
 affect the Rf value for a given chemical
 Thickness ,change in solvent, uniformity of the layer of
adsorbent can greatly change the Rf value.
 Solvent impact retention factors
 Thickness of layer
 Temperature
 Mobile phase
 Too much of sample.
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28.  As a check on processes :- checking distillation fractions
and for checking the progress of purification by
molecular distillation.
 In organic chemistry :
• For checking the purity of sample
• As a purification process
• Examination of reaction
• For identifying organic compounds like acids, alcohols,
alkaloids etc,.
 For separation of inorganic compounds
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29.  Foreign alkaloid substances present in alkaloidal drugs.
e.g.: atropine sulphate, codeine
 Foreign steroids present in steroidal drugs
 E.g.: betamethasone valerate
 Separation of amino acids
 In Quantitative analysis
 Biochemical analysis: TLC is extremely useful in isolation or separation of
biochemical metabolites or constituent from its body fluids, blood plasma,
serum, urine etc.
 One of the most important application of TLC is in separation of multi
component pharmaceutical formulations.
 In food and cosmetic industry, TLC method is used for separation and
identification of colours, preservatives, sweetening agent, and various cosmetic
products.
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30.  Due to its simplicity, sharpness of separations, high sensitivity,
speed of separation and ease of recovery of separated
compounds, TLC has found increasing applications in all
branches and this technique has been proved especially
versatile process for the analytical investigation of substances
that available in very small quantities.
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31. o Gurdeep R. Chatwal, Sham K Anand, ( 2107), Fifth
revised edition , Instrumental methods of Chemical
Analysis, 2.599-2.615.
o Asuthoshkar, Third revised edition, Pharmaceutical
Drug Analysis, 473-491.
o Michael judge (June 10 2018) Factors that affect
Thin layer chromatography Sciencing.com.
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