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1. THIN LAYER CHROMATOGRAPHY
Presented By : Sabir Hussain
M. Pharm 1st Sem
Roll No. 230624010
Dept. of Pharmacy Tripura University
Guided By : Dr. Rajat Ghosh, PhD
Assistant Professor
Dept of Pharmacy, Tripura University
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2. Contents :
History
Introduction
Chromatography
Principle of TLC
Stationary phase
Glass plates
Preparation of glass plates
Mobile Phase
Spotting
Developing chamber
Application
References
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3. History :
The first reported use of a thin layer was in 1938
by two Russian scientists, N.A. Izmailov and M.S.
Schreiber.
They separated plant extracts on a slurried
adsorption medium spread to a 2-mm-thick layer
by spotting an alcoholic plant extract in the
center of the layer and observing rings as the
solution spread.
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4. Chromatography :
Chromatography is an important biophysical
technique that enables the separation,
identification, and purification of the
components of a mixture for qualitative and
quantitative analysis.
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5. TLC Definition :
• TLC is defined as the method of separation identification of a mixture
of compound into individual components by using finely divided
adsorbent solid/liquid spread over a glass plates as a stationary phase
and Liquid as a mobile phase.
• On completion of the separation, each components appears a spots.
Each spot has a retention factor(Rf )
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6. Introduction :
TLC is a liquid chromatography consisting of :
 A mobile phase (developing solvent)
 A stationery phase (a plate or strip coated with a form of
silica gel).
Analysis is done under normal atmospheric pressure and
room temperature.
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7. Principle :
• The principle of TLC is where mixture components are
separated between a fixed stationary phase and a liquid
mobile phase by differential affinities between the two
phases.
• The separation is through adsorption.
• Component with less affinity towards stationary phase
travels fast.
• Component with more affinity towards stationary phase
travels slow.
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8. Stationary Phase :
• TLC plates also known as Chromatoplates can be prepared in the
lab but mostly purchased.
• Silica gel and alumina are among the most common stationary
phases, but others are available as well.
• Many plates incorporate a compound which fluoresces under
short-wave UV (254 nm).
• The backing of TLC plates is often composed of glass, aluminum,
or plastic.
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9. Glass backed TLC plates
Aluminium backed TLC plates
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10. Glass Plates :
• Glass plates which are specific dimensions like 20 cm X 20 cm (Full
plate), 20 cm X 10 cm(Half plate), 20 cm X 5cm (Quarter plate) can be
used. These dimensions are used sincethe width of the commercially
available TLC spreader is 20 cm.
• Microscopic slides can also be used for some applications like
monitoring the progress of a chemical reaction. The development
time is much shorter like 5 minutes.
• Glass plates of different dimensions can also be used when the TLC
plates are prepared without the use of TLC spreader. In general, the
glass plates should be of good quality and should withstand
temperatures used for drying the plates.
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11. Preparation and Identification of TLC plates :
• Pouring technique: The slurry is prepared and poured on to a
glass plate which is maintained on a leveled surface.
• The slurry is spread uniformly on the surface of the glass plate.
After setting, the plates are dried in an oven is used for
spotting.
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12. Dipping technique:
• In this two plates (either of standard dimensions or microscopic
slides) are dipped in to slurry and are separated after removing from
slurry and later dried.
• The disadvantage is that a larger quantity of slurry is required even
for preparing fewer plates.
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13. Spraying technique :
• Resembles that of using a perfume spray on a cloth. The suspension
of adsorbent or slurry is sprayed on a glass plate using a sprayer.
• The disadvantage is that the layer thickness cannot be maintained
uniformly all over the plate.
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14. Mobile Phase :
 It is a developing liquid which travels up the stationary phase, carrying the
samples with it. It depends on;
Nature of the substance to be separated i.e polar or non polar.
Nature of stationary phase used
Mode of chromatography
 Solvent used should be of high purity.
 Solvents used:- petroleum ether
benzene
carbon tetrachloride
chloroform
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15. Spotting :
• 1% solution of sample or standard is spotted using a
capillary tube or micropipette. The spots should be kept
at least 2cm“4 above the base of plate and the spotting
area should not be immersed in mobile phase in a
developing chamber.
• The width of the band must be as narrow as possible.
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16. Developing Chamber :
• It is used for the purpose of TLC plate run in
mobile phase.
• After the mobile phase is poured into the
chamber it is kept closed with lid.
• This is done to equilibrate the atmosphere of
empty space in chamber with the mobile solvent.
• This is also known as saturation of TLC chamber,
Edge effect occurs when the solvent front in the
middle of TLC plate moves faster than that of
edge edge of plate.
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17. Development of TLC plates :
Different development techniques are used for efficient
separations. They are -
1. One dimensional development (ascending or descending
technique).
2. Two dimensional development
3. Horizontal development
4. Multiple development
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18. One dimensional development :
• Like conventional type, the solvent flows against gravity.
• The spots are kept at the bottom portion a kept in a chamber
with mobile phase solvent at the bottom.
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19. Two dimensional development :
• This technique is similar to 2-Dimensional TLC.
The paper is developed in one direction and after
development, the paper is developed in the
second direction allowing more compounds or
complex mixtures to be separated into individual
spots.
• In the second direction, either the same solvent
or different solvent system can be used for
development.
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20. Detecting Agent :
• After the development of chromatogram, the spots should be visualized.
• Detecting of coloured spots can be done visually.
• But for detecting of colourless spots, any one of the following techniques
can be used.
• Non specific method: Where the number of spots can be detected but not
exact nature of compound
Example
1. lodine Chamber Method: Where brown or amber spots are observed
when the paper is kept tank with few iodine crystals at the bottom.
2. UV Chamber for fluorescent compounds: When compounds are viewed
under UV chamber at 245 nm or at 365 nm fluorescent compounds can be
detected.
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21. Specific Method :
• Specific spray reagents or detecting agents visualizing agents used to
find out the nature of compounds for identification purposes
• Examples;.
• Ferric chloride for phenolic compounds
• Ninhydrin in acetone for amino group
• Dragen droff’s reagent for alkaloid
• 3,5-Dinitro benzoic acid for cardiac glycosides
• 2,4-Dinitrophenyl hydrazine for aldehyde and ketones
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22. Advantages :
• Less cost required.
• Less time required.
• High sensitivity.
• High resolution.
• Simple equipment required.
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23. Disadvantages :
• Since the separation takes place in open chamber so it can affect by
humidity.
• Uneven migration of solvent.
• Over large spot formation.
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24. Application :
• Separation of mixtures of drug of chemical or biological origin’
• Separation of carbohydrates (sugars), vitamins, antibiotics, proteins
alkaloids, glycosides, amino acids etc.
• Identification of related compounds in drugs.
• Purity of samples.
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25. References :
• Instrumental Methods of Chemical Analysis, By Dr. H.KAUR 2nd Edition
• Vogel's - Textbook of quantitative chemical analysis (5th Edition)
• https://chem.libretexts.org/Courses/SUNY_Oneonta/Chem_221%3A_
Organic_Chemistry_I_(Bennett)/2%3ALab_Textbook_(Nichols)/02%3A
_Chromatography/2.03%3A_Thin_Layer_Chromatography_(TLC)/2.3E
%3A_Step-by-Step_Procedures_for_Thin_Layer_Chromatography
• https://www.researchgate.net/publication/277703425_Thin_Layer_C
hromatography
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26. THANK YOU