This presentation provides an in-depth overview of Thin Layer Chromatography (TLC), a widely used analytical technique in chemistry and pharmaceutical sciences for separating and identifying compounds. It explains the principle, procedure, and applications of TLC, along with its advantages and limitations. Topics Included- Principle and working mechanism of TLC Components of TLC: stationary phase, mobile phase, and sample Step-by-step experimental procedure Applications in pharmaceutical analysis and quality control Advantages, limitations, and comparison with other chromatographic techniques

1. Thin Layer Chromatography (TLC)
Under the supervision of Assistant
Professor – Mrs. Ritu Rani Yadav
Modern Pharmaceutical Analytical Techniques (MPH 101T)
-Abhni Gupta
-M-Pharm 1st
Year (Pharmaceutics)
PSIT- Pranveer Singh Institute of
Technology (Pharmacy),Kanpur

2. Chromatography
 Chromatography is a process for separating components of a mixture and to
identify the component in a mixture .
 The components of a mixture are differently distributed between two phases
Stationary Phase
(Statisic Part )
Mobile Phase
(Moving Part)
Solid or Gas Liquid or Gas
Chroma + Graphien
Color Writing

3. Introduction of Thin Layer Chromtaography
 TLC is a form of liquid chromatography Consisting of :
-A mobile Phase (Developing Solvent)
-A stationary Phase(A plate or strip Coated with a form of silica gel )
Analysis is performed on a flat surface under atmospheric pressure and room
temperature
The two most common classes of TLC
Normal Phase
Reversed Phase
Normal Phase Reversed Phase
-In which the stationary phase –Polar (Eg-Silica
gel)
-Mobile Phase – Organic Solvent/Mixture of
organic solvent (Less Polar than the Stationary
Phase)
-In which the stationary phase –Silica bonded with an
organic substrate (such as long chain aliphatic acid )
-Mobile Phase – Mixture of water and Organic Solvent
(More Polar than the Stationary Phase)

4. Principal of TLC
The basic principal of TLC is- ADSORPTION
 The component with more affinity
towards the S.P travels slower
 The component with lesser affinity
towards the S.P travels faster

5. Diagramatic way of Procedure

6. Steps involved in Methodology/Procedure
Stationary
Phase
Glass Plate
Preparation
and
Activation of
TLC plate
Application
of sample
Development
Tank
Mobile
Phase
Development
Technique
Detectors and
visualizing
Technique

7. 1. Stationary Phase
 Adsorbents Mixed with water or other solvents Slurry
Name of Adsorbent Compositon Adsobant : Water
Silica gel H Silica gel without
binder
1:1.5
Silica gel G Silica gel + Caso4 1:2
Alumina G Al203 + Binder 1:2
Cellulose powder Cellulose without
Binder
1:5

8. The glass plate have specific dimentions like- Full Plate ( 20×20) c.m.
Half Plate (20×10) c.m.
Quarter Plate (20×5) c.m.
The Glass plate Should be of good quality & withstand high temperatures
(Glass plate)
2. Glass plate

9. 3. Preparation and activation of TLC plate
Slurry ,Whuch is a mixture of stationary phase and water is prepared by using ratio
mentioned earlier
TLC plate can be prepared by using any one of the following techniques-
1. Pouring
Technique
2. Dipping
Technique
3. Spraying
Technique
4.Spreading
Technique

10. 4. Spreading Technique
Spreading is the best technique where a TLC spreader is used.
The slurry after preparation is pour inside the reservoir of TLC spreader.
The Thickness of the adsorbent layer is adjusted by using a knob in the spreader.
Normally a thickness of 0.25 c.m. is used for analytical purpose and 2mm thickness for
preparative purpose.
Then the spreder is rolled on a plate .
The plates are allowed for setting ( air drying ).

11. This is done to avoid crakes on the surface of adsorbent .
After setting, the plates are activated by keeping in an oven at 100 degree
Celsius for 1 hour .
Activation of TLC plate
 After spreading Air dry (5 to 10 minutes)
 Activated by Heating at about 100 degree Celsius for 30 min
 Then plates may be kept in desiccators

12. 4. Application of sample
 Using capillary tube or micropipette
 The spots can be placed at rendom or equidistance from each other.
 the spots should be kept at least & 2cem above the base of the plate.
 Spotting area should not be emerged in to the mobile phase in the devlopment tank
 Atleast four spots can be spotted
5.Development tank
Better to develop in glass beakers, jars to avoid more wastage of solvents
When standard method is used, use twin trough tanks
Do chamber saturation to avoid "edge effect"

13. 6. Mobile Phase
 The pure solvent or mixture of solvent are used .
 The solvent or mobile phase used depends upon various factor.
 Some factors are :-
 Nature of substance to be separated
 Nature of stationary phase
 Mode of chromatography
 Separation to be achieved
Eg- Pet. Ether ,carbon ,acetone, benzene ,toluene, ethyl acetate

14. 7. Development technique
The different development technique are used for efficient
separation.
1. Ascending Technique
2. Descending Technique
3.Two dimensional development technique
4.Horizontal technique

15. 8. Detectors or visualizing agent
 After development of TLC plate spots should be visualize.
 Coloured spots can be detected visually.
 But for detecting colourless spot following technique can be used.
1.Nonspecific
Method
a. Iodine
chamber
Method
b. Sulphuric
acid spray
reagent
method
c.UV chamber
for fluoscent
compound
d. Using
Fluoroscent
stationary
phase
Iodine crystal at the
bottom
70-80%v/v
H2SO4+few mg
k2cr2o7
254nm,at 365nm
When compound
are non-fluoroscent
2. Specific
Method
Spray reagent or detecting agent
Eg. Fecl3 (for phenolic compound
and tannins)

16. R.F. Value (Retention factor)
 It is used for identification of spots.
“ It is the ratio of distance travelled by the solute to the distance travelled by solvent “
Rf = Distance travelled by solute
Distance travelled by solvent
Rf value – (0-1 or less than 1 or 0.3-0.8)
 Rf value is specific and constant for every Compound in a perticular combination of stationary
and mobile phase .
 Rf value of a sample and reference compound is same the compound is identified by it’s
standard.
 when the Rf value differ, the Compounds may be different from it’s Reference or standard.

17. Applications/Advantages/Disadvantages
Applications Disadvantages Advantages
Purity of sample The TLC procedure can not be used for lower
detection limit experiments because it has a high
detection limit.
The separated spots of TLC can be further
visualized without any trouble.
Examination of reaction The plates used in TLC do not possess a more
extended stationary phase. Result reproduction is
challenging in TLC
This chromatography process is cost-effective as
compared to other methods.
Identification of compounds TLC is limited to qualitative analysis, and it can
not be used for quantitative analysis
lt can be used for a number of compounds, and it
does not take much time
Biochemical analysis The process here does not take place in a closed
system. Therefore, aspects like temperature and
humidity can affect the results.
TLC makes it simple to analyze any given
compound's purity standards.
In pharmaceutical industry . The separation length is also restricted as
compared to other chromatography methods
Several compounds can easily get
isolated through TLC.
Separation of multicomponent pharmaceutical
formulations
In food and cosmetic industry