3. CHROMATOGRAPHY
Chromatography is a process of separation in which
the components to be separated are distributed between
two immiscible phases a stationary phase and mobile
phase.
4. THIN LAYER
CHROMATOGRAPHY
Thin layer chromatography (TLC) is an important technique for
identification and separation of mixtures of organic compounds .
TLC can be used to help determine the number components in a
mixture, the identity of compounds ,and purity of a compound.
TLC is performed using thin sheets of glass ,aluminium or plastic
coated with a layer of stationary phase usually silica gel is used.
5. PRINCIPLE
It is based on the principle of adsorption chromatography or
partition chromatography or combination of both ,depending on the
adsorbent, its treatment and nature of solvents employed.
The components with more affinity towards stationary phase
travels slower.
Components with less affinity towards stationary phase travels
faster.
6. PREPERATION & ACTIVATION
OF TLC PLATE
Pouring (simplest method )
Dipping (used for small plates )
Spraying( difficult to get uniform layers)
Spreading( best technique)
7. STEPS IN TLC
TLC consist of three steps
Spotting
Development
Visualization
8. SPOTTING
In TLC the samples must be carefully applied to the plate to
minimize spreading.
Samples sizes from 0.1 mg to 50mg are the best for TLC.
Smaller amount are difficult to visualize, while the larger spots
result in over loaded and variable results.
Sample should be dissolved in relatively volatile solvents.
9. DEVELOPMENT
The spotted plate is placed in a sealed development tank filled with
vapour of mobile phase .
Its lower side immersed in the solvent to a level below the applied
sample spots.
The solvent rises due to capillary flow in a process called
development.
Development times can range from 3-60 minutes .
10. VISUALIZATION
Visible-the bands or spots can be seen immediately.
Fluorescence –observed under UV light.
Reaction with a chromogenic reagent .
11. CALCULATION OF Rf VALUE
The basic parameter used to describe migration in TLC is the Rf value
where,
Rf=Distanced travelled by solute
Distance travelled by solvent
Rf value vary from 1 to 0
12. APPLICATION OF TLC
To check the purity of given samples.
Identification of compounds like acids ,alcohols, proteins etc .
Used in cosmetic industry.
It is used for testing of Drug.
Ink analysis.
13. CONCLUSION
TLC is the method used for analyzing mixtures by separating the
compounds in the mixture .On a TLC plate there is a competition
between the stationary phase , silica gel and the mobile phase ,ethyl
acetate's can be used to help determine the number of components
in a mixture, to identify the compounds ,and the purity of a
compound.
