Thin layer chromatography.

1. THIN LAYER CHROMATOGRAPHY
Presented By
Mr. Sandip Badadhe

2. Learning objectives:
After this presentation learner should be
able to
 Explain principles, Experimental
Details and applications of TLC.
 Perform TLC Experiment for Mixture
separation and Purification.

3. Principle
• Based on the principle of separation.
• Separation is either by Adsorption or by
Partition.
• Different relative affinity of compounds
towards SP and MP.

4. Experimental Details
1) Choice of adsorbent
2) Preparation of thin layers in plates
3) Activation of adsorbent
4) Purification of silica Gel G Layer
5) Sample application
6) Development Tank
7) Solvent system (Mobile Phase)
8) Plate development
9) Detection of components
10)Evaluation of chromatogram

5. What are requirements of TLC?

6. Choice of adsorbent
Adsorbent Nature Activity Separation
Mechanism
Components to
be separated
Silica Gel Acidic Active Adsorption Acidic and
Neutral
Alimina Basic Active Adsorption Basic and
neutral
Kiesleguhr Neutral Inactive Partition Strongly
hydrophilic
Cellulose
Powder
Neutral None Partition Water Soluble

7. Preparation of thin layers in plates
I. Pouring- Pour adsorbent slurry on plate, Then
Plate is tipped back and forth.
II. Dipping- Plates are dipping at a time back to
back in CHCl3 slurries of adsorbent.
III. Spraying- A small point sprayer for Spread of
the slurry on glass plate.

8. Preparation of thin layers in plates
IV. Spreading- The slurry with the help of
applicator.
V. Precoated plates- Ready to use thin layers
Plates are now available.

9. Activation of adsorbent
• Drying the thin layer plates, for 30 minutes in
air and then in an oven at 110 C for another
30 minutes.
• For very active layers, can be heated to 150 C
for about 4 hours.

10. Purification of silica Gel G Layer
preliminary development of plate with
methanol- conc.HCl ( 9:1 v/v)
plates are again dried and activated at 110c

11. Sample application
Agla micro syringe is generally used for
quantitative work
capillary tubes may be used for qualitative
work.

12. Development Tank

13. Solvent system (Mobile Phase)
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 reverse phase)
4. Separation to be achieved- Analytical
or preparative.

14. Solvent system (Mobile Phase)
Selection is done by stahl’s triangle method

15. Plate development
• Generally Ascending method is used to greater
extent
• Descending TLC and Horizontal TLC is also
possible.
• These two techniques have no advantages
over the ascending techniques in terms of
efficiency of separation and speed of analysis.

16. Detection of components
A. Allow solvent toevaporate
from surface of TLCplate.
C. Mark spots with a pencil while
viewing under UV.
UV
B. View results under UV light. look
for grayish spots on the
fluorescent greenbackground
UV

17. Evaluation of chromatogram
A. Qualitative Evaluation
B. Quantitative Analysis
B(i) Direct Method: performed directly on the
adsorbent layer
B(ii) Indirect Method: the separated constituents
are quantitatively removed from the adsorbent
and subsequently estimated after elution.

18. Evaluation of chromatogram
B (1) Direct Method:
(i) Measurement of Spot-areas
(ii) Densitometry
(iii) Spectrophotometry
B(2) Indirect Method: These methods are based on
elution techniques, followed by micro-analysis of
the resultant eluate either by
Colorimetry Fluorimetry ; Radiometry ; Flame-
photometry ; UV-Spectrophotometry ;
Gravimetry ; Polarography

19. Applications of TLC
• To check the purity of the given samples.
• Identification of compounds like acids,
alcohols, proteins, alkaloids, amines,
antibiotics, and more.
• To evaluate the reaction process.
• To purify samples
• To keep a check on the performance of other
separation processes.