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1. THIN LAYER
CHROMATOGRAPHY (TLC)
AND
COLUMN CHROMATOGRAPHY
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2. Overview for today’s experiment
 You will have to separate three components of paprika.
 The three components can be easily identified because they are colored
(absorb visible light).
 They have different polarities.
 They can be separated using column chromatography.
 You can monitor the separation using thin layer chromatography.
What is chromatography….

3. Chromatography
• Very useful technique in organic chemistry
based on differential adsorption.
• Used to separate components in a mixture
(solid or liquid).
• It depends on the polarity of the ingredients
involved --- intermolecular forces!!
• Thin layer chromatography (TLC) is used to
analyze components and purity of a mixture.
• Thin layer chromatography (TLC) is also used
to monitor the progress of a reaction.

4. Chromatography
What do we need to perform a
chromatographic separation?
• Adsorbent: Silica gel (silicon dioxide), also
called “stationary phase”.
• Eluent: solvent used to move your compound
trough the silica gel, also called the mobile
phase.
• Your compound mixture to be separated.
• Patience and chemical intuition.

5. Chromatography
• More polar molecules “stick” to the adsorbent longer.
• Less polar molecule separate more easily from the
adsorbent.
• When this happens, separation occurs.
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
SiOH
Stationary phase To be separated
O
Eluent (mobile phase)

6. Chromatography
• More polar solvent move the molecules more efficiently
• Less polar move the molecules less efficiently
• Separation occurs
Most polar
Least polar
Alkanes
Toluene
Diethyl ether
Chloroform
Acetone
Ethyl acetate
Ethanol
Methanol (CH3OH)

7. Column Chromatography
Load the silica gel plus eluent into the
column…this is called “column packing”

8. Column Chromatography
Using a Pasteur pipette, load your compound that was dissolved
in a minimum of solvent onto the silica.
Your test solution will then add the eluent.
Do not let your column run dry!!

9. Thin Layer Chromatography
Typical TLC chamber
We will use beaker with watch glass or aluminum foil

10. Thin Layer Chromatography
Spotting TLC plate
•Use different capillary for each
solution.
• make solution of approx. 1-2 mg
of sample in 1 ml of solvent.
• Spot 2-3 times
•Try to make small spots

11. Insert filter paper to saturate atmosphere with solvent
Keep the lid on!!
Thin Layer Chromatography
preparation of chamber

12. Mark a line about 1 cm
from the bottom with pencil
It is important to use pencil

13. Place TLC plate in chamber Let things develop!
Don’t let the solvent front run off
The top of the plate!!

14. Pull it out and mark the solvent front
before it evaporates
Mark spots with pencil!

15. Good, bad and ugly
• First TLC shows
”overloading" due
to too much sample.
• Second shows good
separation.
• Third shows almost
not enough compound,
but OK

16. What to do today?
Using diethyl ether, you will extract a mixture of
three compounds from paprika ( Capsanthin,
Capsorbin and -carotene).
 The three compounds have different polarities,
thus can be separated using chromatography.
You will spot the mixture on a TLC plate, develop
using 15% Et2O and 85% heptane.
Calculate the Rf for each spot. Record data in
your notebook with the color of the spots.
Using the procedure in your handout you will
perform a column chromatography, this time you will
increase the polarity of the solvent (eluent)
gradually. Asses with TLC

17. THANK YOU