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THIN LAYER CHROMATOGRAPHY (TLC)

AND
COLUMN CHROMATOGRAPHY

M.PRASAD NAIDU
Msc Medical
Biochemistry,
Ph.D Research scholar.

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.
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.
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.
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)
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 (CH
3
OH)
Column Chromatography
Load the silica gel plus eluent into the
columnthis is called column packing
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!!
Thin Layer Chromatography
Typical TLC chamber
We will use beaker with watch glass or aluminum foil
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
Insert filter paper to saturate atmosphere with solvent
Keep the lid on!!
Thin Layer Chromatography
preparation of chamber
Mark a line about 1 cm
from the bottom with pencil
It is important to use pencil
Place TLC plate in chamber
Let things develop!
Dont let the solvent front run off
The top of the plate!!
Pull it out and mark the solvent front
before it evaporates
Mark spots with pencil!
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