chromathography

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• THIN LAYER CHROMATOGRAPHY (TLC)
• AND
• COLUMN CHROMATOGRAPHY

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Overview for todays 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.

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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.

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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.

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Chromatography

• More polar molecules stick to the adsorbent
  longer.
• Less polar molecule separate more easily from
  the
• adsorbent.
• When this happens, separation occurs.

Eluent (mobile phase)
Stationary phase
To be separated
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Chromatography

• More polar solvent move the molecules more
  efficiently
• Less polar move the molecules less efficiently
• Separation occurs

Least polar
Alkanes Toluene Diethyl ether Chloroform Acetone E
thyl acetate Ethanol Methanol (CH3OH)
Most polar
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Column Chromatography
Load the silica gel plus eluent into the
columnthis is called column packing
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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!!
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Thin Layer ChromatographyTypical TLC chamber
We will use beaker with watch glass or aluminum
foil
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Thin Layer ChromatographySpotting 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

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Thin Layer Chromatographypreparation of chamber
Insert filter paper to saturate atmosphere with
solvent
Keep the lid on!!
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Mark a line about 1 cm from the bottom with pencil
It is important to use pencil
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Let things develop!
Place TLC plate in chamber
Dont let the solvent front run off The top of
the plate!!
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Pull it out and mark the solvent front before it
evaporates
Mark spots with pencil!
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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

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What to do today?

• Using diethyl ether, you will extract a mixture
  of three compounds from paprika ( Capsanthin,
  Capsorbin and b-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

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Thank you