Paper Chromatography
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• Paper chromatography and TLC are examples
of adsorption chromatography.
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Paper chromatography
• Cellulose support is in the form of sheet of paper
which has large amount of water bound to it.
• Partitioning occurs between the bound water and
developing solvent.
• The solvent used is water.
• There is one effluent and substances are
distinguished by their positions in the paper after
the solvent has moved a given distance.
• Uses: To identify unknown samples
• Isolation of components of mixtures.
Dr. Nikhat Siddiqi
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• Paper chromatograms can be developed by
either ascending or descending solvent flow.
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Ascending Paper Chromatography
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Descending Paper Chromatography
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Descending chromatography
• Faster
• For quantitative separations of substances
with very small Rf values which require long
runs the solvent can run off paper.
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Two Dimensional Paper
Chromatography
Dr. Nikhat Siddiqi
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Detection of spots in the paper
• By colour
• Fluorescence
• Chemical reaction after the paper is sprayed with
various reagents
• Radioactivity.
• Identification is based on comparision with
standards of known Rf or by elution.
• Elution is accomplished by cutting out the spot
and soaking the paper in appropriate solvent.
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Thin Layer Chromatography (TLC)
• Originally used to separate lipids.
• Substances used include inorganic substances
like silica gel, aluminium oxide, diatomacious
earth, magnesium silicate organic substances
like cellulose, polyamide, polyethylene
powder.
• Used for separation of small molecules.
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• Stationary phase is a layer (0.25-0.5 mm) of
sorbent spread uniformly over the surface of a
glass or plastic plate.
• A slurry of sorbent is made in a solvent for
that particular sorbent.
• The plate is coated with the slurry and
allowed to dry at 100-120 oC in an oven.
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• The TLC plate is placed in a chamber
containing the solvent and developed by
ascending chromatography.
• After the solvent has reached the top the
plate is removed from the chamber and dried.
• Spots are located as on paper by colour,
fluorescence or by spraying.
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Thin Layer Chromatography (TLC)
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Detection
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Commonly used sprays are
Ninhydrin for amino acids
Rhodamine B for lipids
Antimony chloride for steroids and terpenoids
Sulfuric acid plus heating for organic substances
Potassium permanganate in sulfuric acid for
hydrocarbons
• Anisaldehyde in sulfuric acid for carbohydrate etc.
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Elution
• By scraping off the sorbent and eluting the
powder with a suitable solvent.
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