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Adsorption Chromatography

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• Adsorption chromatography refers to the use of a stationary phase or support such an ion-exchange resin, that has a finite number of specific binding sites for the solute molecules.

• Specific interaction between the solute molecules and binding sites on the surface of stationary phase.

• The attractive forces between the solute and support may be ionic, hydrogen bonding or hydrophobic interaction.

• Binding of solute is reversible.

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• Mobile phase is liquid and the stationary phase is a solid.

• Gas-liquid chromatography is an exception.

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• The bed should be homogenous, free of bubbles, cracks or spaces between the walls.

• The liquid leaving the column (eluent) is usually collected as discrete fractions using an automatic collector.

• The separated components are then identified by testing the aliquots of each fractions-spectral measurements, chemical tests, radioactivity etc.

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Operation of a Chromatographic Column

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Loading the Column

• The sample to be analyzed is applied at the top of the column in a concentrated form.

• After the sample is loaded with a graduated pipette it is allowed to percolate the adsorbent.

• A few ml of solvent is applied to wash the sample into the column.

• The column is then filled with eluting solvent.

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Eluting the Column

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Collecting the Eluent

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Detecting of Eluting Components

• Smaller molecules like sugars, amino acids, lipids can be detected by spotting fractions on a TLC plate or by paper chromatography.

• Proteins and nucleic acids by absorption at 280 and 260 nm respectively.

• Enzymes can be measured by measuring their catalytic activity in the fractions.

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