Adsorption Chromatography - KSUfac.ksu.edu.sa/sites/default/files/bch_332_lecture_9.pdf ·...
Transcript of Adsorption Chromatography - KSUfac.ksu.edu.sa/sites/default/files/bch_332_lecture_9.pdf ·...
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Adsorption ChromatographyBCH 332 lecture 9
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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.
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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 of a specific volume.
• The separated components are then identified by testing the aliquots of each fractions-spectral measurements, chemical tests, etc.
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Operation of a Chromatographic Column
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Packing the 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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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 or by Mass spectrometry.
• Proteins and nucleic acids by absorption at 280 and 260 nm respectively. Or by running on gel electrophoresis.
• Enzymes can be measured by measuring their catalytic activity in the fractions.
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