Adsorption Chromatography 1Dr. Nikhat Siddiqi

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. 2Dr. Nikhat Siddiqi

Mobile phase is liquid and the stationary phase is a solid. Gas-liquid chromatography is an exception. 3Dr. Nikhat Siddiqi

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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. 7Dr. Nikhat Siddiqi

Operation of a Chromatographic Column 8Dr. Nikhat Siddiqi

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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. 10Dr. Nikhat Siddiqi

Eluting the Column 11Dr. Nikhat Siddiqi

Collecting the Eluent 12Dr. Nikhat Siddiqi

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. 13Dr. Nikhat Siddiqi

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