How to do Column Chromotography

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Presentation Description

Column Chromatography: A Step-by-Step Guide on How to do it.

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How to do Column Chromatography

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Column Chromatography

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Here are the steps involved in Column Chromatography

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1) Preparation / Packing of an adsorbent column

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2) Solvent system / Mobile phase Column chromatography  uses a single solvent. The commonly used ones are petroleum ether, benzene, carbon tetrachloride, ethyl acetate, acetone, cyclohexane , ether, chloroform, etc.

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The role of the solvent in column chromatography is

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3) Application of samples The study sample can be applied to the top of the prepared column in various ways: Remove most of the mobile phase from above the column using a substrate and drain the remaining solvent into the column bed. Apply the sample carefully with a pipette and allow it to run in the column. To wash the final traces of the sample into the column bed, apply a small volume of the mobile phase. Carefully add the mobile phase to the column to the height of 2-5 cm and maintain it by connecting the column to a suitable reservoir of mobile phase.

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Add sucrose to a concentration of 1% to increase the density of the sample when the solution is layered into the liquid above the column bed; it will automatically sink to the surface of the column. Use capillary tubing or roller pumps to pass the sample directly to the column surface.

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4) Sample elution After sample application, the next step is to remove the material from the column using an appropriate solvent. This can be carried out in following ways:

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5) Collection and analysis of fraction Collect the fractions from the column into a series of test tubes, either manually or with a fraction collector. Analyze each fraction for the presence of the compound. Colored compounds can be detected by visual observation, but colorless compounds are detected by spectrophotometer or colorimetry or UV adsorption method.

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