Column Chromatography

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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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