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Column chromatography

By: Logo
Column chromatography is a chromatography technique used to separate mixture of chemical
substances into its individual compounds. It is a widely used method for the purification or separation of
chemical compound mixture in lab.
Column chromatography can be done using gravity to move the solvent, or using compressed gas to push
the solvent through the column.
Stationary phase
The stationary phase or adsorbent in column chromatography is a solid. The most common
stationary phase for column chromatography is silica gel, the next most common
being alumina. Cellulose powder has often been used in the past.
There is an important ratio between the stationary phase weight and the dry weight of the analyte
mixture that can be applied onto the column. For silica column chromatography, this ratio lies
within 20:1 to 100:1, depending on how close to each other the analyte components are being
Mobile phase
 The mobile phase or eluent is a solvent or a mixture of solvents used to move the
compounds through the column.
There is an optimum flow rate for each particular separation. A faster flow rate of the
eluent minimizes the time required to run a column and thereby minimizes diffusion,
resulting in a better separation.
Preparation of the Column:
Place the column in a ring stand in a vertical position.
A plug of glass wool is pushed down to the bottom of the column.
Prepare slurry of silica gel with a suitable solvent & pour gently into the column.
Open the stop cock & allow some solvent to drain out. The layer of solvent should always cover the adsorbent; otherwise
cracks will develop in the column.
Adding the Sample to the Column:
 Dissolve the sample mixture in a minimum amount of solvent (petroleum ether).
 Remove the solvent by placing the mixture in a rotary evaporator at a low temperature.
 Place the dry powder on a piece of weighing paper and transfer it to the top of the column through the funnel.
Developing the Chromatogram:
Attach a dropping funnel filled with petroleum ether on to the column.
Add petroleum ether continuously from the funnel to the top of the column.
Open the stopcock carefully.
The components of the mixture run down the column forming two separate bands.
Recovering the Constituents:
 Continue running the petroleum ether till both the bands are removed separately.
 Collect the constituents in two different round bottom flasks.
 Evaporate the solvent by placing the mixture in a rotary evaporator.
When a sample contains compounds with similar polarity, the separation between then can
be small and recovering all of your clean sample can become a challenge
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