COLUMN CHROMATOGRAPHY

Column chromatography is a preparative technique used to separate and isolate pure compounds in multi-components reaction mixtures.
The solvent to be used in bulk chromatography is determined using thin layer chromatography TLC.
The solute spots in the left hand plate have hardly moved from the start line because the solvent system was not polar enough.
Whereas the spot have too far in the right hand plate because the solvent system was too polar.

The centre plate demonstrates a good separation of products in the mixture.
Select a straight column equipped with the tap and clamp it firmly in the stand. If the column has a sintered glass frit at the base, it’s ready to use.
Otherwise a glass wool plug must be place in the column to stop the solid phase clogging the tap.
When using organic solvents, the tap must not be greased because the grease will be leached by the solvent. A plastic tap is ideal.
There are a number of ways of preparing columns for chromatography, but they all have the same aim. To produce a column that is free of air bubbles and voids.

In this common method, first add a little solvent to wet the fret and leave sufficient solvent above it to stop it running dry.
Even if the tap is opened, the fret will only allow running out very slowly.

Next, make up slurry of conical flask by adding solvent to a solid phase such as silica gel.
The amount of solid must be sufficient to fill the column a third full.
Swerve the flask to suspend the solid and release any air bubbles.
Then pour the slurry into the column through a funnel.
Add a little more solvent to give a reservoir above the solid.
Then use a Pasteur Pipette to recycle the solvent washing down the inside of the column while the solid settles.
Any air bubbles can be released by gently tapping the sides of the column.

Use compressed air from a laboratory airline or from a cylinder to force the liquid to just above the top of the column of solid.
Then introduce the crude reaction mixture. This should have been dissolved in a minimum of the chosen solvent or if it not sufficiently soluble, in another compatible solvent.
Run the sample solution down the glass walls to prevent disturbing the top of the column.
Use a little solvent to rinse out the flask and to wash the sample off the wall of glass column.

Connect up the compressed air and force out the excess solvent so that the reaction mixture in the top layer of the silica gel column.
Remove the air line and close the tap to stop the top of the column running dry.
Then to provide reservoir of eluent, completely fill the vessel with solvent taking care not to disturb the surface of the silica gel
With some solid phases, it can be helpful to top the column with a thin layer of sand to minimize any disturbance of the solid surface when solvent is added.

Refit the airline and open the tap.
If a suitable solvent has been chosen, the initial eluent will contain no sample fractions and can be collected in a conical flask.
Well before the initial fractions are due to come off the column, briefly close the tap, then start collecting the eluting fractions in numbered tube or flasks.
The volume in each fraction will vary according to the scale of the separation.
It is undesirable to keep closing the tap during the elution and with good organization; it should be possible to collect the fractions without doing so.

If all the components of the mixture are colorless and there is no other way of judging the progress if the separation, fractions should be collected from the beginning of the elution.
When the elusion is complete, close the tap and analyze the fraction by thin layer chromatography.
If it’s necessary to use more than one plate, it’s important to overlap the fractions making the last fraction on one plate the first on the next.
Well separated bands of single spots indicate a successful separation.
Combine all the fractions with the same component into a pre-weighed round bottom flask.

Dry off the solvent using a Rotary Evaporator.
Then re-weigh the flask and record the yield.