Running the column

Carefully fill the column with the eluent. It is best to start by slowly running the solvent down the sides of the column to avoid disturbing the packed column. Fill the column to just below the ground glass joint and attach the bellows, securing with a Keck clip. Open the tap and start pumping the solvent through the column. You should run the column at a steady rate, switching the tubes over as they get to about 2 cm from the top. Run the column continuously, ideally without stopping the flow to swap tubes. Stop the column each time the solvent level gets close to the sand, remove the bellows (release the pressure first) and top up with more eluent. You will need to run ~25-30 fractions. This should use pretty much all your eluent. The eluent used for loading the silica (which was collected in the conical flask) is fine to use – it has only passed through clean silica. Once you have finished the column, remove the bellows (pressure!) and close the tap leaving an empty tube underneath in case of any further drips.

Identifying fractions

TLC is used to identify which fractions contain products. (For this separation, the intense colour of one of the compounds may give you a clue, but this is an unreliable method!). Using the standard TLC plates, mark the plates up with space for 6 channels per plate. On each plate use the first to run some of the mixture (from earlier) and then number the remaining channels (i.e. 1-5, 6-10, 11-15 etc). Spot 2-3 drops from each test tube in turn onto the plates. You do not need to use a new spotter for each fraction! Dab the spotter onto a piece of tissue to draw out any remaining liquid then sample the next tube. Run each TLC plate in the same solvent conditions as your column.

Visualise your plates under a UV lamp and circle the spots. You should see each compound elute in turn over a couple of fractions. These groups of fractions can then be combined and isolated using rotary evaporation.