Preparing & loading the column³

The following instructions relate to a 50 g of silica in a 25 mm diameter (Ø) column, being used to separate 500 mg of compound (1:100 silica ratio), collecting 20 mL fractions in test tubes.

Preparing the column

In order to hold the sand/silica within the column, a cotton wool plug is fitted above the tap. The column should be securely clamped in two places around the body of the column and a 250 mL conical flask placed underneath to collect any solvent. Open the tap fully whilst loading the column.

Add around 1 cm depth of sand to the column. The sand doesn’t retain the compounds and allows anything eluting from the silica to flow into the tap immediately. The sand layer ensures that the stationary phase itself is contained only in the purely cylindrical part of the glass column; a uniform column aids resolution.

You will need to prepare about 1 L of eluent for the loading and running of this column. The dry silica (50 g) is weighed into a conical flask (in a fumehood – avoid breathing the dust) and some of the eluent is added and the silica slurried. This slurry is carefully poured into the column, taking care to have a flat, level interface between the sand and silica. Remaining silica can be washed in with more of the column eluent and silica stuck to the sides of the column can be washed down using a pipette. Run the solvent down to the level of the silica then close the tap. Bellows can be used to pump the solvent down the column, but must be secured with a Keck clip. Also, check there isn’t silica stuck inside the ground glass joint before trying to attach the bellows to the column.

Once the solvent level has reached the top of the silica and the tap has been closed, the column is ready to be loaded and run.

Loading the column

It is important that once the compound is loaded, the column is then run until all fractions have been collected without interruption. Stopping a column with compounds loaded leads to the bands of compounds spreading as they will diffuse in the solvent and reduce the resolution.

Swap the conical flask from underneath the column for the first fraction test tube. Adjust the height of the column so that the lower end of the column supports the test tube, preventing it from falling, but so that the tube can easily be removed and replaced. In order to load the compound onto the column, dissolve the sample in a small amount of the eluent in the sample vial and carefully pipette onto the column, running the solution down the sides of the column in order to avoid disturbing the silica. Rinse the vial and sides of the column with further small quantities of eluent to ensure all the compound is on the top of the silica.

Run the column down to the layer of the silica again, collecting the run off into the first fraction tube. Turn the tap off when the solvent reaches the top of the silica and add 1-2 cm depth of sand to the top of the column. The sand layer prevents the silica being disturbed when solvent is added to the column.

³There are many different ways to prepare and run flash columns. This method describes how to ‘wet-pack’ the silica and we have selected one method for the loading of compounds onto the column.