Performing Column Chromatography

Close the stopcock at the bottom of the column and insert a cotton ball, pushing it down with a glass rod. Secure the column to a ring stand with clamps.

Pour a fine layer of sand to fill the curved bottom section and form a level surface on the cotton. Clean the sand off the sides with solvent using a Pasteur pipette and gently tap the column to ensure the sand level is horizontal.

Add 25 milliliters of solvent (acetone/ethanol mixture) to silica powder and stir until a slurry forms. Pour the slurry into the column using a glass funnel, continuing to stir until fully added, then wash any remaining gel from the beaker sides into the column.

Open the stopcock to allow solvent to drain until the solvent line is just above the silica gel, keeping the column wet at all times. Tap gently on the column during packing to eliminate bubbles and ensure even settling.

Using a glass pipette, slowly and carefully load a quarter milliliter of the methyl red and bromocresol green mixture onto the top of the column, allowing it to run gently down the side without disturbing the silica layer. Verify all sample is absorbed before proceeding.

Add approximately one centimeter of sand as a protective layer over the loaded sample. Fill the column with the eluent solvent to prepare for running the chromatography.

Open the stopcock to begin elution and start collecting fractions in numbered test tubes as compounds begin to exit. Continuously monitor the solvent level and add more eluent before the column dries, adding carefully down the side to avoid disturbing the column.

Perform TLC or other analytical methods such as UV absorbance spectroscopy to identify compounds in each fraction. Combine fractions containing the same compound and evaporate solvent to obtain pure, isolated products.