Biro Column Chromatography


When we synthesize a compound in chemistry, we’re often left with a mixture in our flask. This could include: 

  • The desired product  
  • Unreacted starting material 
  • Side products 
  • Byproducts 

Whether you’re in a research lab or industry, you need your product to be pure. One method of separating your desired compound from all the impurities is column chromatography.  

Standard setup for column chromatography:

The steps are as follows: 

  1. Load the column with stationary phase (often this is done by pouring in a mixture of the stationary and mobile phase). 
  1. Add your sample mixture and let it soak into the top of the stationary phase. 
  1. Add mobile phase and allow it to move down through the column. 
  1. As the sample moves down the column, different molecules move at different speeds. 
  1. If you have a good combination of solvent and stationary phase, you can get different components of your mixture to completely separate out into bands. 

Stationary phase

The stationary phase is what the molecules in your mixture stick to. It’s a solid material with lots of chemical functional groups on the surface. Depending on the material, these can be hydrophobic, or hydrophilic.  

Molecules will travel down the column if they are more attracted to the solvent. Molecules that interact strongly with the stationary phase will stick to the stationary phase. If they interact more strongly with the solvent, they’ll travel with the solvent down the column.   

Try and write down the different types of intermolecular interactions that you’ve learned about.


Silica (SiO2) is a common stationary phase. You can find silica in sand on the beach. But the silica in columns is a bit different. It has lots of holes in it, called pores. The surface of all those holes is covered in Si-OH groups. We call these silanol groups. The holes give the silica a really high surface area. This means lots of space for molecules to stick to! 

Experiment aim

To investigate the best silica stationary phase for separating colours in black ink.  


  1. Start by preparing your solution. Break a black felt-tip pen and remove the central ink sponge. Squeeze out a few drops of ink into a small beaker, or vial. You might need to add a bit of water to the sponge first. 
  1. Now prepare your column. We will use a ball-point pen casing as our column.  
  1. Pack the end of your column with cotton wool. Add water and check that the water is dripping through ~1 drop per 5-10 seconds. 
  1. Fill the column with silica beads.
  1. Clamp your column in a stand, or tape it to the side of a beaker. 
  1. When the water reaches the top of the sand, add a small amount of ink to the top of the beads. 
  1. Let the ink flow slightly into the beads (or use a pipette bulb to push it into the beads). 
  1. Add more mobile phase (water) and push the solvent through the column until you start collecting ink. 

Repeat the experiment with silica powder (stored in water)

Biro setup with silica bead stationary phase


  1. Which material is the best at separating the colours in black ink? Why? 
  1. Was it possible to remove all the dyes from the column? Why do you think this is? 
  1. In chemistry, we normally use organic solvents for column chromatography. What might be some problems with this? 
  1. What might be some challenges with large scale chromatography in industry? 

Link to downloads – access instructions for teachers, technicians and students, as well as a risk assessment form.