Column Chromatography

Tablets like Nurofen and Paracetamol are formulations. This means that they are complex mixtures of different chemicals, carefully designed for a specific use. As well as the active ingredient, they may also contain flavourings to mask any bitter taste, slippery coatings to make them easier to swallow, and starch to act as a bulking and binding agent. But what if we want to isolate the active ingredient? One way of doing this is through column chromatography.

What is Column Chromatography?

Chromatography is a separation technique used to separate soluble mixtures. In column chromatography, a mixture is dissolved in a solvent and poured down a column packed with a solid material. Different components within the mixture flow out of the column at different rates depending on their adsorption to the solid material. In this way, we can isolate and separate the different components.

• This article is about column chromatography in chemistry.
• We'll start by exploring how the basic principles of chromatography apply to column chromatography before running through the method.
• We'll then look at the advantages and uses of column chromatography.

Principles of column chromatography

All types of chromatography follow the same basic principles.

1. We use a solvent, called the mobile phase, to dissolve a sample of a soluble mixture. The solvent carries the mixture through a solid called the stationary phase.
2. Some components of the mixture are carried through the solid by the solvent more quickly than others. We say that components that travel faster have a greater affinity to the mobile phase. This separates the mixture out into its component parts.

Let's now turn our attention to how this applies to column chromatography.

Stationary phase

In column chromatography, the stationary phase is made up of a fine silica powder packed into a long glass column. The column is open at one end and has a tap at the other. A layer of mineral wool is placed at the bottom of the column to stop the silica powder being washed away.

Mobile phase

In column chromatography, the mobile phase is any suitable solvent. It is also known as the eluent. You dissolve your sample mixture in the solvent and pour it down the column so that it travels through the stationary phase.

Retention times

In other types of chromatography such as Paper Chromatography and Thin-Layer Chromatography, we can calculate retention factors. These are measures of how far each component in the mixture travels through the stationary phase compared to the overall distance travelled by the solvent. But in column chromatography, we instead calculate retention times.

Relative affinity

A substance with a greater affinity to the mobile phase moves faster through the solid in the column than those with greater affinities to the stationary phase. Relative affinity is all to do with bonding between the substance and either the stationary or mobile phase.

Let's look at the structure of the stationary phase, silica powder. It is also known as silicon dioxide. Each particle of silica has a layer of -OH groups on the outside, as shown below:

Fig. 2 - Silica structure

These -OH groups mean silica can form hydrogen bonds with suitable substances. Hydrogen bonds are a type of intermolecular force. These bonds hold the substance in place and stop it being carried down the column as quickly by the solvent. We can therefore predict the following:

• Substances that can form hydrogen bonds will bond more strongly to the silica powder and so have a greater affinity to the stationary phase and a lower affinity to the mobile phase. They'll move more slowly down the column and give higher retention times. We say that they are adsorbed more.

• Substances that can't form hydrogen bonds bond less strongly to the silica powder. There are still other intermolecular forces between them and the silica powder, but these are weaker than hydrogen bonds, so the substance moves more quickly down the column. The substance has a greater affinity to the mobile phase and a lower affinity to the stationary plate. Such substances are more soluble in the solvent and give faster retention times.

For example, amino acids can form hydrogen bonds because they contain an N-H group. However, alkenes can't. Amino acids therefore have a stronger affinity to the stationary phase than alkenes and so have higher retention times. The alkene will travel through the column faster than the amino acid.

Method for Column Chromatography

How do we go about column chromatography? It involves the following steps.

1. Place a layer of mineral wool at the bottom of the column and then fill the column with silica powder; this is your stationary phase.
2. Saturate the silica powder with solvent; this is your mobile phase.
3. Pour your sample mixture into the top of the column.
4. Open the tap at the bottom of the column whilst continuously pouring solvent into the top of the column. Allow the solvent to carry the sample through the silica powder and collect the effluent that flows out of the bottom.

Here is what your column should look like after you've poured the mixture into the top.

Fig. 3 - Column chromatography setup

The mixture should separate out into different components that move through the column at different speeds. Make sure you swap the beaker for a new one as each component reaches the end of the column.

Fig. 4 - Column chromatography method

Analysing Column Chromatography

Look at the example above. The green sample mixture splits into two different components, one blue and one yellow. We, therefore, know that the mixture is made up of two different substances. We can also see that the yellow component moves faster through the column than the blue component. This means that the yellow component has a shorter retention time and a greater affinity to the mobile phase than the blue component. We say that the blue component is adsorbed more than the yellow component - it has a greater affinity to and bonds more strongly to the stationary phase.

What next?

Once you have collected the components of the sample in different beakers, you can analyse them further. For example, you may carry out mass spectrometry or thin-layer chromatography on one of the components to work out its identity. You could purify it by removing the solvent. One way of doing this is through distillation. Or you could simply carry out some basic test-tube reactions to get some clues about the component's structure and reactivity.

Advantages of Column Chromatography

Now we know how column chromatography works, we can consider some of its advantages.

• You can analyse large amounts of a sample. This is useful when separating mixtures.
• The stationary phase is generally low cost and easy to dispose of.
• It has a wide variety of potential applications, depending on the solvent used.

Uses of Column Chromatography

Column chromatography isn't just a cool way of splitting mixtures into stripes of different colours - after all, if we wanted to make rainbows, we could simply use a prism to split light! Column chromatography actually has a range of real-world applications. These include:

• Isolating active ingredients, which we mentioned at the start of the article.
• Separating mixtures such as a mix of amino acids.
• Isolating metabolites from biological samples.
• Removing impurities.