Have you ever wondered how medicines like Nurofen and Paracetamol are made? These tablets are made up of different chemicals, carefully designed to have a specific use. They contain an active ingredient, but also other things like flavorings, coatings to make them easy to swallow, and starch to bulk them up. But what if we want to isolate just the active ingredient? This is where column chromatography comes in.

Column chromatography is a way to separate different components from a mixture that is dissolved in a fluid. It is a type of chromatography that uses a solid material called the stationary phase. The mixture is poured down a long glass column packed with a fine silica powder or alumina. Different components in the mixture flow out of the column at different rates depending on their adsorption to the stationary phase. This way, we can isolate and separate the different components.

The stationary phase is a static solid, liquid, or gel that the solvent carries the mixture through. In column chromatography, the stationary phase is made up of a fine silica powder packed into a long glass column. The mobile phase is the solvent used to dissolve the sample in chromatography. In column chromatography, the mobile phase is any suitable solvent. You dissolve your sample mixture in the solvent and pour it down the column so that it travels through the stationary phase.

Column chromatography is not used to identify substances, but rather to isolate them. It is a lot harder to produce consistent retention times because they depend on lots of different factors. These include the length of the column, the size of the particles in the stationary phase and the gas flow in the room. Instead, we use other techniques like Mass Spectrometry and Thin-Layer Chromatography to identify substances. In summary, column chromatography is a separation technique used to isolate single components from a mixture dissolved in a fluid. It is a type of chromatography technique that uses a solid material called the stationary phase. The mixture is poured down a column packed with the stationary phase and different components flow out at different rates. Column chromatography is used to isolate substances and is not used to identify them.

Relative affinity

In chromatography, relative affinity describes how well a component bonds to either the stationary or mobile phase. It determines how quickly the component moves through the stationary phase.

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:

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Silica, the solid material used in column chromatography, can form hydrogen bonds with certain substances. Hydrogen bonds are a type of intermolecular force that hold the substance in place and stop it from being carried down the column as quickly by the solvent. This means that substances that can form hydrogen bonds will bond more strongly to the silica powder and have a greater affinity to the stationary phase. They move more slowly down the column and give higher retention times. On the other hand, substances that can't form hydrogen bonds bond less strongly to the silica powder and move more quickly down the column. They have a greater affinity to the mobile phase and a lower affinity to the stationary phase, giving faster retention times.

For example, amino acids can form hydrogen bonds because they contain an N-H group. This gives them a stronger affinity to the stationary phase than alkenes, which cannot form hydrogen bonds. As a result, amino acids have higher retention times and move more slowly down the column, while alkenes move more quickly.

Method for column chromatography

Column chromatography is a technique used to separate mixtures of compounds. 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.
5. 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: [Column chromatography setup. Anna Brewer, StudySmarter Originals]^[1]

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.

Analysing column chromatography

In the example above, we can see that the green sample mixture splits into two different components, a blue and a yellow component. This indicates that the mixture is made up of two different substances. We can also observe that the yellow component moves faster through the column than the blue component, indicating that the yellow component has a shorter retention time and a greater affinity to the mobile phase than the blue component. This means that the blue component is adsorbed more than the yellow component, as 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 is a versatile separation technique that has numerous real-world applications. It can be used to isolate active ingredients, separate mixtures such as amino acids, isolate metabolites from biological samples, and remove impurities. The technique involves using a column packed with silica powder as the stationary phase and a solvent as the mobile phase. The sample is poured into the top of the column and the solvent is continuously poured on top to separate the sample into its individual components. While basic, gravity-assisted chromatography is preferred for its low energy costs, a variant of this technique called flash column chromatography is used to speed up the process by forcing the mobile phase through the stationary phase under medium pressure. Overall, column chromatography is a cost-effective and scalable method for separating components in a mixture.

Column Chromatography

How do you prepare column chromatography?

To prepare column chromatography, place some mineral wool at the end of a glass column attached to a tap. Fill the column with a solid such as silica powder. Saturate the solid with your solvent, then pour the sample mixture on top of the solid. You are now ready to carry out the practical.

What is column chromatography and how does it work?

Column chromatography is a separation technique used to separate single components from a mixture dissolved in a fluid. To carry it out, you pour a solvent, known as the mobile phase, over a mixture placed in a glass column filled with a solid, known as the stationary phase. It works because different components in a mixture have different affinities to the stationary and mobile phases. This means that they travel down the column at different speeds.

Where is column chromatography used?

Column chromatography has a variety of applications, from purifying substances to separating proteins and isolating active ingredients from plant derivatives.

What is the principle of column chromatography? 

To carry out column chromatography, you pour a solvent, known as the mobile phase, over a mixture placed in a glass column filled with a solid, known as the stationary phase. It works because different components in a mixture have different affinities to the stationary and mobile phases. This means that they travel down the column at different speeds.

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