Thin-Layer Chromatography

What is thin-layer chromatography?

Chromatography in general is a method which we use to separate different components within a mixture. It is almost 120 years old. The way chromatography works is that it is dependent on the characteristics of different components and their affinity to different media. There are several types of chromatography, such as gas chromatography, partition chromatography, column chromatography, thin-layer chromatography and many others.

We will be focusing on thin-layer chromatography in this article. Thin-layer chromatography (which we also commonly refer to as TLC for short) is used to separate non-volatile mixtures, using a TLC plate that has an adsorbing material. TLC works on the principle that an unknown compound is distributed from the solid phase, known as the stationary phase, which is fixed on a thin plastic or glass plate. This is submerged within a liquid, the mobile phase, which moves over the thin plate. This mobile phase is the eluting solvent which moves up the TLC plate. We can measure this to analyse and determine an unknown compound. The mobile phase can be a polar or non-polar solvent, and this is chosen depending on the unknown compound that we are trying to identify.

Principles of thin-layer 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 up a static medium called the stationary phase.
2. Some components of the mixture are carried up 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 and produces a chromatogram.
3. We then use the distances travelled by the components to work out Rf values. These help us identify the component.

Let's now look at how these basic principles apply to thin-layer chromatography.

Stationary phase

In thin-layer chromatography, the stationary phase is - as the name suggests - a layer of silica gel or alumina on a thin plastic or metal plate. We'll look at why silica gel is used in just a second.

Mobile phase

In TLC, the plastic or metal plate is then placed vertically in the solvent, known as the mobile phase.

When it comes to the mobile phase, there are many different possibilities - you could use alcohols, alkenes, or even just distilled water! It all depends on how the solvent dissolves your mixture. But in TLC, we often add in an additional substance that fluoresces in UV light. This comes in handy when viewing the practical's final result, known as the chromatogram.

Retention factors

We can use the chromatogram produced in TLC to calculate Rf values. These are based on the ratio between the distance travelled by each component in the mixture and the total distance travelled by the solvent. To calculate these, you divide the distance travelled by the component by the solvent's distance travelled.

Rf values are important because each component has a specific Rf value under a specific set of conditions. Variable affecting this include temperature, the mobile phase, and the stationary phase. But if you replicated the experiment exactly, you would get the same Rf value for the same component. We can then compare Rf values to ones in a database to identify the components present in the mixture.

Relative affinity

Components with a greater affinity to the mobile phase will move faster up the plate than those with a greater affinity to the stationary phase. They are more soluble in the solvent.

For example, a component with a greater affinity to the mobile phase might travel three-quarters of the way up the plate in a certain time, whereas a component with a greater affinity to the stationary phase might only travel one-third of the way up the plate. But what determines their relative affinity?

To understand this, we need to look at the structure of silica gel. It is a type of silicon dioxide. You can see that it consists of a lattice of silicon and oxygen atoms, but on the outside of the structure is a layer of -OH groups:

Fig. 1 - Silica gel. Anna Brewer, StudySmarter Originals

Because the surface of the silica gel contains -OH groups, it can form hydrogen bonds with suitable compounds or molecules. Hydrogen bonds are a type of intermolecular force (see Intermolecular Forces). These bonds hold the compound in place, stopping it moving as quickly up the plate. We can therefore infer the following:

• Substances that can form hydrogen bonds will bond more strongly to the silica gel, so have a greater affinity to the stationary phase and a lower affinity to the mobile phase. They'll move more slowly up the plate and give lower Rf values. We say that they are adsorbed more.
• Substances that can't form hydrogen bonds, bond less strongly to the silica gel. There are still other intermolecular forces between them and the silica gel, but these are weaker than hydrogen bonds, and so the substance moves more quickly up the plate. The substance has a greater affinity to the mobile phase and a lower affinity to the stationary plate. Such substances give higher Rf values.

Alumina works in much the same way. It also contains -OH groups on its outer surface that can form hydrogen bonds with suitable substances.

Thin-layer chromatography diagram

Fig. 2: Thin-Layer Cromatography. Taken from: Wikimedia Commons.

So, what exactly does a thin-layer chromatogram look like? If we look at the image on the right, we can see what is known as a chromatography tank. Inside the tank we can see the solvent, which is the mobile phase, the TLC plate which is the stationary phase and finally the solvent front, which shows how far the mobile phase has travelled.

Now, you may be thinking, what are all the coloured dots? These dots represent the different, separate components of a mixture. As the solvent front travels upwards, the mixture is separated and this is why they are all at different points on the plate. When two dots are on the same level, it shows that those two components from a mixture are identical.

Method for thin-layer chromatography

Now we know how TLC works, we can look at how you actually go about it. We'll start by outlining the steps before explaining them in more detail.

1. Take a plastic or metal plate and spread a thin layer of silica gel or alumina, i.e., your stationary phase, across it.
2. Draw a thin pencil line across the bottom of the stationary phase and place a spot of your soluble mixture on the centre of the line.
3. Stand the plate vertically in a beaker containing a small volume of your solvent, the mobile phase. Make sure you only touch the plate at the very edges, and that the solvent level is below the pencil line with the spot of mixture.
4. Line the sides of the beaker with filter paper soaked in the solvent and cover the beaker with a lid. Now leave the setup until the solvent has travelled almost all the way to the top of the plate.
5. Once the solvent has almost reached the top of the plate, remove the plate from the beaker and mark the position of the solvent front with another pencil line. Your chromatogram is now ready to be viewed and analysed.

Here's a typical setup for thin-layer chromatography:

Analysing chromatograms

At the end of your experiment, the setup should look a little like this:

Fig. 4. The end results of TLC.

You can see that the original solute has split into a vertical line of spots, each directly above the starting spot of your solute. Each spot is a different distance from the pencil baseline. Each spot represents a different component found in the solute.

However, your chromatogram might look a little different. Right at the start of the article, we talked about how chromatography can be used to analyse colourless components. But how can we see them if they are, well - colourless? This is where the substance that fluoresces under UV light comes in. If we take our chromatogram and shine it under UV light, the part of the chromatogram that the solvent has travelled through will glow. However, the spots of the components that have travelled up the plate won't glow, and so will show up as dark patches. You can circle these with pencil and move on to analysing the chromatogram like usual.

Another alternative is to spray the plate with something that will produce a coloured compound. If, for example, you know that your mixture contains amino acids, you can spray the finished and dried chromatogram with ninhydrin. This reacts with amino acids to form purple and brown-coloured compounds.

Calculating Rf values

Now that we have a chromatogram and can see the spots of the components on the plate, we can calculate an Rf value for each spot. Remember that each spot represents a different component found in your starting mixture.

1. Measure the distance travelled by the solvent, from the bottom baseline right up to the top line you marked with pencil at the end of the experiment.
2. Measure the distance travelled by each component from the solute, from the bottom baseline up to the middle of the spot, either coloured or circled in pencil.
3. Divide the distance travelled by each component by the total distance travelled by the solvent. This is your Rf value.

\[\text{Rf value} = \frac{\text{distance travelled by solute}}{\text{distance travelled by solvent}}\]

Here's an example, using the chromatogram produced earlier.

Fig. 7. Calculating Rf values.

Take the red spot in the above example. Let's calculate its Rf value.

We need to divide its distance travelled by the distance travelled by the solvent. These values are 5.8 cm and 16.8 cm respectively:

\[\frac{5.8}{16.8} = 0.345\]

We normally round Rf values to two decimal places, and so this would give us a value of 0.35.

Analysing chromatograms

Chromatograms show us two things:

1. The number of different components in our starting mixture.
2. The identity of each component in our starting mixture.

Number of different components

Remember, each spot represents a different component found in the original solute mixture. In our example above, we have three different spots on our chromatogram. We, therefore, know that we have three different substances present.

Identity of each component

There are two ways of identifying substances in a chromatogram. Firstly, when setting up the experiment, you could also place a small dot of a known substance, such as a particular amino acid or organic molecule, on the pencil line to the side of your solute dot. This known substance will also be carried up the plate by the solvent, producing a visible spot. If any of the spots from your mixture match the known substance's spot, you know that substance is present in your mixture.

Sound a little confusing? Here's what it looks like in practice:

Fig. 8. Using a reference molecule in TLC.

The green spot on the left is from a known substance. One of the spots produced by our mixture matches it exactly. We can therefore deduce that the mixture contains this particular substance.

But there is another way of identifying components. We also mentioned earlier that, provided you keep the conditions the same, a particular component will always produce the same Rf value. Let's say that a particular component has an Rf value of 0.4. If we look in a database, we should be able to find a substance that also produces an Rf value of 0.4 under the same conditions - the same mobile phase, stationary phase, and temperature. These two substances are one and the same.

Advantages of thin-layer chromatography

When you first hear the word chromatography, you might think of experiments done at school using paper chromatography. These are relatively quick, cheap, and easy to carry out. But thin-layer chromatography does have some advantages over paper chromatography.

• It runs faster and can analyse smaller amounts.
• It produces more reliable results as the spots spread out less.
• The plates are generally sturdier than the paper used in paper chromatography.

Uses of thin-layer chromatography

Finally, let's look at some of the uses of thin-layer chromatography. At the start of this article, we looked at how we can use this technique to see if a reaction has gone to completion. But there are many other applications, including:

• Testing for purity. Thin-layer chromatography separates mixtures out into their different components and is therefore an easy way to see if a substance is pure, or if it is a mixture of different components.
• Identification of compounds, for example of essential oils or organic molecules.
• Biochemical analysis of blood plasma, serum, and urine.