Chemistry
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Thin-Layer Chromatography

Thin-Layer Chromatography

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Have you ever wondered how you can tell if a reaction has gone to completion?

If you're doing a chemical reaction, it's not always easy to tell if it's done. Sometimes there's no colour change or you're working on such a small scale that you can't see what's happening. That's where chromatography comes in. Chromatography is a way to separate mixtures. If you use it on a reaction mixture, you can see all the different parts of it. By comparing those parts to a database, you can figure out which parts are leftover reactants and which have reacted. In this article, we're going to focus on one type of chromatography called thin-layer chromatography (TLC). We'll explain the basics of how it works, show you how to do it and read the results, give you an example, and talk about why people use it. So let's get started!

Principles of thin-layer chromatography

All types of chromatography work in a similar way. We start by dissolving a sample of a mixture in a solvent, called the mobile phase. Then we put the mixture on a stationary material, which doesn't move, called the stationary phase. As the solvent moves up the stationary phase, it carries the mixture with it. Some parts of the mixture move faster than others, depending on how well they like the mobile phase. This separates the mixture into its different parts and creates a picture called a chromatogram. By measuring how far each part moved, we can figure out its Rf value and identify what it is. But let's focus on thin-layer chromatography (TLC) now and see how these principles work specifically for it.

Stationary phase

The stationary phase is a static solid, liquid, or gel. The solvent carries the soluble mixture up the stationary phase in chromatography. 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. The mobile phase is the solvent used to carry the mixture analysed through the stationary 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

Once we have the chromatogram for TLC, we can use it to calculate Rf values. These values tell us the ratio between how far each part of the mixture moved and how far the solvent moved. To get the Rf value, we divide the distance that the part of the mixture moved by the distance the solvent moved.

Rf values are really useful because each part of the mixture has a specific value for a specific set of conditions. Things like temperature, the solvent used, and the stationary phase can all affect the Rf value. But if you do the experiment the same way each time, you'll get the same Rf value for the same part of the mixture. By comparing the Rf values to a database, we can figure out what each part of the mixture is.

Relative affinity

In chromatography, relative affinity refers to how strongly a component is attracted to either the stationary or mobile phase. This determines how quickly the component moves through the stationary phase.

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. This is because they are more soluble in the solvent.

But what determines a component's relative affinity? To understand this, we need to look at the structure of the stationary phase, such as silica gel or alumina.

Silica gel, for example, is a type of silicon dioxide with a lattice of silicon and oxygen atoms. On the outside of the structure is a layer of -OH groups. These groups can form hydrogen bonds with certain compounds or molecules. Hydrogen bonds are a type of intermolecular force that hold the compound in place and slow its movement up the plate.

Substances that can form hydrogen bonds will bond more strongly to the silica gel, so they 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. This is because they are adsorbed more to the stationary phase.

On the other hand, substances that cannot form hydrogen bonds bond less strongly to the silica gel. While there are still other intermolecular forces between them and the silica gel, these are weaker than hydrogen bonds. Therefore, the substances move more quickly up the plate. They have a greater affinity to the mobile phase and a lower affinity to the stationary plate, giving higher Rf values.

Alumina works similarly to silica gel. It also contains -OH groups on its outer surface that can form hydrogen bonds with certain substances.

Method for thin-layer chromatography

To perform thin-layer chromatography, you need to follow a series of steps. First, spread a thin layer of silica gel or alumina across a plastic or metal plate. Then, 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.

Next, stand the plate vertically in a beaker containing a small volume of your solvent, the mobile phase. Make sure that you only touch the plate at the very edges and that the solvent level is below the pencil line with the spot of mixture. Line the sides of the beaker with filter paper soaked in the solvent and cover the beaker with a lid. Wait until the solvent has traveled almost all the way to the top of the plate.

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. This chromatogram is now ready to be analyzed.

Certain steps in this process are critical to the success of the experiment. For example, drawing the baseline in pencil is important because pencil is insoluble and won't be carried up the plate with the mobile phase. If an ink were used instead, it could dissolve in the solvent and travel up the plate, producing misleading results.

It is also essential to handle the plate by its edges to avoid smearing fingerprints over its surface, which could again produce misleading results. Lining the beaker with filter paper soaked in the solvent and placing a lid on the beaker prevents the solvent from evaporating, which reduces streaking. Finally, marking the position of the solvent front with another pencil line is necessary because the solvent evaporates off after the experiment.

Viewing chromatograms

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

The end results of TLC

After performing thin-layer chromatography, you will be left with a vertical line of spots, each representing a different component found in the original solute. However, these spots may not be visible to the naked eye if the components are colorless.

To make the spots visible, you can shine UV light on the chromatogram. The part of the chromatogram that the solvent has traveled through will fluoresce under UV light, while the spots of the components that have traveled up the plate will not fluoresce and show up as dark patches. You can circle these spots with pencil and analyze the chromatogram as usual.

Another option is to spray the plate with a substance that will produce a colored compound. For example, if you know that your mixture contains amino acids, you can spray the finished and dried chromatogram with ninhydrin. This will react with the amino acids to form purple and brown-colored compounds, making the spots visible to the naked eye. These techniques make it possible to analyze the components of a solute, even if they are colorless. 

Calculating Rf values

Once you have a chromatogram with visible spots representing the different components of your solute, you can calculate an Rf value for each spot. The Rf value tells you how far each component has traveled in relation to the solvent.

To calculate the Rf value for a component, measure the distance traveled by the solvent from the bottom baseline to the top line you marked with pencil at the end of the experiment. Then, measure the distance traveled by the component from the bottom baseline up to the middle of the spot, either colored or circled in pencil. Divide the distance traveled by the component by the total distance traveled by the solvent, and that will give you the Rf value.

For example, if the red spot in the chromatogram traveled 5.8 cm and the solvent traveled 16.8 cm, the Rf value would be 0.35. Rf values are normally rounded to two decimal places and don't have any units. It is important to note that the total distance traveled by the solvent must always be equal to or greater than the distance traveled by each component, and so your Rf value must always be less than or equal to 1.

Analysing chromatograms

Chromatograms show us two things: The number of different components in our starting mixture. The identity of each component in our starting mixture.

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.

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:

Using a reference molecule in TLC

Thin-layer chromatography has many advantages over other types of chromatography, including paper chromatography. It runs faster, can analyze smaller amounts, and produces more reliable results with less spread out of the spots. The plates used in thin-layer chromatography are also sturdier than the paper used in paper chromatography.

Thin-layer chromatography has many uses in various fields. It can be used to test for purity, identify compounds such as essential oils or organic molecules, and perform biochemical analysis of blood plasma, serum, and urine. It is a versatile and useful technique for separating and analyzing mixtures.

Additionally, TLC has various uses such as testing for purity, identifying compounds like organic molecules and essential oils, and performing biochemical analysis of blood plasma, serum, and urine. It is an efficient and useful technique for separating and analysing mixtures, and has applications in various fields.

Thin-Layer Chromatography

What is thin-layer chromatography?

Thin-layer chromatography is a separation technique used to separate, analyse, and identify components within a soluble mixture.

How do you do thin-layer chromatography?

To do thin-layer chromatography, carry out the following steps: Take a plastic or metal plate and spread a thin layer of silica gel or alumina, your stationary phase, across it. 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. 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. 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. Once the solvent has almost reached the top of the plate, remove the plate from the beaker and mark the position of the solvent with another pencil line. Your chromatogram is now ready to be viewed and analysed.

Who discovered thin-layer chromatography? 

Thin-layer chromatography was discovered by Nikolai Izmailov.

What is thin-layer chromatography used for? 

Thin-layer chromatography is used to see if reactions have gone to completion, to identify substances, to determine purity, and in biochemical analysis.

What is the principle of thin-layer chromatography? 

In thin-layer chromatography, a solvent carries a mixture up a solid plate filled with silica gel or alumina. The solvent is known as the mobile phase and the plate is known as the stationary phase. The mixture separates out into its separate components based on their affinities to the mobile and stationary phases. Components with a greater affinity to the mobile phase will move further up the plate in a given time period than those with a greater affinity to the stationary phase.

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