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Thin Layer Chromatography Practical

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Thin Layer Chromatography Practical

Imagine one day you come across an unknown liquid, and you want to know what it is. Luckily, you are a scientist who has access to lab facilities. You know that in the lab you have a database of known Rf values for different substances, a pencil, a thin layer of paper and some solvent. So, what can you do with all of this? All of these materials alongside the situation points to carrying out thin-layer chromatography, and that is what we will be exploring in this article.

  • We will be exploring what thin-layer chromatography is.
  • Then we'll look at the principle of thin-layer chromatography.
  • We'll see what a thin-layer chromatography diagram looks like.
  • Finally, we will explore the method of 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.

Non-volatile: a substance that does not vaporise easily.

Adsorbing: adhesion of a molecule to a surface.

Thin-layer Chromatography principle

The principles of thin-layer chromatography basically lay out what characteristics are required for it to work and what the technique depends on. For thin-layer chromatography in particular, we rely on components having various solubilities and adsorption between the mobile and stationary phase.

Thin-layer chromatography uses the relationship between a solid stationary phase and liquid mobile phase. Solid phases are also known as adsorbents, due to the physical properties that they have. Some common solid phases are silica gel and alumina, both of which are polar. Alumina is more polar than silica and comes in an acidic, neutral or basic composition. Silica is only available as an acid and it is cheap.

Thin-layer chromatography diagram

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.

Methods of thin-layer chromatography

We have now gone over what thin layer chromatography is, the principles of thin-layer chromatography and what a thin layer chromatography diagram look like. So, now let us look at exactly how we can perform a thin-layer chromatography practical.

1. First we need to wear gloves and carefully draw a line 1 cm above the plate using a pencil, then mark 5 equally distanced spots on that line.

Gloves prevent any contamination and a pencil is used as it will not dissolve in the solvent.

Glass capillaries, Wiki Commons, https://commons.wikimedia.org

2. Then use a capillary tube to put small drops of the different solutions that you are testing on the spots you previously marked on the plate and allow this to dry.

Small drops are required to prevent the different solutions from mixing, and to make the spot concentrated. Allow it to dry then use a capillary tube to put another small drop.

3. Add 10 cm3 of solvent in the TLC chamber. This container must be covered with a lid.

We have to ensure that the solvent level is not too high, or it might dissolve the different solutions on the plate.

4. Now place the plate in the container and makes sure the solvent is below the pencil line, then place the lid and make sure it is tight.

By placing a lid, we ensure that nothing escapes from the solvent.

5. Now, wait for the solvent to reach around 1 cm from the top of the plate. Then remove the plate and mark the level which the solvent has reached. This is known as the solvent front. Then allow the plate to dry in a fume cupboard.

It's important to ensure that it dries in a fume cupboard, since the solvent fumes can be toxic.

6. To further identify the different spots that do not have colour, place the plate under UV light and circle the spots with a pencil.

Now we can finally analyse the different components.

Analysing a chromatogram

After carrying out the practical, we need to figure out what each component is. This can be determined using Rf values. There is a large database that contains known Rf values for different components. We use the chromatogram to find the Rf value, then use the database to determine the unknown components. We use the distance travelled by the mobile phase to determine the Rf value. The longer the distance travelled, the less the unknown compound has adsorbed to the stationary phase and allows us to explore the relative solubility of the unknown compound.

We use this equation to find the Rf value:

Rf = Distance travelled by the component ÷ Distance travelled by the solvent front

First, we measure how far the component has travelled. We do this by measuring the distance from the pencil line to the middle of the spot of the component. Next, we measure how far the solvent has travelled from the pencil line, which is also known as the solvent front. We then plug these figures into the equation above. The value we get is the Rf value of the component. We can then compare it to known values and determine what the component is.

ComponentRf Value
β-carotene0.98
Chlorophyll a0.59
Chlorophyll b0.42

Let us say the solvent front has travelled 1.6 cm and the component has travelled 1.57 cm.

To calculate the Rf value, we just need to do the following:

1.57 ÷ 1.6 = 0.98

Using the known values from the table above, we can determine that the unknown component is β-carotene.

Uses of thin-layer chromatography

We can also explore the different uses of thin-layer chromatography:

  • To identify different components in a mixture.
  • To determine the extent of a reaction.
  • To determine the purity of a substance.

Thin-Layer Chromatography Practical - Key takeaways

  • Chromatography in general is a method in which we separate different components within a mixture.
  • For thin-layer chromatography, we rely on adsorption.
  • The principles of thin-layer chromatography are solubility and adsorption.
  • A thin-layer chromatogram has a plate, which is the stationary phase, a solvent, which is a mobile phase and the solvent front which is the distance which the solvent travels.
  • To analyse a thin-layer chromatogram, we determine each component's Rf value.
  • To work out the Rf value, we use the equation: Rf = Distance travelled by the component ÷ Distance travelled by the solvent front.
  • Thin-layer chromatography is used to identify different components in a mixture, determine the extent of a reaction and determine the purity of a substance.

Frequently Asked Questions about Thin Layer Chromatography Practical

The principles of thin-layer chromatography are solubility and adsorption.

We first put the different components on a plate and put the plate in a solvent. Once the solvent has travelled near the top of the plate, we take it out and let it dry. We then analyse it to determine the unknown components.

It is an important technique that helps determine different components within a mixture.

It is mostly used in synthetic chemistry to identify different components within a mixture and determine their purity.

Final Thin Layer Chromatography Practical Quiz

Question

What are some uses of thin-layer chromatography?




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Answer

To identify different components in a mixture

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Question

Why do we use capillary tubes?



 

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Answer

Small drops are required to prevent the different solutions from mixing

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Question

Why do we use gloves?




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Answer

Prevents any contamination

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Question

Why do we use a pencil?




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Answer

Used as it will not dissolve in the solvent

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Question

What does adsorbing mean?



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Answer

Adhesion of a molecule to a surface

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Question

What is a type of chromatography?




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Answer

Column chromatography

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Question

Calculate the Rf value if the solvent front has travelled 1.1 cm and the component has travelled 0.96 cm

 


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Answer

0.87

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Calculate the Rf value if the solvent front has travelled 1.72 cm and the component has travelled 1.53 cm

 

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Answer

0.89

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Calculate the Rf value if the solvent front has travelled 1.35 cm and the component has travelled 0.96 cm

 


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0.71

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Calculate the Rf value if the solvent front has travelled 1.55 cm and the component has travelled 1.25 cm

 

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Answer

0.81

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Calculate the Rf value if the solvent front has travelled 1 cm and the component has travelled 0.65 cm

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Answer

0.65

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Question

What do the different dots on a chromatogram signify?


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Answer

These dots represent the different, separate components of a mixture

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Question

Calculate the Rf value if the solvent front has travelled 1.8 cm and the component has travelled 1.3 cm

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Answer

0.72

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Question

Calculate the Rf value if the solvent front has travelled 1.2 cm and the component has travelled 0.87 cm


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Answer

0.72

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Question

Calculate the Rf value if the solvent front has travelled 0.9 cm and the component has travelled 0.45 cm


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Answer

0.5

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