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Have you ever wondered how you can tell if a reaction has gone to completion?
For some reactions it is obvious - there might be a clear change in colour, for example, or perhaps a change in pH. But for some reactions, it isn't as simple. The products and reactants may all be soluble and colourless, or perhaps your reaction is taking place at too small of a scale to observe any happenings. But with chromatography we can tell whether a reaction has finished or not.
Chromatography is a separation technique used to separate soluble mixtures. If we carry out chromatography on a reacting mixture, it will split apart into all the different components within it. We can then compare the results to a database, analyse and identify the individual components, and see if there are any reactants remaining in the mixture or if they have all reacted. This is just one example of a real-world application of chromatography.
While there are many sorts of chromatography, we will be looking specifically at thin-layer chromatography here.
Thin-layer chromatography is a separation technique used to separate and analyse components in a mixture.
All types of chromatography follow the same basic principles.
Not too sure about these terms? Check out Chromatography for a more detailed explanation.
Let's now look at how these basic principles apply to thin-layer chromatography.
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.
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.
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.
In chromatography, relative affinity describes how well a component is attracted to either the stationary or mobile phase. It 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. 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:
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:
Alumina works in much the same way. It also contains -OH groups on its outer surface that can form hydrogen bonds with suitable substances.
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.
Here's a typical setup for thin-layer chromatography:
Why do we have to draw the baseline in pencil? Here's the rationale behind some of these steps, which may seem arbitrary.
At the end of your experiment, the setup should look a little like this:
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.
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.
Here's an example, using the chromatogram produced earlier.
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:
We normally round Rf values to two decimal places, and so this would give us a value of 0.35.
The solute can't travel further than the solvent. This means that the total distance travelled by the solvent must always be equal to or greater than the distance travelled by each component, and so your Rf value must always be less than or equal to 1.
You should also note that Rf values don't have any units.
Chromatograms show us two things:
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:
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.
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.
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:
Thin-layer chromatography is a separation technique used to separate, analyse, and identify components within a soluble mixture.
To do thin-layer chromatography, carry out the following steps:
Thin-layer chromatography was discovered by Nikolai Izmailov.
Thin-layer chromatography is used to see if reactions have gone to completion, to identify substances, to determine purity, and in biochemical analysis.
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.
What is the stationary phase in TLC?
A thin metal or plastic plate covered in a layer of silica gel or alumina.
What is the mobile phase in TLC?
How do you view colourless components on TLC chromatograms? Give two methods.
Give three advantages of TLC over paper chromatography.
A mixture contains an alcohol and an alkene. Predict which molecule will travel further up the plate in TLC.
Substance A has an Rf value of 0.8. Substance B has an Rf value of 0.4. Predict which substance has a higher affinity to the mobile phase.
In TLC, substance A travels 12.3 cm up the plate. The total distance travelled by the solvent is 15.8 cm. Calculate substance A's Rf value.
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