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One of the first-ever practical experiments you probably carried out in chemistry at school is a simple example of paper chromatography: separating a coloured ink. You draw a pencil line across the bottom of a sheet of paper, place a dot of ink on the line, and place the paper upright in a beaker of solvent. The solvent travels up the paper, carrying the ink with it, and the ink separates out into all of its different coloured components.
This is a simple but effective way of separating mixtures. You'll learn exactly how it works here.
Paper chromatography is an analytical technique used to separate and analyse mixtures of soluble substances. It is a type of chromatography.
All types of chromatography follow the same basic principles.
If you haven't already done so, we'd recommend looking at Chromatography for a more detailed explanation of these ideas.
Let's now look at some of those ideas in terms of paper chromatography.
The stationary phase is a static solid, liquid, or gel. In chromatography, the solvent carries the soluble mixture through the stationary phase.
In paper chromatography, the stationary phase is - as the name suggests - paper. However, it's a bit more complicated than that. Paper is made of cellulose, a polymer of glucose. Cellulose fibres bind to water vapour in the air, alongside any water that was around when the paper was made. You can actually think of the stationary phase as being a complex matrix made of water and paper, not just the paper itself.
The mobile phase is the solvent used to carry the mixture analysed through the stationary phase.
The stationary phase - the paper - is placed in a solvent. This is our mobile phase. In paper chromatography, we typically use a nonpolar solvent. The solvent travels up the paper, carrying the different components within the mixture with it.
The components of the sample mixture travel up the stationary phase at different speeds. This means that in a given time period, the different components will all travel different distances. We'll look at why this is in just a second.
You'll see these components as spots on the chromatogram, which is the name for your paper once the experiment has finished. The ratio between the distance travelled by each component and the total distance travelled by the solvent gives us Rf values.
To calculate an Rf value, divide the distance travelled by the component - in other words, the distance from the starting pencil line to the coloured spot -by the distance travelled by the solvent.
Rf values are important because each component has a fixed Rf value under a specific set of conditions. If you repeated the experiment again, keeping the mobile phase, the stationary phase, and the temperature exactly the same, 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 in the mixture.
What determines how quickly a substance travels up the paper? This is all to do with relative affinity.
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 and travel a greater distance in a given time period.
Let's look more closely at the mobile and stationary phases in paper chromatography to work out why some components have a greater affinity to one or the other.
Remember how the stationary phase is a matrix of cellulose and water? This means that it is polar and can experience permanent dipole-dipole forces. The water molecules can also form hydrogen bonds with suitable substances. In contrast, the mobile phase in paper chromatography is typically nonpolar. It can only form weak van der Waals forces. From this we can deduce the following:
Check out Intermolecular Forces for more on permanent dipole-dipole forces, hydrogen bonds, and van der Waals forces.
That's enough of the technical details - how do you actually carry out paper chromatography?
What's the importance of, say, drawing the line in pencil? Here are some of the reasons behind particular steps in the method.
At the end of the experiment, the setup should look a little something like this:
The dot of ink has travelled up the paper and separated into several spots. Each spot represents a different component found in the original mixture. Each component moves up the paper at a different speed, depending on its relative affinity to the stationary phase and its relative affinity to the mobile phase.
We can now use these results to calculate Rf values for each spot.
Earlier in the article, we mentioned Rf values. These are values that show the ratio between the distance travelled by each component and the total distance travelled by the solvent.
Let's look at calculating Rf values for the chromatogram we showed above.
Let's calculate the Rf value for the green spot. The green spot has travelled 3.0 cm whilst the solvent front has travelled 9.8 cm. Divide 3.0 by 9.8 to get your answer:
We tend to round Rf values to two decimal places. this gives us an overall answer of 0.31
Remember that the distance travelled by a substance all depends on its relative affinities to each of the stages. A substance with a greater affinity to the stationary phase will travel more slowly up the paper and will travel less far in a given time period. This means that it will have a lower Rf value. In contrast, a substance with a greater affinity to the mobile phase will travel more quickly up the paper and will have a higher Rf value.
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 acts as a reference molecule. It 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 red 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.
Two-way paper chromatography uses two different solvents, one after the other, on the same sample. It is useful for separating out components with similar Rf values.
To carry out this technique, place a small spot of your mixture at one edge of the base pencil line. Place the paper in a beaker with your first solvent, removing it when the solvent front has almost reached the top of the paper. Mark the position of this first solvent front.
Your paper should look a little something like the diagram below.
You'll notice that two components produce one merged spot - they haven't clearly separated. This is because they have similar relative affinities to the stationary and mobile phases and so have travelled at similar speeds up the paper.
Now, rotate your paper by 90° so that the separated spots now lie along the bottom of the paper. Choose a different solvent and repeat the experiment again. It is very unlikely that the two substances that produced the merged spot will also have similar affinities to the stationary and mobile phases in this solvent. Therefore, they will travel at different speeds up the paper and separate out into clear, distinct spots.
Paper chromatography is a relatively simple technique. However, it does have its advantages.
Paper chromatography is more than just a way of making pretty coloured patterns. It has a variety of different uses, many of which it shares with other chromatography techniques. These include:
Paper chromatography is an analytical technique used to separate and analyse mixtures of soluble substances.
In paper chromatography, a sheet of paper known as the stationary phase is placed in a solvent, known as the mobile phase. A small spot of a soluble mixture is placed on the paper. The solvent carries the mixture up the paper. Different components of the mixture have different relative affinities to the stationary and mobile phases and so travel up the paper at different speeds. This separates the components out.
Paper chromatography is used for separating mixtures, obtaining pure compounds, and analysing drugs.
Different components within a mixture have different affinities to the stationary phase - the paper, and the mobile phase - the solvent. This means that they travel up the paper at different speeds. Some will travel much further than others in a given time period. This separates the components.
An example of paper chromatography is separating the different dyes within an ink.
What is the mobile phase in paper chromatography?
What is the stationary phase in paper chromatography?
What do you use to draw the base line in paper chromatography?
In paper chromatography, the starting solvent level should be _____.
Below the pencil line containing the spot of mixture.
In paper chromatography, components that travel faster up the paper have a greater affinity to the ______.
In paper chromatography, more soluble components have a _____ affinity to the mobile phase.
What are the units for Rf values?
What are the advantages of paper chromatography?
What are the uses of paper chromatography?
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