Paper Chromatography

Principles of paper 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 solid 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 some of those ideas in terms of paper chromatography.

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.

Mobile 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.

Retention factors

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.

Relative affinity

What determines how quickly a substance travels up the paper? This is all to do with 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 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:

• If any of the components within the sample mixture are polar, or contain chemical groups that can form hydrogen bonds, they will bond more strongly to the polar cellulose-water structure than to the nonpolar solvent. This means that they have a greater affinity to the stationary phase and will travel more slowly up the paper. These components give lower Rf values.
• However, nonpolar components will bond more strongly to the nonpolar solvent than to the polar paper. They are more soluble. They, therefore, have a greater affinity to the mobile phase and will travel more quickly up the paper. These components give higher Rf values.

Method for paper chromatography

That's enough of the technical details - how do you actually carry out paper chromatography?

1. Draw a pencil line along the bottom of a sheet of chromatography paper.
2. Place a spot of the mixture you want to analyse on the middle of the line.
3. Place the sheet of paper in a beaker filled with a shallow layer of your solvent of choice. Make sure the level of the solvent is below the pencil line.
4. Place a lid on the beaker and leave the solvent to travel up the paper, carrying the components of the mixture with it.
5. When the solvent level almost reaches the top of the paper, remove the paper from the beaker and mark the position of the solvent with another pencil mark. Your chromatogram is now ready to be analysed.

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

The end of a paper chromatography experiment. Anna Brewer, StudySmarter Originals

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.

Calculating Rf values

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.

1. Measure the distance between the base pencil line and one of the coloured spots on the chromatogram. This is the distance travelled by the component that produced that spot.
2. Measure the distance between the base pencil line and the pencil line you used to mark the solvent front. This is the distance travelled by the solvent.
3. Divide the distance travelled by the component by the distance travelled by the solvent. This gives you your Rf value.
4. Repeat for all of the coloured spots.

$\mathrm{Rf}\mathrm{value}=\frac{\mathrm{distance}\mathrm{travelled}\mathrm{by}\mathrm{solute}}{\mathrm{distance}\mathrm{travelled}\mathrm{by}\mathrm{solvent}}$

Calculating Rf values for a paper chromatogram. Note that the solvent has evaporated - this is why we drew a pencil line to marks its position. Anna Brewer, StudySmarter Originals

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:

$3.0÷9.8=0.306$

We tend to round Rf values to two decimal places. this gives us an overall answer of 0.31

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 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.

Identifying components in paper chromatography. Anna Brewer, StudySmarter Originals

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.

Advantages of paper chromatography

Paper chromatography is a relatively simple technique. However, it does have its advantages.

• It is cheap and easy to run, with a simpler setup than other types of chromatography.
• It only uses small amounts of the sample mixture.
• It can analyse organic and inorganic compounds.

However, compared to other chromatography techniques such as gas chromatography and thin-layer chromatography, paper chromatography is less accurate. This is one of its main disadvantages.

Uses of paper chromatography

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:

• Separating mixtures. For example, in the early 20th century, paper chromatography was widely used to separate plant extracts.
• Obtaining pure compounds and removing impurities.
• Analysing drugs.
• Testing wastewater.