Paper chromatography is an easy and effective way to separate mixtures. It's a type of chromatography used to separate and analyze mixtures of soluble substances. You might have done this experiment in your chemistry class at school, where you draw a pencil line at 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 carries the ink up the paper, and the ink separates into its different colored components. In this article, we will explore the principles of chromatography, specifically paper chromatography, and how they work.
The stationary phase in paper chromatography is cellulose paper, which is a complex matrix made of water and paper. The mobile phase is a nonpolar solvent that carries the soluble mixture through the stationary phase. The components of the mixture travel up the stationary phase at different speeds, creating spots on the chromatogram. To identify the components, we use an Rf value, which is the ratio of the distance traveled by the component to the total distance traveled by the solvent.
The components travel at different speeds because of relative affinity, which describes how well a component is attracted to either the stationary or mobile phase. Components with a greater affinity to the mobile phase will move faster up the paper than those with a greater affinity to the stationary phase. This is because the stationary phase is polar and can experience permanent dipole-dipole forces, while the mobile phase is nonpolar and can only form weak van der Waals forces.
Polar components will bond more strongly to the polar cellulose-water structure than to the nonpolar solvent, so they have a greater affinity to the stationary phase and will travel more slowly up the paper, giving lower Rf values. Nonpolar components will bond more strongly to the nonpolar solvent than to the polar paper, so they have a greater affinity to the mobile phase and will travel more quickly up the paper, giving higher Rf values.
In conclusion, paper chromatography is a useful analytical technique to separate mixtures of soluble substances. Understanding the principles of chromatography and relative affinity helps to interpret the results and identify the components in the mixture.
To carry out paper chromatography, you'll need chromatography paper, a pencil, a beaker, a shallow layer of solvent, and the mixture you want to analyze. First, draw a pencil line along the bottom of the chromatography paper. Then, place a spot of the mixture you want to analyze in the middle of the line. Next, place the paper upright in the beaker with the solvent, making sure the level of the solvent is below the pencil line. The solvent will travel up the paper, carrying the components of the mixture with it. 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.
It's important to draw the line in pencil because pencil is insoluble, preventing it from being carried up the paper with the mobile phase. Using ink would dissolve in the solvent and travel up the paper, producing confusing results. The solvent level must be below the spot of your mixture to prevent the spot from fully dissolving in the solvent and being washed away. Handle the paper by its edges to avoid getting fingerprints on it, which could dirty the paper and give misleading results. A lid or filter paper soaked in the solvent can be used to keep the environment saturated with solvent and prevent evaporation.
Following these steps will give you a clear and accurate chromatogram, which can be analyzed to determine the components of the mixture.
At the end of the experiment, the setup should look a little something like this:
The end of a paper chromatography experiment.
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.
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. 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. Divide the distance travelled by the component by the distance travelled by the solvent. This gives you your Rf value. Repeat for all of the coloured spots.
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.
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.
The number of different components in our starting mixture. 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.
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.
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.
Paper chromatography is a versatile technique with various uses, including separating mixtures, obtaining pure compounds, removing impurities, analyzing drugs, and testing wastewater. The stationary phase is a sheet of paper, and the mobile phase is a solvent. To identify components, you can calculate their Rf values and compare them to a database. Two-way chromatography is a variation that uses two different solvents to separate out components with similar Rf values. Paper chromatography is a cheap, simple, and uses small sample sizes. With its wide range of applications, paper chromatography is an essential tool in many scientific fields.
What is paper chromatography?
Paper chromatography is an analytical technique used to separate and analyse mixtures of soluble substances.
How does paper chromatography work?
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.
What is paper chromatography used for?
Paper chromatography is used for separating mixtures, obtaining pure compounds, and analysing drugs.
What is the principle of paper chromatography?
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.
What is an example of paper chromatography?
An example of paper chromatography is separating the different dyes within an ink.
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