1. A colourful separation technique

Most leaves around us are green. But the single colour we see is actually due to a mixture of multiple pigments, each with a unique shade. Paper chromatography is the science of separating the pigments and the art of creating a new canvas with their colours.

The canvas is a piece of absorbent paper, on which we place the mixture of pigments. The painter is the solvent, which seeps into the paper and carries the pigments at different speeds, separating them out to unveil a gradient of colours.

Paper chromatography is a technique to separate mixtures of soluble substances.

2. The two phases of paper chromatography

An absorbent paper dotted with the mixture prior to separation

While the set-up may look confusing, there are only two key components in paper chromatography.

The first is the absorbent paper. We call it the stationary phase as it does not move. It simply acts as a running track for the substances in a mixture to race up.

The second is the solvent that acts as the mobile phase. It rises through the paper by capillary action, dissolving and carrying the mixture along the way. While the solvent is usually water, it can be any liquid that can dissolve the mixture completely.

3. The principle behind paper chromatography

A chromatogram with separated spots

When the solvent moves up the paper to dissolve a mixture, substances race up at different speeds depending on how much they are attracted to the two phases.

Substances that are more soluble in the mobile phase travel further up. Conversely, those with a stronger attraction to the stationary phase are held back more, moving a shorter distance.

As every substance moves a unique distance, the mixture will be separated into multiple spots of pure substances. We call the colourful paper with a line of separated spots a chromatogram.

However, the solvent will always be a step ahead of the substances. We call the upper edge of the solvent the solvent front.

Chromatography separates substances by how soluble they are in the solvent and how much they are attracted to the stationary phase.

4. On the line, on your marks

To make important measurements using a chromatogram, we must have marked out the start line using a pencil. This is where we spot the mixture at the start.

We cannot use a pen because it will dissolve in the solvent and interfere with the results. On the other hand, the graphite of pencil is insoluble in most solvents, making it a very safe option!

Another tricky part is the position of the start line. It must be higher than the depth of the solvent so that the mixture does not dissolve directly without climbing up the paper.

5. How to calculate Rf value in chromatography?

We can identify the substances present by measuring the distance they travelled relative to the distance travelled by the solvent. We call this measurement the Rf value. The formula is:

Rf = distance travelled by a substance ÷ distance travelled by the solvent

We measure the distance a substance has travelled by measuring from the start line to the centre of its spot. For the distance the solvent has travelled, we measure from the start line to the solvent front.

Substances that have moved further will have a larger Rf value that is closer to 1. For example, the light yellow spot containing substance 4 has the largest Rf value of 7 ÷ 8 = 0.875 in water. We can compare this experimental value with the literature — the cheem word for database — to identify the compound.

6. Show yourself, colourless substances

Some substances in a mixture are colourless and do not show up on the chromatogram.

To see them, we can either use ultraviolet radiation or spray a reagent that reacts with the substances to form coloured products. An example is ninhydrin, which reacts with colourless amino acids to form blue coloration.