Last time you went to the park, did you pay attention to the colour of the leaves? Different plants have slightly different coloured leaves. Some are reddish, while others are dark greenish. Have you ever wondered why that is? Leaves contain unique pigments that absorb light and harness the energy for photosynthesis. Different plants have different proportions of these pigments, giving them a distinct colour. In this article, we will learn about chlorophyll chromatography, a method used to separate the pigments found in plants.

What is the meaning of chlorophyll chromatography?

To understand the meaning of chlorophyll chromatography, it is essential first to grasp the concept of chromatography.

In this process, two main phases need to be in interplay, a mobile phase and a stationary phase. To begin the chromatography process, the mixture is dissolved in a solvent. This is the mobile phase since it can transport the chemical compounds dissolved in it through a second substance known as the stationary phase.

The different components of the mixture have other properties, such as size, charge, solubility, and pH, that make them travel at different speeds through the stationary phase. Hence, they are forced to separate from one another. The specific mobile and stationary phases dictate whether chemicals go faster or slower and how they are separated based on the component's properties.

Chlorophylls and carotenoids

Plants can harness the sun's energy and use it to fixate carbon dioxide (CO₂) into simple sugars. This is done through a process you may be familiar with, photosynthesis.

Mesophyll cells in the leaves often have the highest number of chloroplasts and hence are the prominent photosynthesising cells in the plant. Inside chloroplasts, there are photosynthetic pigment proteins whose job is to absorb light. These pigments are integral to the light-dependent stage of photosynthesis.

These light-absorbing pigments can be classified into two main groups based on the colours of the light they absorb, chlorophylls and carotenoids.

Carotenoids are made of three pigments; yellow xanthophyll, orange carotene, and grey pheophytin.

As mentioned earlier, each of these pigments absorbs a different wavelength of light. Plants in different environments have evolved to make different proportions of these pigments to maximise light absorption.

Chlorophyll chromatography method

Separation of chlorophyll and carotenoid pigments are done using paper chromatography. This chromatography technique is called 'paper chromatography' since the stationary phase in this technique is a sheet of paper.

In paper chromatography, the dissolved chemical compounds are separated based on their varied migration rates over sheets of paper. This allows for identifying dissolved chemicals based on how soluble they are in a specific solvent. Photosynthetic pigments such as chlorophyll, carotene, and xanthophyll can be separated using the paper chromatography method.

The retention factor (Rf) is used in paper chromatography to compare and identify the separated chemical substances. A compound's Rf value equals the distance travelled on paper by the compound divided by the distance travelled by the solvent.

$$Rf=\dfrac{\text{Distance travelled by compound}}{\text{Distance travelled by solvent}}$$

The \(Rf\) value tells us about the compound's solubility and size. A low Rf value implies that the compound is less soluble and has a greater size.

Chlorophyll chromatography solvent

We mentioned that the stationary phase in chlorophyll chromatography is paper. But what about the mobile phase? In other words, what chlorophyll chromatography solvents are used to help create this phase?

Some pigments will dissolve in one solvent but not in another. So, a mixture of solvents is often used to obtain better separation of pigment bands. In chlorophyll chromatography, ethanol (C₆H₂O) and acetone (C₃H₆O) are the solvents typically used to dissolve the pigments.

Chlorophyll chromatography procedure

In this section, we will examine how chlorophyll chromatography is carried out and its procedure.

Materials and equipment

1. Safety goggles

2. Pestle and mortar

3. Chromatography paper or coffee filter paper

4. A 100ml Beaker

5. Small capillary tube or a pipette

6. Pencil

7. Ruler

8. Sellotape

9. A handful of leaves (e.g., spinach leaves)

10. Scissors

11. Ethanol

Procedure

1. Finely chop the leaves.

2. Fill a mortar with chopped leaves to a depth of about 2 cm.

3. Add a pinch of sand and six drops of ethanol to the mortar. The sand will help break down the leaves, and ethanol will dissolve the pigments.

4. Grind the ingredients for at least three minutes with a pestle.

5. Draw a pencil line 3 cm from the bottom of a strip of chromatography or coffee filter paper.

6. Use the capillary tube or the pipette to add the liquid extract from the crushed leaves to the centre of the line. Keep the spot as small as possible.

7. Allow the first drop to dry before adding another. Repeat this process until you have added five additional drops of solution, allowing each to dry before applying the next. The goal is to create a highly concentrated small region on the paper.

8. Attach the paper to the pencil using sellotape and place it over the beaker, so the chromatography paper is vertical and barely clear of the beaker's base.

9. Add some ethanol to the beaker so that the ethanol reaches the paper but is still below the pencil line and the spot.

10. Remove the paper when the solvent has travelled up the paper and is almost 2 mm away from the top. Then immediately draw a line to mark how far the solvent has travelled and draw circles around each pigment mark.

11. Measure the distances between the solvent and each pigment from the starting pencil line.

12. Calculate the Rf value for each pigment.

An example of chlorophyll chromatography calculations

Let's try to calculate the Rf of pigments on chromatography paper. In the example below, there are four distinct pigment bands.

Here are the distances travelled by the solvent and the pigments: