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Chlorophyll Chromatography

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.

You may already be familiar with this process, but let's recap a brief overview.

Chromatography is a process in which different chemical compounds in a mixture are separated based on certain properties.

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.

Photosynthesis occurs in the chloroplast, which is found inside the plant cells.

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.

Sunlight is a mixture of electromagnetic waves with different wavelengths and frequencies; the visible part is only a tiny section of the electromagnetic spectrum. The wavelength of the electromagnetic waves in the visible spectrum determines the light's colour.

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

Chlorophylls are the most critical photosynthetic pigments, absorbing blue and red lights. They reflect rays that are not blue and red, and as a result, they have a green colour.

There are two chlorophyll pigments: chlorophyll a and chlorophyll b. Chlorophyll a has a bluish-green pigment, while chlorophyll b has a yellowish-green pigment.

Carotenoids are the accessory pigments of photosynthesis that help with light absorption but are not as essential as chlorophylls. These pigments mainly absorb purple light, which has more energy.

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.

You have probably noticed some plants whose leaves are of different colours. Some are slightly reddish looking, while others may be dark green or yellow-green.

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.

Chromatography is a Greek phrase that combines the terms "chromo" and "graph", which together mean "colour writing". This is because the first chromatography technique was used in the late 19th century to separate pigments in a mixture.

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.

It is a low-cost but effective analytical method that takes only a small amount of material.

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.

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.

If a mixture of compounds is separated using solvents, as mentioned above, then different spots will show up on the chromatography paper. However, a pure compound will show only a single spot - no matter the solvent used.

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:

Pigment/solventDistance from the origin (cm)
Solvent9.9
Chlorophyll b3.8
Chlorophyll a5.3
Xanthophylls7.6
Carotenes9.7

Rf value can be indicative of a substance's solubility in the solvent and/or size. Pigments with small Rf values are either less soluble in the solvent, large in size and/or have a greater affinity for the stationary phase (paper) than those with larger Rf values.

Chlorophyll Chromatography - Key takeaways

  • Chromatography is a process in which different chemical compounds in a mixture are separated based on certain properties.
  • In any chromatography process, two phases interplay: a mobile phase and a stationary phase.
  • Photosynthetic pigments found in chloroplasts can be classified into two main groups based on the colours of the light they absorb, chlorophylls and carotenoids.
  • The retention factor (Rf) is used in paper chromatography to compare and identify the separated chemical substances.

Frequently Asked Questions about Chlorophyll Chromatography

In chlorophyll chromatography, photosynthetic pigments such as chlorophyll, carotene, and xanthophyll can be separated using paper chromatography based on their solubility in the solvent and size.

Yes, chlorophyll pigments can be separated by paper chromatography based on their solubility and size.

In chlorophyll chromatography, a mixture of ethanol and acetone is typically used to dissolve the pigments. 

Some pigments will dissolve in one solvent, but not in another. So, often a mixture of solvents is used to obtain better separation of pigment bands.

By comparing the Rf values calculated with the standard Rf values, we can identify the pigments on the chromatography paper. Just bear in mind that the standard values must be based on the same solvents used in the experiment. 

Final Chlorophyll Chromatography Quiz

Question

How many phases are in interplay in a chromatography process?

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Answer

2

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Question

The two phases in chromatography are _______ and ________.

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Answer

Mobile phase

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Question

Which cells have the highest concentration of chloroplasts?

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Answer

Mesophyll cells

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Question

All photosynthetic pigments are green. 

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Answer

False

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Question

What are the two main classes of photosynthetic pigments?

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Answer

Chlorophylls and Carotenoids

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Question

Which pigments are in the chlorophyll class?

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Answer

Chlorophyll a

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Question

Which pigments are in the carotenoids class?

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Answer

Carotene

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Question

Which type of chromatography is used to separate photosynthetic pigments?

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Answer

Paper chromatography 

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Question

What is the Rf value?

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Answer

A compound's Rf value equals the distance travelled on paper by the compound divided by the distance travelled by the solvent.

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Question

Only one solvent is used as the mobile phase in chlorophyll chromatography.

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Answer

False

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Question

What are the two solvents most commonly used as the mobile phase in chlorophyll chromatography?

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Answer

Ethanol

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Question

Pigments with small Rf values are either less soluble in the solvent.

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Answer

True

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Question

Carotenoids mainly absorb purple light.

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Answer

True

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Question

Chlorophylls absorb _____ and _____ lights. 

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Answer

Red

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