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Photosynthetic Pigments

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Biology

There are various methods by which you can investigate aspects of photosynthesis. These methods are used in research by biologists to shed light on the many different reactions that occur during photosynthesis.

One method of investigating photosynthesis involves chromatography, which analyses the range of photosynthetic pigments in leaves. Photosynthetic pigments are small molecules that are capable of absorbing energy from light.

Remember that photosynthesis occurs in the chloroplasts of a plant, and the chlorophyll in these chloroplasts gives the leaves of a plant their green colour. This is because chlorophyll, the most common pigment found in leaves, absorbs blue and red light and reflects green light.

Chloroplasts are an essential part of the light-dependent reaction during photosynthesis. Without chloroplasts and the chlorophyll contained within them, the plant wouldn't absorb light energy, and photoionization could not occur. If you're curious about how light energy is converted into chemical energy via photosynthesis, check out our articles on light-dependent and light-independent reactions.

What is chromatography?

Chromatography is a laboratory technique used by biologists and biochemists. It allows for the separation of a mixture, which is achieved by passing it in a solution or suspension in which the mixture's components can move at different rates. It is often used to separate a product from any unreacted solution.

There are two primary types of chromatography: thin-layer chromatography (sometimes shortened to TLC) and column chromatography.

The function of both methods are the same, but each uses different equipment to separate a mixture into its various components. You are most likely to use thin layer chromatography during your studies; however, at university-level or a professional laboratory, you will make use of both. The differences between the two are illustrated in Figure 1 below.

How do you investigate photosynthesis using chromatography?

For your A-level course, you will be asked to use chromatography to investigate the pigments isolated from the leaves of different plants. You might, for example, be asked to compare the leaves of shade-tolerant vs shade-intolerant plants or leaves that are different colours from the same plant. To do this, you will need to carry out the experiment as detailed below.

Equipment:

  • Leaf samples

  • Filter paper

  • Distilled water

  • Pestle and mortar

  • Ruler

  • Capillary tube

  • Chromatography solvent

  • Acetones

  • Pipette

  • Pencil

Method:

  1. Using the pencil, draw a straight line around 1 cm above the bottom of the filter paper. It is important to use a pencil for this step, not a pen, as the ink will get into your solution and mess up your results.

  2. Take a section of the leaf, and place it into the mortar. Add 20 drops of acetone, and grind up the leaves with the acetone using the pestle. This will release the pigments in the leaf.

  3. Using the capillary tube, extract the pigment and drop it onto the centre of the pencil line on the filter paper.

  4. Suspend the filter paper with the pigment in the solvent; however, make sure that the liquid level does not lie above the pencil line.

  5. Leave the paper until the solvent has risen close to the top. Make sure that the solvent does not soak through the paper.

  6. Remove the paper from the solvent, and draw a pencil line marking where the solvent has moved up. You should notice that pigment has separated into different compounds, which should all be placed at different heights above the first pencil line.

Photosynthetic pigments [+] Thin Layer Chromatography Example [+} Study Smarter

Figure 1. Example of Thin Layer Chromatography. Source: Theresa Knott, CC BY-SA 3.0, via Wikimedia Commons

Although this won't look exactly like your experiment, it will be set up similarly.

How do you calculate an Rf value?

Once you have collected your filter paper with the different pigments separated, you will calculate the Rf value of each spot. Rf stands for retention factor, and this formula is used to help identify the pigments present on your chromatography sample.

The equation for the Rf value is as follows:

When calculating the Rf value for each spot, always measure to the centre of each spot, not the edge!

When you have your set of Rf values, you can then compare them to the known Rf values (provided by a teacher) to identify which pigments are present in your leaf. If you need to find your own database, make sure that they are specifically for paper chromatography. Use the same solvent as you since these variables will affect the distance travelled by certain pigments!

Which photosynthetic pigments are in chloroplasts?

You are likely to find the following pigments in a leaf during chromatography:

  • Chlorophyll a and Chlorophyll b: These pigments are green in colour. Chlorophyll a, the more abundant of the two pigments, absorbs blue and red light and reflects green light. Chlorophyll b is the less abundant of the two; however it can absorb a wider wavelength of light energy than chlorophyll a. Chlorophyll b absorbs blue and red light and reflects green light, like chlorophyll a; however, the green light reflected is a different shade of green. At a molecular level, chlorophyll has a porphyrin ring, which helps absorb light energy more efficiently.

  • Carotenoids: These pigments reflect orange, yellow and red light waves. In the chloroplast, carotenoid pigments cluster next to chlorophyll molecules to efficiently hand off any absorbed photons.

  • Xanthophylls: like carotenoids, xanthophylls pass along light energy to chlorophyll a and act as antioxidants. Xanthophylls reflect yellow light waves.

  • Anthocyanin: a pigment that absorbs blue and green light. Anthocyanin molecules are stored in the vacuole of the plant cell.

Chlorophyll c is another important photosynthetic pigment; however, you will not find it in your chromatography experiment. This is because it is not found in plants but in microorganisms capable of performing photosynthesis!

Why are photosynthetic pigments important in photosynthesis?

Photosynthetic pigments are crucial to photosynthesis as they absorb photons (waves of light) at certain wavelengths. As you will see in the diagram below, different pigments can absorb and reflect different wavelengths of light, which allows a plant to get as much energy as possible from a single light source.

After absorbing photons, the electrons contained in the pigments become excited and increase their energy level. Accessory pigments such as carotenoids, anthocyanins, and xanthophylls can then pass on this energy to primary pigments like chlorophyll a, which can oxidize and donate an electron to the electron transport chain in the light-dependent reaction.

Photosynthetic Pigments - Key takeaways

  • We can investigate the different photosynthetic pigments present in leaves via chromatography, which involves isolating the pigments in a solution and then using filter paper to separate each pigment.

  • We can identify different pigments using an Rf value, which you can calculate from the distance each pigment has travelled on the filter paper. You can work out the Rf value using the following equation:

    Depending on the kind of leaf you experiment on, you will find a range of pigments that include Chlorophyll a and Chlorophyll b, Carotenoids, Xanthophylls, and Anthocyanin. Chlorophyll a is likely to be the most abundant pigment.

  • Photosynthetic pigments are crucial to photosynthesis as they absorb photons (waves of light) at certain wavelengths.

  • Different pigments can absorb and reflect different wavelengths of light, which allows a plant to get as much energy as possible from a single light source.

Photosynthetic Pigments

Photosynthetic (or other ones investigated) pigments will have different relative solubulities. This means that the pigments will move at a different rates in the media (e.g. paper). 

Chromatography is a lab technique that separates a mixture by passing it through a solution that allows the different mixture components to move at different rates.

Chlorophyll a, Chlorophyll b and carotenoids.

Photosynthetic pigments are found in chloroplasts or in photosynthetic bacteria. 

They are pigments that can absorb light from the sun for photosynthesis. 

Final Photosynthetic Pigments Quiz

Question

What is chromatography generally used for?

Show answer

Answer

Chromatography is generally used to separate a product from any unreacted solution. 

Show question

Question

Where in the plant cell are photosynthetic pigments found?

Show answer

Answer

In the chloroplasts.

Show question

Question

What are photosynthetic pigments?

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Answer

Small molecules in a plant cell that are capable of absorbing energy from light

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Question

Fill in the blanks: Chromatography allows for the _(a)_ of a mixture, which is achieved by passing it in a _(b)_ or solution in which the mixture’s components can __(c)___.

Show answer

Answer

a. separation, b. suspension, c. move at different rates.

Show question

Question

What are the two main types of chromatography?

Show answer

Answer

Thin layer chromatography, Column chromatography

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Question

What does Rf stand for?

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Answer

Retention factor.

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Question

Name three photosynthetic pigments

Show answer

Answer

Any three from: chlorophyll a, chlorophyll b, carotenoids, xanthophylls, and anthocyanin

Show question

Question

What kind of photosynthetic pigment absorbs blue and red light, and reflects green light?

Show answer

Answer

Chlorophyll

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Question

What kind of photosynthetic pigment reflects red, orange and yellow light?

Show answer

Answer

Carotenoids

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Question

What kind of photosynthetic pigment reflects yellow light? 

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Answer

Xanthophylls (Carotenoids is also correct however note that they also reflect red and orange light!)

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Question

What kind of photosynthetic pigment reflects red light and absorbs blue and green light?

Show answer

Answer

Anthocyanin (Carotenoids would also be correct, however note that they reflect yellow and orange light as well as red light) 

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Question

True or false: Chorophyll c is the most abundant pigment typically found in plant cells. 

Show answer

Answer

False: Chlorophyll c is only found in microorganisms that are capable of performing photosynthesis.


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Question

At a molecular level, Chlorophyll has a ______, which helps it to absorb light energy


Show answer

Answer

Porphyrin ring

Show question

Question

What kind of particles do photosynthetic pigments absorb?

Show answer

Answer

Photons

Show question

Question

What are three examples of an accessory pigment?

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Answer

Carotenoids, anthocyanins, and xanthophylls 

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