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LAB Chlorophyl Chromatography

This document provides instructions for a laboratory activity investigating photosynthetic pigments through paper chromatography. Students are asked to extract pigments from leaf samples using acetone and spot the solution onto filter paper. As the chromatography solvent travels up the paper, different pigments will move at different rates and separate into visible spots. Students will then calculate the Rf value of each spot and identify pigments based on color. Questions ask students to sketch their chromatograms, explain why chlorophylls appear green, and discuss factors that influence a plant's photosynthetic activity and appearance of color.

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16 views3 pages

LAB Chlorophyl Chromatography

This document provides instructions for a laboratory activity investigating photosynthetic pigments through paper chromatography. Students are asked to extract pigments from leaf samples using acetone and spot the solution onto filter paper. As the chromatography solvent travels up the paper, different pigments will move at different rates and separate into visible spots. Students will then calculate the Rf value of each spot and identify pigments based on color. Questions ask students to sketch their chromatograms, explain why chlorophylls appear green, and discuss factors that influence a plant's photosynthetic activity and appearance of color.

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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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A LEVEL BIOLOGY

LABORATORY ACVTIVITY: PHOTOSYNTHESIS /25


st
1 Term, AY 2023 - 2024
Date:_______________

Name: ______________________________________________________ Grade 12

Investigating Photosynthetic Pigments with Chromatograph


NAME: _______________________________________________

PRELAB QUESTIONS:

1) What are the requirements for photosynthesis to occur?


______________________________________________

2) Why do some trees appear green during the spring and summer, but then red / orange / yellow in the fall?

3) What are two factors that cause pigments to move at different rates during paper chromatography?

MATERIALS:

• Leaf sample
• Distilled water
• Pestle and mortar
• Filter paper
• Capillary tube
• Chromatography solvent
• Acetone
• Pencil
• Ruler

METHODOLOGY:

1. Make two filter- paper rectangles that are each approximately 10 cm by 3 cm.
2. Using a pencil, draw a base line 1.5 cm above the bottom of the filter paper being used.
a. Do not use a pen as the ink will separate into pigments within the experiment and obscure the
results
3. Cut a section of leaf and place it in a mortar
a. It is important to choose a healthy leaf that has been in direct sunlight so you can be sure it
contains many active photosystems
4. Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments
a. Acetone is an organic solvent and therefore fats, such as the lipid membrane, dissolve in it
b. Acetone and mechanical pressure are used to break down the cell, chloroplast and thylakoid
membranes to release the pigments
5. Extract some of the pigment using a capillary tube and spot it onto the center of the pencil line you have
drawn.
6. Dry the spot for few minutes and repeat step 4 two times to ensure good concentration of chlorophyll in
the center.
7. Suspend the paper in the chromatography solvent so that the level of the solvent is below the pencil line.
Do not disturb the set-up for approximately 15 minutes, or until the solvent is about 1 cm from the top of
the paper.
a. The mixture is dissolved in the solvent (called the mobile phase) and the dissolved mixture then
passes through a static material (called the stationary phase)
8. When the solvent is about 1 cm from the top of the paper, remove the paper and mark the farthest point
of the solvent’s progress (front line) with your pencil before this line evaporates.
a. The pigment should have separated out and there should be different spots on the paper at
different heights above the pencil line, these are the separate pigments
9. Allow the filter-paper strips to dry, and then make a sketch of each chromatographs. Some possible
colors and the pigments they represent are:
• faint yellow / orange - carotenes
• olive green - chlorophyll b
• yellow - xanthophyll
• red - anthocyanin
• blue green - chlorophyll a
10. Calculate the Rf value for each spot.
Rf value = distance travelled by component (pigment) ÷ distance travelled by the solvent
11. Be mindful to always measure to the center of each spot.

RESULTS AND OBSERVATIONS


QUESTIONS:

1) Sketch your two chromatograms in the image here. Label the colored lines with their distance
traveled from the origin, and the likely identity of that pigment.

2) Why are chlorophyll a & b green? (or, why do they appear green to us?

3) The spinach leaf looks green, but your chromatogram demonstrated that other pigments are in
the leaf as well. Why can you not readily see the other pigments in the leaf?

4) Would you expect a plant to grow well in only green light? Explain

5) In which colors of light would you expect a plant to obtain maximum photosynthetic activity and
why?

6) Why are plants generally green?

7) Complete the table below:

*END*

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