Above Image: An array of leaves with varying pigments © nakornkhai, iStockphoto.com
Students will be introduced to the concept of pigment chromatography and will collaborate to perform an inquiry in which they will separate plant pigments using paper chromatography.
Inquiry Skill Development
- To identify the major pigments found in plant leaves and understand each pigment’s function
- To understand how mixtures can be separated using the process of chromatography
Prior Knowledge and Skills
- Understanding of the components of matter (elements, atoms and molecules) and the concept of mixtures
- Basic understanding of the process of photosynthesis and the role of chlorophyll in this process
- Familiarity with the concept of a “testable question” and a “hypothesis”
- Students develop appropriate testable questions
- Students conduct the chromatography activity carefully and use allotted time effectively
- Students groups complete all sections of Inquiry Summary
- Students participate actively during class discussions
- 15-20 minutes of small group discussion
- Background reading may be done outside of class time
- 10 minutes to set up materials and equipment
- 20 minutes to run chromatography
- 20 minutes for data analysis
- 15-20 minutes class time to discuss the results
Clear plastic cups - 2 per group
Isopropyl alcohol* - approximately 10 mL per group
Coffee filters, cut into ~2.5 cm strips* - 2 per group
Pencils, wooden dowels/skewers or coffee stir sticks - 2 per group
Coins (pennies or nickels) – 1 per group
Tape* - 2 pieces per group
Safety glasses – 1 pair per student
Fresh leaves from plants* (these can be from your Tomatosphere™ plants or other plants, such as spinach, red cabbage, beet greens, etc.) – at least 2 different types of leaves per group
Plant Pigments (Tomatosphere™ backgrounder) - 1 per student
BLM 1: Plant Pigment Chromatography Inquiry Summary – 1 per group
BLM 2: Plant Pigment Chromatography Instructions – 1 per group
*Indicates consumable materials
Tips for Success
- Organizing the working groups prior to the lesson is highly recommended to save time.
- Background reading and/or video viewing may be assigned prior to class in preparation for the inquiry in-class discussions.
- Demonstrate how to calculate the Rf value (Retardation or Retention Factor) for the pigments using the final chromatography strips
Introduction to the Inquiry
Have the students read the Plant Pigments (Tomatosphere™ backgrounder) as a pre-class reading assignment.
Organize students into working groups of 2 to 4. Distribute one copy of BLM1: Pigment Chromatography Inquiry Summary to each group.
Have each student group develop a testable question for an inquiry that compares the pigments from two leaf samples using paper chromatography (e.g., compare the types or quantities of pigments, etc.). Have each group of students also develop a hypothesis based on their inquiry question.
Optional Minds-On activity: Have students watch the video Creating Testable Questions or read the science processes section on Asking Testable Questions on Tomatosphere™ if they need practice with developing testable questions.
Note: To avoid mix-ups regarding the identity of the leaves, you may wish to hand out one leaf at a time and have the groups prepare and label the chromatography strip for one leaf before you hand out the second leaf.
Conduct the Inquiry: Plant pigment chromatography
Distribute the materials to each group. Each group will be working with TWO leaves - ONE Tomatosphere™ leaf from each of the TWO treatment groups. If you are not using the Tomatosphere™ plants, give each group two different types of leaves.
Provide each group a copy of BLM 2: Plant Pigment Chromatography Instructions or project the instructions on a screen using the technology available in your classroom.
Familiarize yourself with the chromatography process and tips for success as outlined below:
- Using a pencil, have students mark a line on the paper strip 2.0 cm from one end. This is the origin - it represents where the pigments start from at the beginning of the experiment (Figure 1). If pens or markers are used, the pigments in the ink will also dissolve and move along the paper, so students MUST use PENCIL. Also, have student use pencil to print the name of the sample at the other end of the paper strip. For Tomatosphere™ samples indicate which seed group the leaf is from (e.g. group A or B leaf) (Figure 1).
Note: There should only be one solid band of pigments present.
- Have students place one of the two leaves on top of the paper strip so that the leaf covers the pencil mark (Figure 2).
- Students should gently roll a coin back and forth across the leaf to transfer a “smudge” of pigments from the leaf onto the paper strip (Figure 2). Allow pigments to dry for one minute, reposition the leaf and repeat this pigment transfer process 3 to 4 more times until a dark strip of pigments is transferred (Figure 3).
- Instruct students to label their cups so that they know which leaf sample is being tested in each cup (Figure 4). Once students have prepared their cups and strips, fill each cup with isopropyl alcohol to a depth of approximately 1.0 cm (Figure 4). This is the solvent. The alcohol will dissolve the plant pigments allowing the pigment molecules to travel up the filter paper.
Note: Depending on the height of the cup, students may need to adjust how the paper strip is taped to the pencil or coffee stir stick. Instruct students to try this out before you start the experiment so they know where to tape the paper strip. Make sure students are careful not to jostle or move the paper once the experiment is running.
- Have students tape each prepared paper strip to a pencil or coffee stir stick (Figure 5).
- Instruct students to lower the paper strip into the cup carefully. The pigments from the leaf sample must be above the isopropyl alcohol. Remind students that they must be careful not to dip the pigment band in the alcohol; the pigments will dissolve away into the alcohol and they will lose their sample! The coffee stir stick should rest of the rim of the glass to hold the paper strip upright. The paper strip should not rest on the side of the cup (Figure 6).
Students should repeat steps a) to f) for the second leaf sample. To save time it is best to prepare all the samples and then run them at the same time in separate cups. The chromatography will take approximately 30 to 40 minutes to complete (the alcohol has travelled about ¾ of the way up the paper strip).
After the chromatography is complete, have students carefully remove the strips of filter paper and lay them out on BLM1: Pigment Chromatography Inquiry Summary to dry. The paper strips should be removed from the alcohol before the solvent reaches the top of the strips of the paper.
Note: The colours will fade as the paper dries, so this is much easier to do as soon as possible, and of course, the solvent front will disappear quite quickly as the alcohol evaporates.
Students should immediately mark the position and note the colour of each band (e.g., light green, dark green, etc.) on BLM1. They should also note the position of the pencil line and how high up the filter paper strip the solvent travelled (i.e., the highest point on the paper that is wet) – this is called the solvent front.
Have students complete their observations and conclusions on BLM1. The chromatography strips (chromatograms) should look something like the ones below.
At the end of the experiment, review the results and conclusions students recorded on BLM1. The students should see three or four distinct, differently coloured bands on their chromatograms:
- Xanthophylls will travel the least distance (closest to the origin) and are yellow in colour.
- Chlorophyll b is found just above the xanthophylls and is yellow-green in colour.
- Chlorophyll a travels the furthest distance (closest to the solvent front) and is blue-green in colour.
Have students discuss the results in relation to the testable questions and hypotheses they developed at the start of the activity.
Have students complete the extension activity on the bottom of BLM1. As a class, share and compare the results of the Rf calculations.
- The exact distance that each pigment band travelled in each group’s experiment may vary, but the Rf values for the same pigments should be very close if using the same solvent.
- Afterwards discuss the relationship between the distances that the pigment molecules travelled versus their relative sizes.
- Repeat the activity using different types of leaves, or leaves from different parts of the same plant.
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