CHEMISTRY NOW: Chlorophyll Chromatography (Grades 9-12)
Students will be able to design and carry out an investigation to separate the pigments from a leaf by paper chromatography. Students will identify a mixture by separating it into the different compounds. Students will compare pigments found in different leaves to see similarities and differences.
CHEMISTRY NOW: Chlorophyll Chromatography
Subject Area: Chemistry
Grade Level: High School Chemistry
Lesson Title: Chlorophyll Chromatography
National Science Education Standards:
Science as Inquiry: 9-12
Physical Science Standards:
Properties and Changes of Properties: 5-8
Structure of Atoms: 9-12
Structure and Properties of Matter: 9-12
Chemical Reactions: 9-12
Suggested Prior Knowledge: concepts of solutions, mixtures, separation of mixtures, solubility, photosynthesis
Purpose: To give students an understanding of the process of chromatography and to allow students to separate a mixture of photosynthetic pigments extracted from leaves.
chromatography- method used to separate a mixture of compounds based on differing solubility of the compounds in the solvent being used
solution – homogeneous mixture of two or more substances
solute– substance dissolved in a solution (the substance there is less of)
solvent – substance dissolving the solute in a solution (the substance there is more of)
absorbent– solid material used in chromatography that will attract and absorb the compounds being separated
eluent (solvent) – material used in chromatography which carries the compounds to be separated through the absorbent
photosynthesis – process by which plants convert energy from sunlight, water and carbon dioxide gas into glucose and carbon dioxide
chlorophyll – one of many pigments used by plants to absorb energy from sunlight in the process of photosynthesis
- Students will be able to design and carry out an investigation to separate the pigments from a leaf by paper chromatography.
- Students will identify a mixture by separating it into the different compounds.
- Students will compare pigments found in different leaves to see similarities and differences.
- safety goggles
- Column Chromatography Kit (commercially available from Flinn Scientific AP7392)
- spinach leaf (fresh) and other leaves
- UV light source (optional)
- other optional materials if Column Chromatography Kit is not used:
o chromatography solvent (commercially available: 90% petroleum ether and 10% acetone) [Note: this solvent can be reused – do not throw it away; save it!]
o chromatography paper
o wooden splint
o large test tube
o 50 ml graduated cylinder
o parafilm or aluminum foil
o stapler or tape
o penny / one-cent coin
1. Review with students the process of photosynthesis and the pigments that plants use to carry out this process. Two of these pigments are chlorophyll a and b (below). Review solutions and mixtures.
2. Discuss with students how we can design an experiment that allows us to visually separate these pigments from a leaf and try to identify each pigment. Begin with a leading question and follow-up:
• What pigments do leaves contain to carry out photosynthesis?
• What kind of experiment can we do to find out how many pigments are in a leaf?
3. Lab protocols should be followed incorporating safety equipment. Goggles must be worn at all times.
4. The basic lab procedure from the Basic Principles of Column Chromatography Kit from Flinn Scientific can be completed as a teacher demonstration or as a class experiment to introduce the column chromatography technique. This procedure uses column chromatography to separate pigments in spinach powder. The kit also includes information on how to analyze the relative polarity of the compounds separated.
5. Once students have familiarized themselves with this technique, guide them in designing an investigation using this technique to explore photosynthetic pigments in different leaves. Some leading questions include:
• Do leaves from various plants contain different pigments or do they all contain the same pigments?
• Do leaves of plants that thrive in different environments (sun vs. shade etc.) contain different pigments?
6. If column chromatography is not used, the following basic procedure for paper chromatography can also be used:
a. Obtain the leaf or leaves to be tested.
b. Obtain chromatography paper strips, pencil, ruler, penny, large test tube, wooden splint and scissors.
c. Cut the chromatography paper into a long thin strip that is slightly narrower than the mouth of the test tube and is long enough to extend from the bottom of the test tube out the mouth. A rectangular strip about 15 cm long and 2 cm wide is generally good.
d. Make a pencil line 1 cm in from one narrow end, as in the diagram below. This end will be the bottom of your strip.
e. Place a piece of the spinach or other leaf over this line and use the edge of the penny to rub across the leaf along the pencil line drawn on the strip so that the pencil line is now covered with pigments from the leaf. It is important that the chromatography strip contain a single, narrow, horizontal green line.
f. Stand the strip of chromatography paper in the empty test tube and staple it to the coffee stirrer so that it hangs freely from the stirrer into the test tube but does not touch the bottom. It should hang very close (0.5 cm or so) to the bottom when the stirrer rests across the opening of the test tube (see diagram below).
g. Remove the chromatography strip from the flask and put enough solvent in the flask to completely cover the bottom of the flask to a depth of less than 1.0 cm. When you hang the strip back into the flask, it should touch the solvent but the solvent should be below the pencil line on the strip.
h. Carefully place the chromatography strip in the flask so that the bottom of the strip is in the solvent yet the solvent level is below the pencil line.
i. Allow the solvent to move up the chromatography strip. This is caused by capillary action. As the solvent is drawn up the strip, it will carry the pigments in the sample at different rates depending on the characteristics of the individual compounds. When the solvent level gets close to the top of the strip (not to the staple and stirrer yet), remove the strip from the solvent to stop the run and make a light pencil mark at the solvent top. It may also help to use a pencil to mark the separated bands on the strip in case the colors fade as the paper dries.
j. Let the strip dry. You should be able to see the pigment spots for each pigment in the leaf separately (see diagram of spinach paper chromatography results below).
k. If you have access to an ultraviolet (UV or “black”) light, have students shine the light on their strips and record their observations. Some pigments will fluoresce under UV light. The fluorescent properties of a particular chlorophyll pigment are functions of the structure of the molecule and its immediate environment. It is a characteristic of chlorophyll that can help students compare and identify the pigments separated. Different pigments may not fluoresce or may produce specific colors as characteristic properties. Chlorophyll pigments emit red fluorescence.
7. Students can then work to design an investigation in which they can use this technique to explore the photosynthetic pigments in various leaves. Guide them to develop a sound question and an investigation that is valid and will help them to learn about these compounds and their properties. Green plants have six closely-related photosynthetic pigments (listed from least to most polar); Carotene (orange), Xanthophyll (yellow), Phaeophytin a (gray-brown), Phaeophytin b (yellow-brown), Chlorophyll a (blue-green) and Chlorophyll b (yellow-green). Chlorophyll a is the most common, present in all plants. Some possible questions to investigate include:
a. Do “shade-loving” plants have different pigments than “sun-loving” plants?
b. Do plants with different colored leaves contain different pigments?
c. Do all plants contain chlorophyll?
d. Do autumn leaves that are red or orange still contain chlorophyll?
8. Guide students as they develop their questions and procedures to be certain that they include all safety concerns and that they focus on a limited number of variables so their results are valid. They can use the spinach investigation from the Flinn Kit or from the basic procedure of paper chromatography as a control.
9. Have the students record the data and any calculations from this experiment. If they do paper chromatography they can calculate Rf values for each separated pigment:
Rf = the distance traveled by the pigment
the distance traveled by the solvent.
10. They should include analysis of the relative polarity of the compounds found in each leaf. This will allow them to draw some valid comparisons between the pigments from different sources.
11. Have students draw conclusions about their results. They can then present their results to the class using slideshow, movies or poster boards.
Student Worksheet for Chromatography Investigation
Experiment Title: ____________________Date: __________Name: ____________
Student Question or Hypothesis:
Analysis of Data: