Plant Pigment Chromatography
Student Laboratory Kit
Materials Included In Kit
Acetone, 50 mL
Blue-green algae extract, 1 g
Chromatography solvent, 80 mL
Spinach powder, 1 g
Capillary tubes, 100
TLC sheets, 10 cm x 20 cm, 2
Additional Materials Required
Beakers, 50-mL, 2
Beakers, 50-mL, 2*
Graduated cylinder, 10-mL*
Marker or wax pencil
Pipet, Beryl-type, or medicine dropper
Watch glasses, 2 or Parafilm®
- Slurry of each of the extract powders.
a. Pour each of the extract powders into separate, labeled 50-mL beakers.
b. Add 8 mL of acetone to each beaker.
c. Swirl for several minutes.
d. Cover to prevent evaporation of the acetone.
- Prepare TLC plates for each lab group.
- Cut the TLC sheets into plates approximately 6.5 cm x 3 cm.
- Be careful not to scrape any of the silica gel from the plates. Note: Some silica gel will chip from the edges of each plate—this is not a problem.
Acetone and the chromatography solvent are flammable and dangerous fire risks; toxic by ingestion and inhalation. This lab should be performed only in an operating chemical fume hood or well-ventilated area. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash hands thoroughly with soap and water before leaving the laboratory. Please consult current Safety Data Sheets for additional safety, handling and disposal information.
Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Acetone, chromatography solvent, spinach extract and blue-green algae extract may be disposed of according to Flinn Suggested Disposal Method #18a. TLC plates may be disposed of according to Flinn Suggested Disposal Method #26a.
- Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The pre-laboratory assignment may be completed before coming to lab, and the data compilation and calculations may be completed the day after the lab.
- Good technique is important to achieving clean separations in thin-layer chromatography. Common sources of student error include “overloading” the TLC plates by placing too much extract on the initial spot and band broadening that occurs because the initial spot is too large. Another common problem is “staining” that occurs when the pigment spots are submerged below the solvent level.
- Allowing enough time for the development of the TLC is critical. The plate must be left in the development beaker long enough for the solvent to be drawn up near the top of the plate. Do not stop the development until the solvent front nears the top of the plate. Underdevelopment will lead to incomplete separation. Typical time for a 6.5 cm TLC plate is 10 minutes.
- The chromatography solvent contains 80% petroleum ether and 20% acetone. Do not discard leftover chromatography solvent—the solvent may be recycled. Save it for use by another class or for another chromatography experiment. Do not leave the chromatography solvent uncovered for long periods of time—one component may evaporate faster than the other, changing the polarity of the mixture.
Many plant pigments fluoresce under ultraviolet light. Once students’ TLC plates have dried, shine a UV light source very close to the plate—you should see many or all of the pigments fluoresce! You may even find pigments that were not visible under room light. Note: A black-light bulb will not work.
- Have students prepare extracts from other plants. Simply soak leaves from various plants in acetone to extract pigments. They may need to tear and/or grind the leaves (with a mortar and pestle and a small amount of clean sand) to extract the pigments. Additional chemicals and materials are available from Flinn Scientific, Inc.
Correlation to Next Generation Science Standards (NGSS)†
Science & Engineering Practices
Asking questions and defining problems
Developing and using models
Planning and carrying out investigations
Analyzing and interpreting data
Using mathematics and computational thinking
Engaging in argument from evidence
Obtaining, evaluation, and communicating information
Disciplinary Core Ideas
MS-LS1.A: Structure and Function
HS-PS1.A: Structure and Properties of Matter
HS-PS1.B: Chemical Reactions
Cause and effect
Scale, proportion, and quantity
Systems and system models
Structure and function
Energy and matter
HS-PS1-1. Use the periodic table as a model to predict the relative properties of elements based on the patterns of electrons in the outermost energy level of atoms.
HS-PS1-2. Construct and revise an explanation for the outcome of a simple chemical reaction based on the outermost electron states of atoms, trends in the periodic table, and knowledge of the patterns of chemical properties.
HS-LS1-5. Use a model to illustrate how photosynthesis transforms light energy into stored chemical energy.
MS-LS1-2. Develop and use a model to describe the function of a cell as a whole and ways parts of cells contribute to the function.
MS-PS1-2. Analyze and interpret data on the properties of substances before and after the substances interact to determine if a chemical reaction has occurred.
MS-PS1-1. Develop models to describe the atomic composition of simple molecules and extended structures.
Answers to Prelab Questions
- Read the Background section and the Procedure—why is a pencil rather than a pen used to mark the “starting line” in step 3?
Pen ink may contain pigments that will dissolve in the solvent and interfere with the separation.
- The plant extracts in this experiment will be drawn up the capillary tube and the solvent will flow upward through the TLC plate, each by capillary action. What is capillary action and what causes it?
Capillary action refers to the spontaneous rise of a liquid in small tubes or fibers, or to the wetting of a solid (e.g., paper or fabric) in contact with a liquid. Capillary action is responsible for the rise of sap in plant fibers and the flow of blood through capillaries. Capillary action is due to attractive forces (adhesion) between the molecules of the liquid and the walls of the vessel.
- What is the solvent front and how is its migration on the TLC plate measured?
The solvent front is the leading edge of the solvent as it travels up the TLC plate. The distance the solvent front migrates is measured from the pencil line where the plant extract is spotted to the maximum height the solvent rose.
Answers to Questions
- Compare and contrast the pigments observed on the spinach TLC plate with the pigments on the blue-green algae TLC plate.
Both samples exhibited chlorophyll a. Both also contained a xanthophyll, although they appeared to be different pigments. The blue-green algae contained phycobilins. The spinach contained carotenoids and chlorophyll b.
- What factors are involved in the separation of the pigments?
The polarity of the pigments varies because of differences in the structures of the pigment molecules. The different polarities cause the pigments to adsorb differently on the TLC plates. The polarity of the solvent and the affinity for the adsorptive surface affect the separation of the pigments. The time and distance allowed for the separation to occur also affect the separation of the pigments.
- Refer to step 8 in the Procedure: Why was it necessary to keep the chromatography solvent level in the beakers below the sample extracts spotted on the TLC plates?
If the chromatography solvent is above the extract spots on the TLC plates, some of the extract will dissolve directly in the solvent and will not travel as a distinct band on the TLC plates.
- Which pigment directly captures light energy? What are the roles of the other pigments?
Chlorophyll a is the only pigment that is able to directly capture energy from light and convert it to chemical energy. The other pigments expand the range of wavelengths of light absorbed, but that energy must be transferred to chlorophyll a.
Bregman, A. A. Laboratory Investigations in Cell Biology, 2nd ed.; John Wiley & Sons: New York, 1987; pp 119–123.
Bold, H. C.; Wynne, M. J. Introduction to the Algae, 2nd ed.; Prentice-Hall: Englewood Cliffs, NJ, 1985, p 39.
Green, N. P. O.; Stout, G. W.; Taylor, D. J. Biological Science: Organisms, Energy, and Environment: 2nd ed.; Saper, R., Ed.; Cambridge University: Cambridge, MA, 1990; pp 255–257.
Russo, T.; Meszaros, M. W. Vial Organic; Flinn Scientific: Batavia, IL, 1996; pp 25–33.
Wilkins, M. B., Ed. Advanced Plant Physiology; Pitman: Marshfield, MA, 1984; pp 221–224.
||Plant Pigment Chromatography—Student Laboratory Kit
||Thin Layer Chromatography Sheet, 20 x 20 cm
||Acetone, Reagent, 500 mL
||Chromatography Solvent, 500 mL
||Beakers, Polymethylpentene (PMP), 50 mL
||Cylinder, Polymethylpentene, 10 mL
||Parafilm® M, 20" x 50', Roll