Discover Chlorophyll Variety in Different Plants Using Paper Chromatography

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Areas of Science Chemistry
Time Required Short (2-5 days)
Prerequisites None
Cost Low ($20 - $50)
Safety No issues
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Do you like to analyze stuff and finding out what it is made of? An analytical chemistry method, called chromatography, allows you to separate mixtures of compounds and to identify each individual compound within the mixture. Chromatography is used by many scientists, for example food scientists, forensic scientists, or organic scientists to analyze all kinds of mixtures such as food, blood, or medicine. In this project, you will be using paper chromatography to analyze the pigments from different plant leaves. What do you think you will find?


The objective of this project is to use paper chromatography to analyze the leaf pigments found in three different types of plants.

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Author: Amber Hess
Editor: Andrew Olson, Science Buddies
Edited by Svenja Lohner, PhD, Science Buddies

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MLA Style

Science Buddies Staff. "Discover Chlorophyll Variety in Different Plants Using Paper Chromatography." Science Buddies, 20 Nov. 2020, Accessed 21 Jan. 2021.

APA Style

Science Buddies Staff. (2020, November 20). Discover Chlorophyll Variety in Different Plants Using Paper Chromatography. Retrieved from

Last edit date: 2020-11-20


samples of green leaves of plants for discovering chlorophyll


Plants need to absorb light in order to create their own food in form of glucose. They do this with the help of a special pigment mixture located in their leaves, which include chlorophyll a, chlorophyll b, and carotenoids. Chlorophyll absorbs photons, which excite electrons in its central magnesium (Mg) atom. These electrons are channeled away from the chlorophyll to be used as energy to create glucose. Figure 1 shows the chemical structure for chlorophyll. Chlorophyll a has one carbon and three hydrogen atoms in place of the "R" group, and chlorophyll b has a carbon, a hydrogen, and an oxygen atom. The red highlighted part of the image is called the porphyrin ring. Each "point" in the diagram (i.e. each tip of the pentagons, hexagons, and jagged edges) represents a carbon atom. Hydrogen atoms are attached to most of the carbon atoms, but they are not shown in this structure.

Diagram of the molecular structure of chlorophyll

Chlorophyll comes in two forms depending on their chemical structure, but both forms contain a porphyrin ring. A porphyrin ring has a magnesium atom at its center and is surrounded by nitrogen atoms.

Figure 1. The molcular structure of chlorophyll includes a central magnesium atom and a porphyrin ring (highlighted in red).

Due to its specific molecular structure, chlorophyll is able to absorb red and blue light from the sun, but it does not absorb green light, it reflects it. This is why tree leaves are normally green. In the fall, the tree shuts down chlorophyll production to save energy, and recycles it for later use. Thus the green-reflecting chlorophyll disappears, and we are left with the orange and red carotenoids (pigments that capture light energy and transfer it to chlorophyll), and so the leaves turn red and orange.


In this project, you will be using paper chromatography to investigate chlorophyll and other pigments in plant leaves. Paper chromatography is a technique, that is used to separate the various components in a complex mixture or solution. In each chromatography apparatus there is generally a mobile phase, which is a fluid that runs along the stationary phase, and a stationary phase, that stays stationary while the mobile phase moves through. You will be extracting chlorophyll and other dye pigments from plant leaves. Then you will make a small spot of this extract onto a strip of chromatography paper. The bottom of this strip will be placed in a beaker of acetone, and the acetone will soak up into the paper. The acetone (solvent) is the mobile phase of the chromatography system, whereas the paper is the stationary phase. These two phases are the basic principles of chromatography. Chromatography works by something called capillary action. The attraction of the acetone to the paper (adhesion force) is larger than the attraction of the acetone to itself (cohesion force), hence the acetone moves up the paper. The chlorophyll and other plant pigments will also be attracted to the paper, to themselves, and to the acetone differently, and thus a different component will move a different distance depending upon the strength of attraction to each of these objects, as shown in Figure 2. As an analogy, let's pretend you are at a family reunion. You enjoy giving people hugs and talking with your relatives, but your cousin does not. As you make your way to the door to leave, you give a hug to every one of your relatives, and your cousin just says "bye." So, your cousin will make it to the door more quickly than you will. You are more attracted to your relatives, just as some chemical samples may be more attracted to the paper than the solvent, and thus will not move up the solid phase as quickly. Your cousin is more attracted to the idea of leaving, which is like the solvent (the mobile phase).

Diagram of a homemade paper chromatography testing box




Diagram of solvent and marker spot travelling up a paper chromatography strip

The distance from the top of the blue pigment to the origin line is divided by the distance of the solvent front to the origin line to solve for an Rf value.




Equation 1:

In our example, this would be:




Figure 4 below shows a water molecule bonding with another water molecule. The negatively charged oxygen atom (red) is attracted to the positively charged hydrogen atom (white) on the other molecule.

Diagram of water molecules bonding

Molecules of water are made of a single oxygen atom and two hydrogen atoms. Oxygen atoms have a negative charge and hydrogen atoms have a positive charge. Hydrogen bonds can form between the hydrogen and oxygen atoms of different water molecules.

Figure 4. Hydrogen bond formation between two adjacent water molecules.


Diagram of water molecules bonding to a fatty acid molecule

The head of a fatty acid molecule can create a hydrogen bond with water molecules. Hydrogen can bond to carbon atoms in the fatty acid and subsequent oxygen atoms in water can bond to the hydrogen attached to the carbon.

Figure 5. Hydrogen bond formation between a fatty acid molecule and a water molecule.

Chromatography Applications


In this science project, you will be extracting color pigments, including chlorophyll, from different plants, and analyze the extracts using paper chromatography. Which types of chlorophyll and other pigments do you think you will find?

Terms and Concepts

  • Carotenoid
  • Chlorophyll
  • Paper chromatography
  • Mobile phase
  • Stationary phase
  • Capillary action
  • Adhesion force
  • Cohesion force
  • Rf value
  • Polarity
  • Hydrophilic
  • Hydrophobic


  • Why do trees leaves turn different colors in the fall?
  • What are the different pigments used for in the plant?
  • What is the process of photosynthesis?
  • What wavelengths of light do plants use?
  • What makes a molecule polar or nonpolar? How does its polarity affect its interactions with other molecules?
  • How do the polarities of the solid phase and liquid phase affect how far chemicals travel on the paper strip/plate?


Here are some basic chemistry and chromatography resources:
  • Andrew Radar Studios staff. (n.d.) Chem4KIDS. Retrieved March 18, 2013.
  • Carpi, Anthony, Ph.D. (2003). Chemical Bonding. Visionlearning. Vol CHE-1 (7). Retrieved March 18, 2013.
  • Clark, Jim. (2007). Paper Chromatography. Retrieved March 18, 2013.
These resources will give you more information about chromatography and teach you about the types of chromatography used in research labs today: You can start learning more about plants and pigments from these resources:

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Materials and Equipment Product Kit Available


    • 100 mL beaker
    • Mini binder clips (2)
    • Wooden splints
    • Pipette

You will also need to gather these items:

  • Various plant leaves:
    • Spinach leaves
    • Iceberg lettuce leaves
    • Marigold leaves
  • Mortar and pestle
  • Test tubes (9)
  • Acetone (nail-polish remover)
  • Water
  • Ruler
  • Pencils
  • Lab notebook


Recommended Project Supplies

Project Kit: $34.95
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Experimental Procedure

Note: To make sure you can compare your results, as many of your materials as possible should remain constant. This means that the temperature, brand of nail-polish remover, size of paper strips, where the plant extract is placed onto the stationary phase, etc., should remain the same throughout the experiment.
  1. Grind up roughly equal samples of each of the different plant leaves and distribute them into test tubes. There should be at least three labeled test tubes for each type of plant (if using the Iceberg lettuce, "Iceberg 1," "Iceberg 2," and "Iceberg 3" are good names for the tubes).
  2. Add enough acetone (nail-polish remover) to suspend the ground-up leaves.
  3. Let the acetone/leaf mixture sit for 24 hours.
  5. Use a pencil to lightly label which leaf extract will be spotted on each paper strip. Label three chromatography strips for each type of plant leave. Tip: do not use a pen for writing on the strips: the ink will run when the solvent passes through the strips.
  6. Draw a pencil line 1 cm from the edge of each strip of paper, as shown in Figure 6 below.
    1. This will be the origin line.
    2. You will spot the plant extract for each strip right on this line, as shown in Figure 6.
  7. Take a pipette and fill it with one of the plant leaf extracts.
  8. Spot the sample in the middle of the origin (see Figure 6, below). You might want to practice a few times in order to get a nice round spot.

    An origin line is drawn on a paper strip used for chromatography
    Figure 6. Each chromatography strip will have an origin line. The plant extract to be tested will be spotted in the middle of the origin line.

  9. Repeat step 8 with the other plant leaf extracts you want to test. Use a new paper strip for each one.
  10. Fill the 100 mL beaker to a depth of 1 cm with the acetone (nail-polish remover).
  12. If necessary, add more of the acetone. The goal is to have the end of the chromatography strip just touching the surface of the solvent solution, as shown in Figure 7 below.
Two paper chromatography strips slowly absorb alcohol from a beaker
Figure 7. Your setup should look similar to this example. The end of the chromatography strip should just touch the acetone. Note: This picture does not show chromatography strips with plant extract. The colors on your paper strips should look different.
Two paper chromatography strips slowly absorb alcohol from a beaker
  1. Let the solvent rise up the strip (by capillary action) until it is about 0.5 cm from the top then remove the strip from the solvent. Keep a close eye on your chromatography strip and the solvent front — if you let it run too long the plant extract may run off the paper and become distorted.
  3. Allow the chromatography strip to dry, then measure (in centimeters) and calculate the Rf value for each plant pigment. Record your results in your lab notebook.
    1. Tip: Use Equation 1, which is given in the Introduction, for calculating the Rf value.
  4. Repeat this experiment for each of your plant extracts three times.


  • What types of pigments do you think are present in each type of leaf? How can you tell? (Consider color, Rf value, etc.)
  • How were the Rf values different for each pigment and leaf? Why? (Make sure to consider molecular structure, polarity, etc.)
  • How do the different pigments help the plant? Why do different plants have different amounts and different types of these pigments?


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  • Do plants' pigments vary with more or different types of light? You might try growing a few plants of the same type under different colored lights, and/or more intense light, and then do the chromatography procedure. If you are very precise when taking samples (i.e., measuring how much of the leaf you grind up for each plant), you could even comment how the amount of each pigment changes, if at all.
  • You can also analyze the amino acids that are found in orange juice and lemon juice with chromatography.
  • For a less advanced chromatography project, see Explore How Chromatography Can Unmix Mixtures

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