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Investigation 1 – Lab









This Investigation is designed to:

  • provide you with a foundation from which to study chlorophyll and photosynthesis,
  • use the process of paper chromatography to separate the pigments in plants, including chlorophyll, and
  • help you understand the relationship between chlorophyll and other plant pigments and photosynthesis.


Student Preparation for the Investigation includes gathering the following materials.

Note: The materials are listed in your SDR. They are also listed below for your reference.

  • (1) mortar and pestle
  • (1) piece of filter paper
  • (1) metric ruler
  • (1) test tube
  • (1) test tube rack
  • (1) 400 ml beaker
  • (1) funnel
  • (1) basket-style coffee filter
  • (1) small binder clip
  • (1) pair of scissors
  • (1) triple beam balance
  • (1) scoopula
  • (2) plastic droppers
  • (1) rubber band
  • (1) piece of plastic wrap
  • (1) pair of gloves
  • (1) pair of goggles per student
  • (1) bunch of spinach, approximately four (4) leaves
  • Ask one student from the group to pour 10 ml of acetone in a 50 ml beaker.
  • Another student from the group should pour 10 ml of ethanol in a 50 ml beaker.

Direct one student from each lab group to collect the materials listed in their SDRs.



  • The spinach leaves get their dark green color from chlorophyll. For this reason, spinach leaves are useful in investigating chlorophyll.
  • Reflect on the PreLab video as you move through the procedural steps.
  • During the Experiment, every procedural step is important. If one step is skipped, data can become invalid. To help you keep on track, read each step thoroughly, complete the step, then check it off (Read it – Do it – Check it).
  • Complete all of the procedural steps in your SDR.



  1. Spinach leaves get their dark green color from chlorophyll. For this reason, you will use the spinach leaves to investigate chlorophyll.
  2. Create a spinach extract using the mortar and pestle. To do this, use the following steps.

A. Use the triple beam balance to obtain a 5 gram sample of spinach leaves.

B. Tear the leaves into small pieces and place them in the mortar.

C. Use the plastic dropper to place 1 ml of acetone on the spinach leaves in the mortar.

D. Using the pestle, take turns grinding the spinach leaves until a fine pulp is formed.

E. Place the funnel in the 400 ml beaker, then place the Photo Inv. 1 Lab Acoffee filter inside the funnel.

F. Using the scoopula, scrape the pulp mixture from the mortar into the coffee filter to remove the pulp from the extract.

G. The mixture will likely be too thick to drain easily into the beaker. You may need to put your hand inside the funnel and squeeze the coffee filter so that the extract flows into the beaker

Note: Try not to touch the filter paper with your hands. The oils from your hands may cling to the paper and affect the chromatography process.

  1. Cut the filter paper so that you have a piece that will fit inside a test tube, approximately 1.2 cm by 13.5 cm.
  2. Cut one end of the paper into a point.
  3. Attach the small binder clip to the end of the strip opposite to the pointed end, as shown in the picture. You may touch the binder clip when you need to move or handle the paper.
  4. Draw a line with your pencil 3 cm from the pointed tip of the filter paper. You may not use a pen on the filter paper.

Note: It is important that you not use pens to draw the line in the filter paper. If pens are used, the pigments in the pen ink will likely travel up the filter paper during the chromatography process and mix with the pigments of the spinach leaves.

  1. Use the plastic dropper to place a drop of spinach extract on the penciled line. Follow these steps:

A. Dip your dropper into the beaker with the extract. A small sample of extract will cling to your dropper.Photo Inv. 1 Lab C

B. Gently tap the tip of the dropper on the line on the paper, releasing a small amount of extract.

C. Blow on or wave the paper until the drop is dry.

D. Repeat these steps 3 or 4 times until a dark area of extract is formed.

Note: A toothpick may be substituted for the dropper pipette so that a smaller amount of extract can be applied. Between the applications of each drop, students should gently blow on the spot until it is dried. Several applications should be made in this manner until enough dried extract has been applied to the filter paper

  1. While one student from the group is placing extract on the filter paper, another student from the group should pour ethanol in the test tube. Follow these guidelines when pouring ethanol.Photo Inv. 1 lab D

A. The level of ethanol should be approximately 1 cm from the bottom of the test tube.

B. When the filter paper is placed in the test tube, it should just touch the ethanol.

9. Do not place the filter paper in the test tube. Fold the piece of plastic wrap and cover the top of the test tube. Wrap the rubber band tightly around the plastic wrap.

10. Allow the test tube to sit for two or three minutes.

11. After the drops of extract have dried on the filter paper, open the test tube and place the strip of filter paper in it. If the tip of the filter paper does not touch the ethanol, remove the paper and quickly add a few more drops of ethanol to the test tube.

12. Cover the test tube with the plastic wrap and fasten it with the rubber band. The paper should now be in the test tube. Make sure the filter paper does not touch the sides of the test tube. If it does, gently remove the paper, reinsert it, and then cover it with the plastic wrap.

13. Leave the test tube undisturbed. Observe the movement of the extract.

14. Clean up your lab table while you wait for the chromatography process to finish.

  1. Record: When the solvent nears the top of the strip (approximately 10 minutes), observe the filter paper. Record your observations. Include a written description and a drawing. You will likely observe a streaking of green into yellow pigment extending up toward the top of the paper.

Photo Inv. 1 Lab E


Be sure to clean up your lab bench after the experiment.