The Life and Times of Percy Julian

For Teachers

Activity 7A.6 (Lab Activity)
Separation by Paper Chromatography

Note to Teachers: There is no corresponding student version of this separation activity on the site. This is an in-class activity.

Lesson Overview Students will use the laboratory separation technique of paper chromatography to separate the different dyes in pen inks.
Learning Objective Students encounter many substances in everyday life that are mixtures, even though some might appear to be pure substances at first glance. Techniques like paper chromatography can separate the mixture into its individual components. 
Lesson Concepts

Chromatography is a family of techniques for separating mixtures. Paper chromatography utilizes differences in solubility of the various component substances in a mixture to separate the mixture, as well as differences in the ability of the component substances to adhere to a separating medium. The mixture in this case is ink, and the component substances are the component dyes or pigments of the ink. The separating medium is filter paper. The solvent is isopropyl alcohol.

  • separation
  • mixture
  • pure substance
  • chromatography
  • paper chromatography
  • chromatogram
  • solubility
  • solvent
Materials Required

Per lab group:

  • Three or four ballpoint or felt-tip pens of different color inks, at least one of which is black
  • One round sheet of filter paper, 18.5 cm in diameter (but see comment under “Notes” below)
  • Isopropyl alcohol
  • 400 mL beaker or an equivalent-volume clear plastic cup
  • Petri dish or plastic wrap
  • Metric ruler
  • Pencil

Students should wear safety goggles and avoid inhaling alcohol fumes.

Skills Required

No special skills are required for this activity, other than reading and following instructions for preparing the chromatography paper.

Time Required

One full class period and overnight to allow the papers to dry. Setup should take 10 minutes and the rest of the class period should be for allowing the solvent to move up the paper. If the solvent has not moved up the paper to the desired mark by the end of the class period, the teacher will have to remove the papers from the solvent whenever separation is complete.

Student Ability Level and Grouping

This activity may be done by middle school students working individually or in pairs.


Have enough materials prepared to distribute to the lab groups. Be prepared to walk your students through the procedural steps below, demonstrating on your own filter paper as you go.

Procedure Notes

Have student follow the following procedure:

  1. Cut out a 5 cm by 16 cm rectangle from the round sheet of filter paper.
  2. On the rectangle of filter paper, draw a line in pencil 1 cm from the edge as shown below. Note: It is important to use a pencil for drawing the lines on the paper, because ink from a ballpoint or felt-tip pen will become part of the moving “front” of alcohol and ink once the paper is placed in the alcohol.
  3. Diagram of filter paper

  4. Using the ballpoint or felt-tip pens, make spots with three or four different colors of ink (one should be black) on the pencil line. Be sure the spots are at least 1.5 cm apart. The ink spots should be small but dense. Add as much ink as possible without tearing the paper. Note: With felt-tip pens, it is better to lay down several spots on the same location, allowing them to dry between applications, than it is to make one big spot the first time. The individual spots will superimpose on each other without spreading out as much as one big spot would.
  5. Fold the rectangle of paper in half, as shown in the picture below. It should look like a paper “tent” and the pencil line with the dots should be on the outside of the tent. Note: Students should write their names on the inside of the tent in pencil at this time for easy identification.
  6. Diagram of filter paper

  7. Pour isopropyl alcohol into the cup or beaker to a depth of 0.5 cm. Note: Some teachers choose to “develop” the chromatography “plates” themselves rather than allowing students to handle isopropyl alcohol. In that case, the teacher carries out steps 6–10. See comment below under “Notes.”
  8. Place the folded sheet of paper in the beaker, with the ink spots toward the bottom, so that it stands up in the beaker like a tent. The paper should be sitting in the alcohol, but the alcohol should be below the pencil line. Note: If the paper sags and won’t stand upright in the beaker, improvise by suspending the filter paper over a pencil and hang that over the beaker. If you do this, you may have to fill the beaker with more alcohol so that the alcohol reaches the paper, but not enough to reach the pencil line.
  9. Cover the beaker with a Petri dish or plastic wrap to minimize evaporation of the alcohol. Note: If the paper tent is suspended over a pencil, you will have to use plastic wrap because the support pencil will extend beyond the edges of the beaker, and a Petri dish will not fit.
  10. Watch as the paper begins to absorb the isopropyl alcohol and move up the paper. Students should write down their observations on the Investigation Record Sheet for this activity as the watch. As the alcohol rises, it will meet and dissolve the ink spots, which will then travel up the paper with the alcohol (the solvent). The different color inks will travel at different rates. Moreover, the different component dyes of each color ink spot will begin to separate from one another as they travel at different rates up the paper, depending on both their solubility in the solvent and on their ability to adhere to the paper. Students will be able to see that pen ink—whether black, blue, green, or red—is a mixture of component dyes or pigments. The component dyes will begin to reveal themselves.
  11. When the isopropyl alcohol has risen to a level 0.5 cm from the top of the paper, remove the paper from the beaker.
  12. Set the papers aside to dry overnight.

Have students record their answers to the following on the Investigation Record Sheet.

  1. After the papers have dried, have students count and record the number of separate colored stains there are above each original ink spot. Note which ink spot produced the greatest number of separate stains and which ink spot produced the least.
  2. Students should compare results: Do all black inks give the same streak (chromatogram)? Does each particular color ink reach the same distance from the start line for each student? How do the streaks from the same ink on different groups’ chromatography sheets compare?
  1. The principles behind chromatography might be a bit advanced for middle school students, but this procedure will clearly show the separation of the components of everyday materials.
  2. Filter paper is usually sold in round sheets of standard sizes and is available from many science supply companies. You will need to use 18.5 cm diameter sheets in order to cut rectangular sheets of the proper size. Newsprint, heavier matte copy paper, or coffee filters can be used in place of filter paper, although they may sag as they soak up the alcohol. If you are buying from a science supply company, they also sell chromatography paper, in rectangular sheets. It is more expensive, but since it is already a rectangle, there is less waste.
  3. If you choose to “develop” the “plates” yourself so students do not have to handle isopropyl alcohol, you may want to use a larger container so that you can develop all the plates at once. Just be sure to cover the container so the alcohol will not evaporate as quickly.
Assessment See Activity 7A.7, Assessment: A Challenging Separation.

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