Stuyvesant High School                                           Department of Biology and Geo-Science

                                      LABORATORY EXERCISE #17

                              HOW DO WE SEPARATE PLANT PIGMENTS
                                 BY PAPER CHROMATOGRAPHY?

Plants have various colored pigments in their chloroplasts to help absorb the light energy from the sun.
The most common pigments are CHLOROPHYLL b, a yellow-green pigment, CHLOROPHYLL a, a blue-
green pigment, XANTHOPHYLL, a yellow pigment, and CAROTENE, a yellow-orange pigment. These
pigments can be easily isolated from green plant leaves by using a technique called PAPER
CHROMATOGRAPHY. Paper chromatography is a techniques used by scientists to separate molecules
on the basis of differences in solubility in specific solvents and their ability to adhere to the filter paper.
Specific molecules, dissolved in specific solvents, travel up the paper at specific rates. This rate of travel
is measured by a ratio of solvent fronts known as the R f. Thus, molecules that are not very soluable in
the solvent and/or adhere strongly to the filter paper will travel up the paper at a slower rate than those
substances that are very soluable in the solvent, and/or do not adhere well to the filter paper.

           Distance a component travels up the paper (In our case, “components” are pigments)
   Rf = -------------------------------------------------------------
          Distance the solvent travels up the paper
   or      Rf = ----------

Thus, by comparing the values of unknown and known molecules, one can actually identify the unknown
molecule. Other types of chromatography have been developed, using substances other than paper.

1. Learn the principles of paper chromatography, which is a very common and very powerful tool in the
2. Separate the pigments in spinach leaves and determine their Rf values.

   1. What is the purpose of chromatography as a laboratory tool?

    2. How may chemicals separated by paper chromatography be identified?

Spinach, collard greens, carrots, test tubes and rack, cork with paper clip hook attached, strip of
chromatography paper, capillary tube, mortar and pestle, gravel, solvent for making the spinach extract,
chromatography solvent, ring stand, funnel, filter paper, small beaker, small metric ruler, scissors, pencil.

Solvents may cause irritation. If you get any of the liquid materials from this lab on your skin, be sure to
rinse the affected area with water. Also, wear safety goggles at all times during this lab.

Work in pairs.

I. Preparation of Pigment Extract
1. Tear some spinach leaves into pieces and place them into the grinding bowl. Add a pinch of gravel
     and a small amount of extract solvent. Grind the leaves until a concentrated “soup” of spinach extract
     is produced. Be careful not to add too much solvent. Ask your teacher to check the consistency of

Regents Living Environment                         1                                    Laboratory Manual
Stuyvesant High School                                         Department of Biology and Geo-Science

   your spinach “soup”. Other plants, such as collard greens and carrots, are rich in carotene and might
   also be added to your mixture.
2. Place the funnel in the ring of the ring stand and position the small beaker just below so the tip of the
   funnel touches the wall of the beaker. Fold the filter paper circle into quarters. Place it in the funnel
   and dampen it lightly with the extract solvent (this prevents the filter paper from absorbing all the
   spinach extract).
3. Slowly pour the spinach extract into the funnel. Carefully collect the filtered drops in the beaker. There
   may not be that much extract produced.

II. Preparation of the Paper Strip
     CHROMATOGRAPHY. Measure 2 cm from the bottom of the paper strip. Lightly draw a pencil line
     across the strip. Use scissors to cut off the two bottom corners of the strip. [See the diagram below]
2. Place the capillary tube into the extract. Notice
     that the extract climbs up the walls. QUICKLY
     touch the tip of the capillary tube to the center
     of the pencil line on the paper strip. TRY NOT
     TO LET THE SPOT SPREAD. A small spot will
     produce a better chromatogram. Blow on the
     drop to dry it. When the drop is dry, touch the
     original spot with more extract. Let the spot dry
     again. Repeat until the spot is a very dark

III. Running the Chromatogram
1. Carefully pour a small amount 1 of chromatography solvent into the test tube. Make sure it will be
      enough to touch the bottom of the chromatography paper when you place the paper strip into the
      tube. Also make sure that the solvent begins BELOW the dot where you placed the leaf extract.
      [Refer to above diagram]. Do not get the solvent on the side of the tube. If you do, let the tube dry.
2. Hang the paper on the hook in the cork.
3. While the test tube is in the rack, gently ease the strip into the solvent at the bottom of the tube.
      MAKE SURE YOUR SPOT DOES NOT TOUCH THE SOLVENT. Gently press down the cork to seal
      the test tube – this is very important in order to obtain good chromatogram results.
4. Watch the solvent creep up the paper strip. Notice how the pigments are beginning to separate.
      When the solvent almost reaches the hook hole, remove the paper strip and use a pencil to mark the
5. Record the distance the solvent traveled from the center of the original pigment dot.
6. Record how far the center of each pigment front traveled.
7. Calculate Rf

Regents Living Environment                         2                                   Laboratory Manual

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