Chlorophyll and accessory pigments are used by leaves to collect energy from light and transduce it to chemical energy be used to synthesize sugar. These pigments can be separated by chromatography in which a solvent system travels through paper by capillary action, carrying the pigments with it. Because each pigment has a different solubility and/or affinity for paper, they move at different rates, and separate along the paper. We will use this technique to detect a variety of pigments, and separate them for further analysis.
- Fresh, unfaded leaves (spinach, etc)
- Solution of 90% petroleum ether + 10% acetone
- Whatman #1 chromatography paper
- 95 or 100% EtOH
- 250 mL d flask
- #8 rubber stopper
- A penny
- Place 20 mL of 90% petroleum ether + 10% acetone solution in a stoppered 250 mL Erlenmeyer flask. Replace stopper and allow the “fumes” to equilibrate at least 20 minutes in the flask while the next steps are performed.
- Cut a strip of chromatography paper slightly longer than the distance from the bottom of the stopper to touch the solvent. Handle only by the edges. The bottom edge of the paper should be cut as straight as possible. Mark the origin with a pencil line 1.5 cm from the end of the paper.
- Lie the chromatography paper strip on a clean piece of paper, and place a fresh spinach (or other) leaf across the origin line. Use the edge of a penny to crush the leaf into the origin line to make a green stripe. Repeat several times until the stripe is quite dark. Allow to dry.
- Hold the paper strip up to the equilibrated flask, and adjust its length by folding at the top so that the bottom will reach just below the surface of the solvent.
- Flip the stopper over to cover the mouth of the flask (keeping flask covered), and attach the paper strip to the center of the stopper with a T pin. Quickly invert the stopper and lower the strip into the center of the flask, so that it hangs straight down and just touches the solvent, drawing it up by capillary action.
- As the solvent moves up the paper by capillary action, it carries the various pigments up from the origin. When the upper most band is about 1.0 to 0.5 cm away from the top of the paper, remove the paper strip and replace the stopper. The chromatogram should be observed and drawn, especially noting the colors of the various bands that are visible. How many bands to you see? Calculate the Rf for each band, by dividing the distance the band moved by the distance from origin to the solvent front.
- Label a clean 10 × 130 test tube for each band observed according to Rf s color. Cut out your different pigment bands with clean scissors and add to the appropriate tube. Once the class specimens have been collected, add 5 mL of 100% ethanol to each tube. Cover with Parafilm® And place in the designated rack for storage until the next lab period.
- Return “clean” chromatography solvent to the reagent bottle for reuse/disposal.