Chlorophyll is the pigment in plants that captures sunlight energy and uses it to drive photosynthesis. While chlorophyll does give plants their characteristic green color, chlorophyll actually comes in many colors and subtypes ranging from green to yellow to orange to red. In this experiment, students use paper chromatography to separate the many pigments from one another. First the pigments are extracted from the plants by simply crushing the plant cells open on the filter paper with the edge of a penny. When the filter paper is then immersed in rubbing alcohol, the pigments are carried upwards through capillary action. The smallest pigments travel more quickly and thus separate from the larger pigments that remain closer to the origin line.
Chlorophyll is an amazing chemical that is the essential ingredient in photosynthesis, the process through which plants capture light from the sun to create glucose. When photons of light hit chlorophyll, the electrons in the central magnesium atom donate an electron to a series of chemical reactions – the electron transport chain – that produce ATP, the cells primary unit of energy currency. The chlorophyll gets this electron back by taking one from water, resulting in the release of oxygen gas as a byproduct.
Chlorophyll comes in two main forms: chlorophyll a and chlorophyll b. Each has a slightly different chemical structure and therefore absorbs light of different wavelengths. In this paper chromatography experiment, chlorophyll a will appear as a bright yellow-green band while chlorophyll b will appear as a dull green band. In addition, there are many other accessory pigments in plants that absorb light and help transfer photons to the chlorophyll. These include carotene (an orange band) and xanthophyll (a yellow band). A final pigment that may be detected is anthocyanin (a red-brown band) that acts as a type of sunscreen for plants, protecting the plant from UV damage.
For more information on paper chromatography and its use in chlorophyll extraction, a fabulous scientific description may be found on the Science Buddies website. Also see this photosynthesis lesson plan by Sara Swisher, Barb Wilson, and Jean Wilson. Finally, Cheryl Massengale has a great write up for chlorophyll extraction including a table illustrating how to calculate the Rf value for each band and use that as a quantitative means of identifying the various pigments.
- Filter paper cut into 9 cm x 2 cm strips (coffee filters work but not as well)
- Beaker or cup no more than 9 cm tall
- Small stick - bamboo skewer, pencil, plastic stirrer, drinking straw, etc.
- Rubbing alcohol
- Assorted fresh plant leaves – spinach, lettuce, or leaves collected from around the neighborhood (unusually colored leaves such as white, purple, red, and yellow are particularly interesting)
- Use a pencil to draw a line across the filter paper strip, 2 cm from one end.
- Place the leaf you want to test on top of the filter paper strip, remembering where the line is.
- Using the edge of a penny, trace the penny across the leaf right on top of where you drew the line. Push down hard enough to leave a green smear on the paper on top of the original pencil mark.
- Repeat 2 more times with fresh sections of leaf to make the smear darker. Try to keep the smear as close to the original pencil line as possible.
- Fill the cup or beaker to a depth of 1 cm with rubbing alcohol.
- Carefully lower the filter paper strip, green smear end in first, into the beaker until the bottom just touches the alcohol. Make sure that the green smear does not actually touch the alcohol, only the tip of the paper should actually be in contact with the alcohol.
- Lay the stick across the mouth of the beaker like a bridge from one edge to the other.
- Tape the filter paper strip to the stick so that the paper is held in place just touching the alcohol but not touching the sides of the beaker.
- Wait. The alcohol should gradually move up the paper, bringing many of the pigments along with it.
- Remove the stick and paper when the alcohol has almost reached the stick. The actual length of time will depend on the type of paper you use.
- Mark how far up the paper the alcohol traveled with a pencil.
- Determine how many bands of pigment you have, what color they are, and measure how far each band traveled from the origin line. Some of the pigments will fade and disappear over time. It may help to trace around each band in pencil while they are still clear so that the strip can still be analyzed if and when the bands fade.
- Calculate an Rf value for each band (Rf value = distance that band traveled divided by distance the alcohol front traveled).
- Identify the pigments based on their colors or by their Rf value. The Rf values allow you to compare one pigment band to another from strip to strip.
- Compare what was found in different types of leaves.
- Try this on purple plants. Do they have chlorophyll too?
- Try this on white-leafed plants. Do they have chlorophyll too?
- Try this on leaves from trees before and after they change color in the fall. What happens to the chlorophyll as the leaves change color? Do new pigments emerge or were they there all along?
- Cover half of a wide green leaf with tin foil. Leave the plant in a sunny window for at least 4-5 days. Remove the tin foil and see what happened to the leaf. Compare chlorophyll extracted from the uncovered half of the leaf with chlorophyll extracted from the covered half. Is there any difference? On a separate leaf that underwent the same treatment, uncover the leaf and leave it in a sunny window. Does the chlorophyll come back?