Key concepts
Plant biology

In the springtime it can be easy to spot flowers in a dazzling array of colors at flower gardens and in plant nurseries. And with Mother's Day coming, stunning flower bouquets seem to be everywhere. Have you ever wondered what makes a flower appear a certain color to us? For example, what pigments make a rose be a deep, rich shade of red? Do different flowers use the same pigments? In this activity you'll get to use paper chromatography to investigate if the pigments from one type of red flower are different from those in another type of red flower.

A flower’s pigments help to attract possible pollinators, such as honeybees, butterflies and hummingbirds. There are two major classes of flower pigments: carotenoids and flavonoids. Carotenoids include carotene pigments (which produce yellow, orange and red colors). Flavonoids include anthocyanin pigments (which produce red, purple, magenta and blue colors). Usually, the color a flower appears depends on the color of the pigments in the flower, but this can be affected by other factors. For example, blue cornflowers have the same pigments as red roses, but the pigments in the cornflower petals are bound to other pigments and metal ions, making cornflowers look blue.

In this activity you'll use paper chromatography to investigate the pigments in flowers. Chromatography is a technique that is used to separate out the components of a complex mixture or solution. In paper chromatography a solution is dabbed onto the bottom of a paper strip, and the strip is then placed in a liquid. The liquid moves up the paper and, depending on how soluble they are in the liquid, the pigments are carried up the paper with the moving liquid. Ideally the components move at different speeds, so they can be separated.

• Paper towels (Thicker ones will work better.)
• Scissors
• Pencil
• Ruler
• Jar, drinking glass or mug
• Measuring cup
• 70 percent isopropyl rubbing alcohol
• Water (distilled water is preferable, but tap water is also suitable).
• Large-mouth glass jar
• Red flower petals. Try to get at least two flower petals from at least three different plants, such as from your own garden, a florist or plant nursery. You could also try flowers that are similar in color, such as more purple or orange flowers. (Pink petals may not work well.) Larger petals, such as those from roses and tulips, work better than smaller petals.
• Piece of scratch paper
• Coin
• Timer or clock

• Cut the paper towels into strips that are each about one inch wide. Make each strip the same height as your large-mouth glass jar. Cut at least one strip for each type of flower you want to investigate.
• Draw a pencil line one inch from the bottom end of each paper strip.
• At the other end of each paper strip, use a pencil to label which flower will be spotted on the strip.
• In a clean jar, drinking class or mug mix one quarter cup of water with one quarter cup of the isopropyl rubbing alcohol. Pour a small amount of this mixture into the large-mouth glass jar, a little less than an inch deep. Adult supervision is recommended when working with the isopropyl rubbing alcohol.

• Place a piece of scratch paper on a hard, flat surface. (Some pigments can stain so you will want to protect the surface with this piece of scratch paper.) Put one of the paper towel strips on top of the piece of scratch paper. Lay a flower petal on the paper strip, over the line you drew.
• Roll a coin on its edge, like a wheel, over the petal and across the pencil line. Push down hard so that the petal is crushed and a strip of pigment is visibly transferred to the paper towel strip. Repeat this about three or four times (using a fresh part of the petal each time) so that a thick line of pigment is transferred to the pencil line. How does the line of pigment look? Is it what you expected?
• Tape the paper towel strip to the pencil so that when the pencil is laid horizontally across the top of the large-mouth glass jar, the strip hangs straight into the jar and the bottom edge of the strip is just barely immersed in the diluted isopropyl rubbing alcohol. The pigment line should not be immersed in the liquid. (To do this you may need to cut off part of the top of the strip.) Use a small piece of tape so that it does not cover much of the strip.
• Lay the pencil across the top of the large-mouth glass jar, as described, and let the liquid rise up the paper towel strip until the liquid is about an inch from the top of the strip. Remove the strip at this point. (This may take about 20 to 60 minutes.) What has happened to the pigment on the strips? Keep a close eye on the strip and where the liquid is—if you let the strip run too long, the liquid can reach the top of the strip and distort your results.
• Allow the paper towel strip to dry out. An easy way to do this is to tape the strip to the overhang of a counter or table so that the strip is dangling in the air.
• Now prepare a paper towel strip for one of the other flowers you want to investigate, rolling a coin over the pencil line as you did before. Put the prepared strip in the jar and, as you did with the first strip, run the new strip until it is similarly done. Do the results look similar to the results from the first flower?
• Repeat this process for any other flowers you want to investigate, using a new, pencil-marked paper towel strip for each one.
• Look at all of your finished paper towel strips together. If you see the same color band around the same height on different paper strips, it is likely that it is the same pigment. Do different red flowers have the same pigments in them? Do any of the flowers you investigated have more than one pigment? If you investigated more purple or orange flowers, do they have different pigments? Do your results make sense to you?
Extra: Try this activity with flowers that vary even more in their coloration. Do other colored flowers have pigments similar to the ones in the red flowers?
Extra: Some plants grow very colorful leaves, such as coleus plants, bromeliads and purple clovers. You could try this activity again, but this time investigate colorful leaves on plants instead of flowers. What pigments make the leaves so colorful? Are these the same as the pigments in similarly colored flowers?
Extra: A more accurate way to identify flower pigments using paper chromatography is by determining their retention factor (Rf factor). The Rf value is the ratio between how far the pigment travels and the distance the liquid travels from a common starting point (the pencil line you drew on the strips). If other conditions are kept the same, the Rf value for a certain pigment should be consistent. You can do this activity again, but this time measure these distances and calculate the Rf value for the pigments. Based on the Rf values, do the pigments look like they're the same in different types of red flowers?

Observations and results
Did you find that most (or all) of the red flowers used the same pigments? Did the pigments create a reddish-purplish band on the paper towel strips?

Carotene pigments (which are carotenoids) produce yellow, orange and red colors whereas anthocyanin pigments (which are flavonoids) produce red, purple, magenta and blue colors. Most red flowers use anthocyanin pigments to produce their red coloring (although some use carotenoids). On the paper strips, the anthocyanin pigments may have appeared as a purplish-reddish band. If different red flowers made similarly colored bands around the same height on the paper towel strip as one another, then they likely have the same pigment. If the bands are different colors and/or at different heights, however, then they're probably different pigments. Carotene pigments are more commonly found in vegetables, and, in fact, they are what make carrots look orange. Yellow and orange flowers can have carotenoids or flavonoids, and blue flowers often have anthocyanin pigments that are modified. Some flowers even have chlorophyll that gives them green coloring.

In paper chromatography the pigments move up the paper with the liquid and are separated based on the solubility of the pigments. So, if a pigment is very soluble in the diluted isopropyl rubbing alcohol, it should be easily carried far up the paper strip whereas a less soluble pigment will generally travel a shorter distance. Because different pigments often have different solubilities, they can be separated from one another on the paper strip.

More to explore
What Pigments are in Fruit and Flowers? from WebExhibits
The Chemical Pigments of Plants ( pdf ), from Joy Alkema and Spencer L. Seager, Weber State University, Ogden, Utah
Paper Chromatography , from Chemguide
Reveal the Red: Exploring the Chemistry of Red Flower Pigments , from Science Buddies

This activity brought to you in partnership with Science Buddies