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• Paper Chromatography

Project Summary

Difficulty	6
Time required	Short (several days)
Prerequisites	None
Material Availability	Specialty items: Chromatography paper
Cost	Low ($20 - $50)
Safety	You will be working with isopropyl alcohol. Adult supervision is recommended

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Sponsor

Sponsored by a generous grant from Monsanto Fund

Objective

The objective of this plant biology science fair project is to analyze the pigments in red flower petals, and to determine if different red flowers use the same or different pigments.

Introduction

Plants contain many complex chemicals. Some of these chemicals are used in the normal metabolism of the plant, such as those involved in photosynthesis. Some are involved in the plant's defense against insects, bacteria, and other pathogens. And some, such as the pigments in a flower, are involved in attracting the attention of possible pollinators, such as honeybees, butterflies, and hummingbirds.

The major types of flower pigments are anthocyanins, flavones, and carotenes. The anthocyanins produce red, purple, and blue colors; the flavones produce pale yellow colors; and the carotenes produce yellow, orange, and red colors.

Usually, the color of the flower depends on the color of the pigments found in the flower. But there was a century-old mystery that defied this logic. Cornflowers, which are blue, appear to have the same pigments as roses, which are red. How could this be explained? It turns out the difference is due to way the pigment molecules are organized inside the flower petal. The pigment that is red in the rose is blue in the cornflower because of the chemical environment within the cornflower (for more information about this, see the article by Steve Conner in the Bibliography, below). This occurrence is a good example of how, in nature, new and unexpected characteristics can emerge from the organization of simple parts.

The goal of this plant biology science fair project is to analyze the pigments found in flowers using paper chromatography. Paper chromatography is a technique to separate molecules, such as a mixture of pigments. The concept is shown in Figure 1, below. A mixture is dabbed onto a piece of chromatography paper near one end, at a point called the origin (See Figure 3). The same edge as the mixture dab is immersed in a solvent, with the origin above the level of the solvent. The paper works like a wick, with the solvent moving up the paper, due to capillary action. The pigment molecules are then carried up the paper with the moving solvent.

The rate at which different pigments move depends on a number of factors, including their size and solubility in the solvent. If a pigment is small and very soluble in the solvent, it can be carried up the paper with the solvent front, which is the leading edge of the solvent, as it travels up the paper. Because different pigment molecules have different chemical characteristics, they are separated from each other on the chromatography paper.

Figure 1. Paper chromatography. Molecules are separated from each other, depending on how fast they migrate with the solvent on the chromatography paper. (Wikipedia, 2008.)

How can you compare how far a pigment has traveled in two different experiments? It is possible to compare the results of different experiments if the distance that the pigment travels is given as a fraction of the distance that the solvent front has traveled. The relationship of the distance moved by a pigment to the distance moved by the solvent front is specific for a given set of conditions. This relationship is called the R_f, and is defined as follows:

Equation 1:

You will use paper chromatography to analyze the pigments in red flowers from different species. Do you think they will have the same pigments, as determined by paper chromatography, or will the pigments be different?

Terms, Concepts and Questions to Start Background Research

• Pigment
• Anthocyanin
• Flavone
• Carotene
• Paper chromatography
• Origin
• Solvent
• Capillary action
• Solvent front
• R_f
• Basic solution

Questions

• What are the names of the two scientists who were awarded the 1952 Nobel Prize in chemistry for their work in paper chromatography?
• Explain the principle behind the separation of molecules using paper chromatography.
• Based on your research, what kinds of pigments are present in red rose petals?
• True or false: the pigments in red rose petals turn blue in a basic solution?

Bibliography

• Texas A&M University. (2008). Paper chromatography. Retrieved September 15, 2008, from http://peer.tamu.edu/podium_poster_presentations/Paper%20Chromatography.ppt#256
• Conner, S. (2008). A century on, a colourful mystery is solved. Retrieved September 18, 2008, from http://findarticles.com/p/articles/mi_qn4158/is_/ai_n14887739

Materials and Equipment

• Chromatography paper; available from Science Kit & Boreal Laboratories (search for "filter paper")
• Scissors
• Ruler
• Pencils (2)
• Flower petals, red; you will need at least 2 flower petals from 3 different plants, which you can get from your own garden or from a florist or plant nursery.
• Isopropyl alcohol
• Distilled water is preferable, but tap water is also suitable.
• 2-cup liquid measuring cup
• Spoon
• Large-mouth glass jar
• Coin
• Lab notebook

Experimental Procedure

To make sure you can compare your results, as many of your materials as possible should remain constant. This means that the temperature, type of water used, size of paper strips, where the ink is placed onto the paper, etc., should remain the same throughout the experiment.

Cutting and Marking the Paper Strips

1. Cut chromatography paper strips about 5 inches long (they should all be the same size).
2. Use a ruler and pencil to draw a line across each paper strip, horizontally, 2 cm from the bottom. This is the origin line, where the sample is spotted (see Figure 2, below).
3. Using a pencil, number each strip at the top so that you can identify it later.

Figure 2. Chromatography strip with a pigment sample.

Spotting the Sample onto the Paper

1. Make a data table in your lab notebook that lists the kinds of flowers you will be testing.
2. To transfer the pigments onto the chromatography paper, you will crush a petal from each flower with a coin. Lay the flower petal on the chromatography strip, over the origin line. Roll the coin, like a wheel, over the petal, pushing down so that a strip of the pigment is transferred to the strip.

Placing the Strip into the Chromatography Chamber

Figure 3. The setup for the paper chromatography jar.

1. Make your solvent out of 50% isopropyl alcohol and water by mixing 1 cup of water with 1 cup of isopropyl alcohol.
2. Pour a small amount of the solvent into your glass jar, about 1 inch deep. See Figure 3, above.
3. Lay a pencil across the top of the jar.
4. Tape a strip of the chromatography paper that has been imprinted with a flower's pigment to the pencil and hang it into the jar of solvent so that the bottom edge is just immersed in the solvent.
   1. Note: Do not immerse the origin.
5. Allow the chromatography paper to remain in the solvent for 1 hour, or until the solvent front approaches the top of the strip.
6. Take the chromatography strip out of the jar.
7. Use a pencil to mark the solvent front.
8. Measure the distance from the origin to the solvent front and from the origin to the top of the pigment band that should now be visible. Record the data in your lab notebook.
Repeat Spotting the Sample onto the Paper, step 2, and Placing the Strip in the Chromatography Chamber, steps 1-8, with the other red-colored flowers.

Analyzing Your Data

1. Now, using Equation 1 from the Introduction, calculate the R_f value for each pigment. See Figure 4, below.

Figure 4. The pigment moves up the paper as the solvent front advances. The Rf value is the ratio of the distance to the top of the band, to the distance to the solvent front, measured from the origin.

2. List the R_f values in a data table, with the names of the flowers.
3. Do different red flowers have the same pigments, as determined by paper chromatography? Or are the pigments from different flowers also different?

Variations

• Try other colors of flowers, such as yellow and pink roses.
• Try other solvents, such as 100 percent water, 100 percent isopropyl alcohol, 10 percent isopropyl alcohol, etc.
• For another Science Buddies science fair project about chromatography, try Paper Chromatography: Advanced Version 1.

• For more science project ideas in this area of science, see Plant Biology Project Ideas.

Credits

David B. Whyte, PhD, Science Buddies

Last edit date: 2008-12-23 10:26:00