# Reveal the Red: Exploring the Chemistry of Red Flower Pigments

 Difficulty Time Required Short (2-5 days) Prerequisites None Material Availability Filter paper can be purchased from the Science Buddies Store. Cost Low ($20 -$50) Safety You will be working with isopropyl alcohol. Adult supervision is recommended

## Abstract

Are all reds the same? Find out in this science fair project! Investigate if the pigments in one type of red flower are different from those in another type of red flower. Flowers contain an assortment of amazing chemicals that produce color. In this plant biology science fair project, you will analyze the colored pigments in different plants' red flower petals using paper chromatography, and compare the pigments in the different flowers.

## Objective

Analyze the pigments in red flower petals and determine if different red flowers use the same or different pigments.

## Credits

David B. Whyte, PhD, Science Buddies

Teisha Rowland, PhD, Science Buddies

### MLA Style

Science Buddies Staff. "Reveal the Red: Exploring the Chemistry of Red Flower Pigments" Science Buddies. Science Buddies, 15 June 2015. Web. 4 Aug. 2015 <http://www.sciencebuddies.org/science-fair-projects/project_ideas/PlantBio_p035.shtml>

### APA Style

Science Buddies Staff. (2015, June 15). Reveal the Red: Exploring the Chemistry of Red Flower Pigments. Retrieved August 4, 2015 from http://www.sciencebuddies.org/science-fair-projects/project_ideas/PlantBio_p035.shtml

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Last edit date: 2015-06-15

## Introduction

Plants contain many complex chemicals that have different functions. 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.

There are two major classes of flower pigments: carotenoids and flavonoids. Carotenoids include carotene pigments, which produce yellow, orange, and red colors, and xanthophyll pigments, which only produce yellow colors. Flavonoids include anthocyanin pigments, which produce red, purple, magenta, and blue colors, and flavones and flavonol pigments, which produce yellow colors. Some flowers may even have chlorophyll, a green pigment usually found in the leaves of plants. Watch the video below to find out more about pigments in plants.

This video gives an introduction to pigments in plants.
Watch this NBC Learn Plant Pigment video http://www.nbclearn.com/chemistrynow/cuecard/53149

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. Blue cornflowers appear to have the same pigments as red roses. How could this be explained? The pigment that is red in the rose is blue in the cornflower because the pigment in the cornflower interacts with other pigments and metals in the cornflower petal. This occurrence is a good example of how, in nature, new and unexpected characteristics can emerge from the organization of a few basic parts.

The goal of this plant biology science fair project is to analyze the pigments found in flowers using paper chromatography. Chromatography is a group of techniques, including paper chromatography, that are used to separate molecules in a complex mixture or solution, such as specific pigments in a mixture of pigments. In each chromatography apparatus there is a mobile phase, which is a liquid the solution is dissolved in, and a stationary phase, which is a material the liquid moves through. The mobile phase is also called the solvent.

In paper chromatography, a liquid like water or isopropyl alcohol is the mobile phase (or solvent), and filter paper is the stationary phase, as shown in Figure 1 below. A mixture is dabbed onto a piece of filter paper near one end, at a point called the origin. The same edge as the mixture dab is immersed in the 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.

How does chromatography separate the different molecules in a solution? The molecules ideally move at different speeds as they travel through the stationary phase. This is done by adjusting the mobile and stationary phases so that they interact with different properties of the solution's molecules, such as their molecular size, electrical charge, or other chemical properties, to distinguish and separate them from each other. In paper chromatography, different pigments can be separated out from a solution based on the solubility of the pigments. If a pigment is very soluble in the solvent, it can easily be carried up the paper with the solvent front, which is the leading edge of the solvent, as it travels up the paper. A pigment that is less soluble in the solvent, or one that interacts with the filter paper more than the solvent, will generally travel a shorter distance. Because different pigment molecules have different solubilities, 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 up the chromatography paper. (Wikipedia, 2008.)

In paper chromatography, you can see the pigments separate out on the filter paper and identify the pigments based on their color and how far they travel. 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 retention factor (Rf value), and is defined as follows:

Equation 1:
$R_f$ is a fraction that is characteristic for each pigment in a given set of experimental conditions.

For example, if a pigment from a solution moves 2.5 centimeters (cm) up the filter paper and the solvent front moves a total of 5.0 cm, then the Rf value for the pigment is 0.5. You can use Rf values to identify different pigment molecules as long as the solvent, pH, and type of paper remain the same.

In this plant biology science project, 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 and Concepts

• Pigment
• Carotenoids
• Flavonoids
• Carotene
• Anthocyanin
• Paper chromatography
• Chromatography
• Solution
• Mobile phase
• Stationary phase
• Solvent
• Origin
• Capillary action
• Solubility
• Solvent front
• Retention factor (Rf)

### Questions

• Which pigments may cause red colors in flowers?
• In paper chromatography, how does the mobile phase travel through the stationary phase?
• How can paper chromatography separate different pigment molecules? How is solubility involved?
• What is a retention factor (Rf) and how can it be used to identify a specific pigment?

## Bibliography

This resource will give you more information about the history of chromatography, and teach you about the types of chromatography used in research labs today:

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## Materials and Equipment

• Filter paper; available from the Science Buddies Store.
• Note: Coffee filters can not be substiuted for filter paper in this project. For more details about choosing the right paper see the Paper Chromatography Resources page.
• Scissors
• Ruler, metric
• Pencils (2)
• Clean jar, drinking glass, or mug
• 100 mL graduated cylinder or measuring cup. A 100 mL graduated cylinder is available online at Amazon.com.
• Isopropyl rubbing alcohol, 70% (at least 60 mL)
• Distilled water (at least 60 mL). Distilled water is preferable, but tap water is also suitable
• Large-mouth glass jar
• Flower petals, red; you will need at least 2 flower petals from at least 3 different plants, which you can get from your own garden or from a florist or plant nursery. Tip: Larger petals, such as those from roses and tulips, work better than smaller petals.
• Piece of scratch paper
• Coin
• Timer or clock
• Lab notebook

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## 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 the chromatography filter paper into strips that are about 2 centimeters (cm) wide and as long as the large-mouth glass is tall (the strips should all be the same size).
1. You will want to make at least three strips for each flower you want to investigate, or a minimum total of nine strips.
2. Use a ruler and pencil to draw a line across each paper strip, horizontally, 2 cm from the bottom, as shown in Figure 2 below. This is the origin line, where the sample will later be spotted.
1. Tip: Do not use a pen for writing on the strips because the ink will run when the solvent passes through the strips.
3. In your lab notebook, assign a number to each different type of flower, starting with the number 1 and going up. Using a pencil, number three strips "1," three other strips "2," and three other strips "3" at the top of the strip, as shown in Figure 2. This is so that you can identify which flower was used with which strip later.
1. If you are investigating more than three different types of flowers, similarly continue to number the test strips.

Figure 2. Chromatography strip with a pigment sample.

### Performing Paper Chromatography on the Paper Strips

1. In your lab notebook, make a data table that lists the kinds of flowers you will be testing. Also make four columns titled something similar to "Color of the Pigment Band," "Distance to the Solvent Front (in cm)," "Distance to the Top of the Band (in cm)," and "Retention Factor (Rf)."
2. In a clean jar, drinking glass, or mug, dilute the 70% isopropyl rubbing alcohol in half with water by mixing 60 milliliters (mL) (1/4 cup) of water with 60 mL of isopropyl alcohol. This water and isopropyl alcohol mixture is your solvent.
3. Pour a small amount of the solvent into your large-mouth glass jar, about 2 cm deep.
1. If needed, prepare more solvent by repeating step 2.
4. Next transfer the pigments from one type of flower onto a strip of chromatography paper.
1. Pick a flower type you want to investigate.
2. Take one of the paper strips you prepared with this flower's number and place it on top of a piece of scratch paper on a hard surface. Note: Some pigments can stain so the paper strip should be prepared on a piece of scratch paper to protect the surface beneath it from getting stained.
3. Lay a petal from the selected flower on the paper strip, over the origin line.
4. Roll a coin, like a wheel, over the petal and across the origin line. Push down hard so that the petal is crushed and a strip of pigment is visibly transferred to the strip.
5. Repeat step d about three to four times (using a fresh, unused part of the petal each time) so that a thick line of pigment is transferred to the strip, on the origin line. Be careful to only transfer the pigment onto the origin line.
1. If the origin line becomes a little wider with pigment, this is OK, but make a note of it in your lab notebook.
2. If you accidentally transfer pigment to an area that is away from the origin, prepare a new strip of paper as you did in the "Cutting and Making the Paper Strips" section and repeat steps 4b-e in the "Performing Paper Chromatography on the Paper Strips" section.
6. Tip: Only prepare one strip with sample at a time so that the sample does not dry out.
5. Tape the strip to the pencil so that when you lay the pencil across the top of the large-mouth glass jar, as shown in Figure 3 below, the strip hangs into the jar of solvent and the bottom edge of the strip is just barely immersed in the solvent. Note: The origin should not be immersed in the solvent.
1. To do this you may want to cut off part of the top of the strip. If you do this and it removes the number you wrote on the strip, be sure to re-write the number at the new top of the strip.
2. Use a small piece of tape so that it does not cover much of the top of the strip.
3. Be sure the strip hangs straight into the solvent.

Figure 3. The setup for the paper chromatography jar.

1. Complete the setup as shown in Figure 3 and let the solvent rise up the strip (by capillary action) until the solvent front is about 2 cm from the top and then remove the strip from the solvent. Check on the strip and the solvent front every 5 to 10 minutes - if you let it run too long the dye may run off the strip and become distorted.
1. This may take about 20 to 60 minutes.
2. If the solvent front has not reached 2 cm from the top of the strip after one hour, take out the strip anyway.
2. Use a pencil to mark the solvent front.
3. Allow the chromatography strip to dry. Tip: 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.
4. After the strip has dried, measure the distance (in centimeters) from the origin to the solvent front and from the origin to the top of the pigment band that should be visible, as shown in Figure 4 below. Record the data in the data table you made in your lab notebook.
1. Also record the color of the pigment band. For example, it may look "red," "purplish red," "pink," etc.
2. Note: If you see more than one pigment band on the strip, record this band's color in your data table in a new column titled "Color of the Second Pigment Band." Also measure the distance from the origin to the top of this pigment band and record the data in your data table in a new column, titled "Distance to the Top of the Second Band (in cm)."
5. Repeat steps 4-9 two more times for the same type of flower.
6. Repeat steps 4-10 two more times, each time with a different type of flower, so that you have run at least three chromatography paper strips for each of the three different flower types you want to investigate.

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.

### Analyzing Your Data

1. Now, using Equation 1 from the Introduction in the Background tab, calculate the Rf value for each pigment for each strip. Record the values in the data table in your lab notebook.
1. Note: If you saw more than one pigment band on a strip, calculate the Rf value for this band as well and record it in your data table in a new column titled "Retention Factor (Rf) of the Second Band."
2. Compare the Rf values and colors of the pigment bands for each different flower. To be the same pigment, the pigment bands should have similar Rf values and be a similar color. Do all of the different red flowers you investigated have the same pigments, as determined by paper chromatography? Or did the different flowers have different pigments? If they used different pigments, was there one pigment that was in most of the red flowers?
1. If one pigment was used by multiple red flowers, which pigment do you think it might be? Tip: You may want to re-read the Introduction in the Background section, and do additional research on carotene and anthocyanin pigments.

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## Variations

• Try this science project with other, similar colors of flowers, such as purple, pink, or orange flowers. Do they have pigments similar to the ones in the red flowers? You could even try it with a greater variety of flower colors, such as yellow, blue, or green flowers. What pigments do you think are in these flowers?
• Some plants grow very colorful leaves, such as coleus plants, bromeliads, and purple clovers. You could try this science project 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?
• Changing the mobile phase, or solvent, in paper chromatography can affect how well the chromatography works. Investigate this by trying other solvents, such as plain water, (undiluted) 70% percent isopropyl alcohol, isopropyl alcohol that has been diluted 90% with water, saltwater, etc. Does a pigment travel different distances depending on the mobile phase you use? What do you think this tells you about the solubility of that pigment in the different mobile phases?
• For other Science Buddies science fair projects about paper chromatography, try

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## Ask an Expert

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