Manduca Project - Chromatography

6/12/03 3:10 PM

Plant and Insect Pigments

Middle and High School Activity by James Pennington and John Kevin Hall Department of Biochemistry and Molecular Biophysics University of Arizona

Purpose: 1) To provide a hands-on example of the practical application of science, scientific thinking, and scientific technique to the high-school science classroom 2) To demonstrate properties of different types of molecules (hydrophobic vs. hydrophilic) 3) To introduce the experimental technique and theory behind two chromatographic methods (paper and thinlayer chromatography) 4) To give the students an understanding of organic extractions as a laboratory technique 5) To explore how the objectives below can be used to answer a scientific question. Objectives: At the end of this exercise, the student will be able to: 1) Demonstrate an understanding of the differences between hydrophilic and hydrophobic molecules 2) Demonstrate an understanding of the theory behind chromatographic techniques 3) Perform paper chromatography and thin-layer chromatography with proper safety conditions under supervision 4) Perform lipid extractions on plant and animal tissues with proper safety conditions 5) Predict the outcome of an experiment utilizing chromatographic techniques 6) Extend the knowledge gained during the lesson to other scientific questions. Arizona state standards met:
Essentials

1SC-E1. Identify a question, formulate a hypothesis, control and manipulate variables, devise experiments, predict outcomes, compare and analyze results, and defend conclusions 1SC-E4. Identify and refine questions from previous investigations 2SC-E3. Provide different explanations for a phenomenon; defend and refute the explanations 2SC-E4. Identify characteristics of scientific ways of thinking 2SC-E5. Explain how scientific theory, hypothesis generation and experimentation are interrelated Proficiency: 1SC-P1. Propose solutions to practical and theoretical problems by synthesizing and evaluating information gained from scientific investigations 1SC-P2. Compare observations of the real world to observations of a constructed model (e.g., an aquarium, a terrarium, a volcano) 1SC-P3. Analyze and evaluate reports of scientific studies 1SC-P6. Identify and refine a researchable question, conduct the experiment, collect and analyze data, share and discuss findings
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Why are Manducas blue?

Manduca sexta larvae, when fed on the artificial diet, are blue in color. Under natural conditions (feeding on plants) however, they are green in color. This green color is made up of two chemical pigments: the blue pigment biliverdin and the yellow pigment lutein. Artificial diet has very little lutein, and because Manducas are unable to synthesize lutein on their own they have more of a blue color than their plant-fed counterparts. Pigments can be fat soluble or water soluble Plant leaves and flowers are colored by many different pigments. Some are fat soluble, some are water soluble. The fat soluble pigments, known as carotenoids, are yellow in color. These include beta-carotene and lutein. They are found primarily in the leaves of plants, but the flowers may also contain small amounts of fat soluble pigments. Hydrophobic pigments Pigments that are made up primarily of carbon and hydrogen are fat soluble. They are "hydrophobic", meaning that they DO NOT have the properties to be surrounded by water molecules; therefore they move away from water. They are quite soluble in other hydrophobic compounds such as fats, oils, and organic solvents. Structure of beta-carotene, a yellow carotenoid In the image at the right, carbon atoms are shown as gray balls, hydrogen atoms as white balls. Notice the molecule is made only of carbon and hydrogen and is lacking any polar groups (i.e. oxygen). This molecule is very similar to the molecule lutein, the yellow pigment that helps make Manduca green. In Manduca, lutein is the only carotenoid absorbed from the diet. This is because special transporters in their gut responsible for absorption of carotenoids only recognize lutein. Beta-carotene

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Water soluble pigments have a considerable amount of oxygen in their makeup. This makes them "hydrophilic", meaning they can be surrounded by water and easily dissolved. Structure of cyanidin Various changes to this molecule result in the different colors of petunia flowers. Note that there are several oxygen groups (red) on this molecule, making it soluble in aqueous solutions. Cyanidin

Pigments in petunia flowers In the case of the petunia flower, the purple color that forms in the flower is from a class of water soluble pigments known as anthocyanidin. However, when fed to Manducas, petunia flowers do not make them purple. Once again, this is because Manducas DO NOT have the ability to absorb the purple pigments. Therefore, the pigments are excreted in the feces.

Manduca feces after feeding on various materials

Chromatography System Theory

So how do we find out how many different types of pigments make up the colors of plants and insects? One option is a method called "chromatography". This is a system in which a mixture of many different molecules can be separated by the speed at which they move through a material. We will use two materials to separate these molecules: paper and silica (fine glass) powder The material that we use to separate molecules in chromatography is a matrix. This means that it has a "woven" quality, like fabrics. In the case of paper, the individual fibers criss-cross together. The spaces between the fibers allow molecules to move through them. Small molecules will easily pass between the fibers and move quickly. Large molecules will have difficulty moving between the fibers and move much slower. How do we get the molecules moving through our matrix? This is done with a solvent. The solvent is a liquid that will dissolve our mix of molecules and move through the matrix, carrying the molecules with them. The properties of the solvent are very important. If you want to separate hydrophilic (water-loving) molecules, you will need a water-based solvent. If you want to move hydrophobic (water-fearing) molecules, you will need an organic solvent. You may also use a mixture of solvents to take advantage of both properties, as in the case of the paper chromatography example.

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Introductory Chromatography: Colored Markers

This exercise is excellent for middle-school level classrooms that do not have access to the necessary scientific equipment to perform the organic extractions required in the next section. This exercise does not use highlydangerous organic solvents and is much more palatable for those students who may object to handling insects (as is necessary in the advanced exercise). Materials "Crayola" brand eight-pack of "classic" washable colored markers bottle of 70% iso-propyl alcohol (found in most drug stores) white vinegar wide mouth glass jar (such as a pickle jar) pencil heavy-grade white construction paper (best if as heavy as possible with a consistent surface). You might also want to experiment with different types of paper. Methods Cut construction paper in 6-inch by 3-inch pieces. With a pencil, draw a line across the bottom of the paper (3" side), about 1cm from the bottom. On this line, place individual dots with the various color markers. It is important not to place too much ink on the paper to prevent smearing. A quick dot with the normal pressure necessary to write with the marker is adequate. Pour isopropyl alcohol and vinegar into jar in a 40:1 ratio (e.g. 20ml alcohol and .5ml vinegar). Ensure that the liquid level will be below the pencil line on the paper. Stand the paper in the jar, spot side down, and allow the solvent to run up through the matrix for approximately 15 minutes or until the pigments have separated. Results

Paper chromatography of colored marker ink. The three primary colors (blue, red and yellow) will separate very well in this system. The compound colors will also separate into primary colors, with some smearing.

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Advanced Chromatography: Plant and Insect Pigments

Introduction This is a more advanced project for high-school level students. Special facilities and equipment found in most high-school teaching labs, are required because dangerous organic solvents are used. In addition, there will be new skills introduced in this project. In this project, a separation between fat and water soluble pigments will be made, and the fat soluble portion will be separated into individual pigments. This will be done for both plant and insect pigments. In addition, the selectivity of pigment by the insect will be determined. Materials one medium flowering purple petunia plant per insect used chloroform methanol di-ethyl-ether hexane glacial acetic acid silica TLC (thin-layer-chromotography) plates (si250 from JT Baker, obtainable through VWR), (If TLC plates are cost prohibitive, chromatography paper, such as Whatman 3MM, can be substituted, but will not yield as clear of a result.) deionized water, a homogenization system (see "Plant Processing", below), several glass 15 ml glass tubes with caps, a centrifuge, (obtainalble through VWR or EDVOTEK) a fume hood. Methods Feeding on plants/artificial diet A group of Manducas should be fed on the artificial diet according to the instructions provided on the Manduca Project website (http://manducaproject.com). For green Manducas, insects should be fed on petunia plants. If these are unavailable, other plants such as tomato, tobacco, capsicum, potato, or datura can be used. It is important to find plants that have NOT been treated with pesticide. Many of the larger chain stores with plant nurseries have treated plants. If you can find a nursery that has control of spraying insecticide, you will have much better luck. Many pesticides, such as BT toxin, are very difficultif not impossibleto wash off. The pesticides would kill the insect you want to raise. Manducas to be plant-fed, can be fed on artificial diet until the 4th instar and then transferred to a plant. One final word of caution: because a fifth instar Manduca can eat a petunia plant in 24 to 48 hours, it is a good idea to limit the number of insects fed on plants so you won't need to have a forest growing in your classroom. Plant processing The flowers and leaves of the plant have to be processed separately. Take one flower and homogenize in a glass homogenizer, teflon and glass homogenizer, mortar and pestle, polytron, or whatever suitable homogenization system is available to you. Place the macerated material into a glass tube. Add 2 ml of water

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and mix well. Repeat the same procedure with one petunia leaf, placing in a separate tube. Centrifuge these tubes at 500xg for 5 minutes. Place the supernatants in new glass tubes and discard the pellet. Insects processing Chilling Manducas on ice Place as many insects as you wish to bleed in a container with crushed ice. This will anesthetize the insects. It will also make them more manageable as they will not move during the procedure. The bleeds for the "green" and "blue" insects should proceed separately.

Folding Manduca Place approximately 2 ml of 150mM NaCl in a small (50ml) beaker for each 10 insects to be bled. After the insects have been chilled for five minutes, fold each insect into a "U" shape between two fingers, so that the legs face the outside of the curve.

Bleeding of insect With the other hand, make a small shallow cut with fine scissors into each of the prolegs at the bottom of the insect and allow the hemolymph to drain into the beaker. Be careful not to cut too deep or squeeze the insect too hard as that will cause the midgut to rupture, releasing its contents through the incisions into the beaker. A fourday fifth instar Manduca will yield approximately 1 ml of hemolymph. After collecting, place 2 ml of hemolymph in a glass tube. It is important to quickly move onto the lipid extraction phase of the procedure from this point. The insects have a natural defense mechanism in their hemolymph, which will result in the hemolymph turning black. Once the lipids have been extracted, this will not be a problem. After performing this operation, euthanize the insect in the freezer.

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Lipid extractions The following procedures should be performed in a fume hood. Into each tube (petunia flowers, petunia leaves, blue hemolymph, and green hemolymph) place 2 ml of methanol and 4 ml of chloroform. Cap the tubes and mix very well. Allow the tubes to sit for 5 minutes at room temperature and centrifuge at 500xg for 5 minutes. A clear phase separation should be present in each tube. With a long Pasteur pipette, carefully remove the bottom (organic) phase which containts the fat soluble pigments of each tube and place into a new tube. The upper (aqueous) layer contains the water soluble pigments. Be careful not to get any of the aqueous phase into the tube. (It is better to leave some of the organic phase in the tube rather than to try to obtain all of it and contaminate it with aqueous material).

Expected Results The initial separation of the petunia flowers should result in a purple upper aqueous phase and a slightly yellow lower organic phase. The leaf extract will give a mostly clear upper phase and green lower phase. Both hemolymph samples will both give clear upper and yellow lower phases, with the green hemolymph producing a greater amount of yellow pigment. TLC (Thin-Layer Chromatography)

Chromatography chamber set up Place a glass chromatography chamber in a fume hood. In the chamber, place 60 ml of di-ethylether, 40 ml of hexane, and 1 ml of glacial acetic acid. Mix these solvents and place a square piece of filter paper in the chamber. Cover the chamber and allow the solvent to saturate the paper.

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TLC plate spotting With a pencil, draw a light line about 1 cm from the bottom of the TLC plate. At even intervals, mark places on the line where you wish to place your samples. Carefully place the extracted organic phases on the line. It is important to keep the spots as small as possible. A little patience goes a long way here. Also, the more you are able to spot, the more easily you will see the pigments migrate. This spotting is most easily performed with microcapillary pipets, although any small pipetting device will work. Running of TLC After spotting, place the plate in the chamber, spot side down. Allow the solvent to migrate to about 75% to the top of the plate. After the migration, remove the plate and allow all of the solvent to evaporate before removing the plate from the hood. If you wish to store the plate for a long period, it is best to place it in an airtight bag and keep it in a dark place. The pigments are easily broken down with light and air.

Expected Results The TLC will show a single slight yellow pigment from flowers, and numerous yellow, green and black pigments from the leaf extract. The blue hemolymph extract will give a single faintif not indiscerniblespot. The green hemolymph will give a single bright yellow spot. Arrows point to areas with yellow pigments.

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Discussion All of the yellow and green pigments from the sources used here are fat soluble and will migrate with the organic phase of the extraction. The purple pigment from flowers is water soluble and will extract with the aqueous phase. Manducas only absorb a single yellow pigment, lutein, from plants. All of the other pigments in the leaf are unable to be absorbed, as well as the purple pigment from petunia flowers. This provides support for the conclusion that it is the yellow pigment lutein which gives plant-fed Manducas their green color. #####

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