Chromatography. Investigation Photosynthetic Pigments. Do all leaves contain the same pigments?

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1 Investigation Photosynthetic Pigments Materials For Group of 2 - Fresh spinach leaves - Wet erase marker - Chromatography paper - 2 Vials with caps - Scissors - Developer solution (Lighter fluid + Acetone, 8:1) - Water - Ruler - Penny - Pencil Cap Glass Vial Chromatography Paper Sample Developer (Solvent) Figure 1: Experimental Setup Name Per. Chromatography Do all leaves contain the same pigments? Introduction Chlorophyll (green), the primary pigment of photosynthesis, often hides or masks the other accessory pigments present in leaves. In autumn, chlorophyll breaks down, allowing carotene (orange), xanthophyll (yellow), and newly made anthocyanin (red), to show their colors. The mixture of pigments in a leaf may be separated into streaks of color by the technique of paper chromatography. Chromatography means color writing. With this technique the components of a mixture in a liquid medium, are separated. The separation takes place by capillarity, which pulls the substances up the paper at different rates depending on their molecular weight (size), causing them to show up as colored streaks. The resulting pattern of separated components on the paper is called a chromatogram, from which the rate of flow ( ) of each component may be calculated. Prelab A. List the names of the various plant pigments on the attached data table in the following order, from top to bottom: carotene (orange), xanthophyll (yellow), chlorophyll b (yellow-green), and chlorophyll a (blue-green). B. In the text or your notes, review a diagram of a plant cell and chloroplast. Find the grana (stacks of thylakoids) in the chloroplast diagram. Answer the following as specifically as possible: (Hint: This series of questions are related to each other and the purpose is for you to show the connection!) 1) Where are plant pigments like chlorophyll located? 2) What is the function of the thylakoid membrane? 3) How does the location of pigment relate to its function? Procedure Each team of 2 will run two different chromatograms; one leaf and one ink. A. Experimental Setup (see Figure 1): Cut two lengths of chromatography paper so that they fit inside the vials, but do not put them in. Cut a triangular point at one end of each strip. Pipet 1ml of chromatography solvent into one vial and 1 ml of water (solvent) into the other. Replace the caps on both vials and the chromatography solvent bottle. DO NOT PLACE THE PAPERS INTO THE VIALS YET!

2 B. Make a sample spot about one centimeter from the pointed tip of one filter paper using a green leaf and a penny. Turn the leaf upside down over the filter paper and use the penny to press the juice from the leaf onto the filter paper. You will obtain best results if you make the spot as small and concentrated as possible by going over it many times, moving the leaf slightly for each press of the penny. Do the same with black wet-erase ink on the other strip. 4) Explain the importance of making the pigment spot be as concentrated as possible? C. Carefully place the chromatography papers inside the appropriate vials (leaf to solvent, ink to water) so the pointed tip just touches the developer. The sample spot must NEVER enter the developer!! Replace the vial caps and watch the developer rise up the paper by capillary action, carrying and separating pigments. Allow the solvent to come within one centimeter of the top of the paper, then remove it and quickly mark the solvent front with a pencil. Allow the paper to dry and observe the streaks of pigment. Identify, circle and label the pigment streaks. They will fade and not be visible in a short while. 5) Recreate your spinach results on the attached Leaf Results page. 6) Recreate another team s results on the Leaf Results page. D. Each pigment has a rate of flow ( ), the speed at which it moves up the paper relative to the speed at which the solvent moves up the paper. It is a decimal ratio: E. For each chromatogram, measure the distance in cm from the sample spot to the farthest edge of each pigment (pigment front), then measure the distance traveled by the solvent from the sample spot to the solvent front. 7) Enter your measurements into the data table. 8) Calculate the value as a decimal for each pigment, and record them in the data table. Analysis 9) Copy the ink results of the other team onto your table and compare. Do both chromatograms show the same number and color of pigments in the same order? (they should) EXPLAIN why! 10) Are the values close to the same for each color? (should be) EXPLAIN why! 11) Copy the leaf results of the other team onto your table and compare. Do both chromatograms show the same number of pigments in the same order? (they should) EXPLAIN why! 12) Which pigment is most visible (darkest) in the spinach chromatogram? This is an indication that there is a greater quantity of that pigment in the leaf. Is this pigment most visible in the leaf s original color (green)? (should be) EXPLAIN why! 13) Which pigment is least visible in the spinach chromatograms? Is this pigment obvious in the leaf s original color? (shouldn t be) EXPLAIN why!

3 Data Table Species Color Pigment Name (Top to bottom) Pigment Distance Solvent Distance Value (Yours) GREEN COMPARE! Note: List the 4 pigment names in the same order they appear on your chromatogram from top to bottom. (Other Team s) GREEN Your Ink BLACK COMPARE! Other s Ink Note: The pigment names are the names of the 3 distinct, primary colors. List them in the same order as on your chromatogram from top to bottom. BLACK

4 Leaf Results Species Your Results Species Other s Results Use colored pencils, markers or crayons to diagram your results above. First, use a ruler to draw the two chromatography paper strips to scale. Then, Label the sample start, solvent front and each pigment with its scientific name and value.

5 Paper Chromatography Chromatography - A technique for separating molecules from solution. Paper Chromatography - separates molecules according to size and molecular weight by the capillary movement of liquid developer along a strip of paper. Larger, heavier molecules have a slower rate of flow ( ) than smaller, lighter ones. C A E Maple Solvent Front Pigment Front for A & B Sample Start Solvent Developer The distance the molecule traveled The distance the solvent traveled Practice Calculate the rate of flow ( ) values for the pigments in the figure above. ALL measurements should be made from the sample starting point to the maximum distance traveled by each pigment (pigment front) and the distance traveled by the solvent (solvent front). D B F Oak This figure represents two chromatographs of pigments from maple and oak leaves. Three pigments are evident on each, but only one of those pigments are shared by both leaves. Identification of known substances is possible because the same pigment molecules will always have the same value.