Paper Chromatography Essay, Research Paper

Paper Chromatography Introduction: Paper chromatography is used to separate and identify pigments and other molecules from cell extracts that contain complex mixtures of molecules. In 1903 Michael Twsett invented this technique. In 1944 Martin and Synge, who went on to win the Nobel Prize in 1954 for their work, perfected it. Paper chromatography was used by Melvin Calvin to separate the order of photosynthesis, and Fred Zanger to find out how to make insulin by determining what orders to put the amino acids in. Trial 1 (Using paper chromatography to separate colors in a marker or dye): Hypothesis The colors in the black marker or dye will separate into different bands across the chromatography paper. The green dye will not have as many separate colors as the black marker. Procedure 1. Obtain a piece of chromatography paper and make a pencil line about 1.5cm from the bottom of the paper. Draw a line with the marker (or dye) over the pencil line.2. Staple the paper into a roll so that it will stand by itself.3. Put about 1cm of water into the bottom of a glass container.4. Put the rolled paper, marker line side down, into the glass container so that it does not touch the sides of the glass container and the marker does not touch the water.5. Wait until the water has moved to about 1cm from the top of the paper and remove the paper from the container.6. While the paper is wet, and the colors are still bright, draw a pencil line separating each color.7. Measure how far each color traveled to determine Rf values. Results Band Number Distance (mm) Band Color Rf Values1. 5 Yellow .112. 10 Orange/Yellow .213. 23 Orange .494. 34 Pink .725. 38 Light Pink .816. 47 Blue/Black 1 Trial 2 (Using paper chromatography to separate pigments in a spinach leaf): Hypothesis The colored pigments in the spinach leaf will separate in colored bands across the paper similar to the way the colors in the black marker separated. The trial using the homemade extract will show the separation of pigments better than the trial where the spinach leaf is rubbed onto the paper using a quarter. Procedure (When using the homemade extract) 1. Make the extract:a. Obtain 1g of spinach leaves and grind them into a paste using a mortar and pestle.b. Transfer the paste into a test tube.c. Add 4ml of Acetone to the mortar and pestle to rinse it out and then pour it into the test tube.d. Cork the test tube and shake it hard for 10sec.e. Let the test tube and mixture set for 10min.f. Add 4ml of water to the test tube and shake for 10sec.g. Add 4ml of petroleum ether to the test tube and shake for 10sec.h. Allow the test tube and mixture to set until a thin line forms of the top of the mixture. This is the extract.2. Obtain a piece of chromatography paper. Draw a line in pencil about 1.5cm from the bottom of the paper.3. Wash the paper strip by wetting it from bottom to top in petroleum ether:acetone solvent (1 part acetone, 9 parts petroleum ether) in a petri dish. Set the paper aside to dry.

4. Pour the petroleum ether:acetone into a glass container and put the lid on.5. Using a capillary pipet spread the plant extract in the test tube across the pencil line on the chromatography paper. You may have to go over the line several times.6. Staple the paper into a roll so that it will stand by itself.7. Put the paper into the glass container making sure that it does not touch the sides.8. Wait until the solvent in about 1cm from the top of the paper and then remove the paper from the glass container.9. While the paper is wet, and the pigments are still visible, draw a pencil line separating each color.10. Measure how far each pigment traveled to determine the Rf values.Procedure (When rubbing the spinach leaf onto the chromatography paper using a quarter) 1. Follow steps 2-4 from above.2. Using a quarter, rub the spinach leaf onto the chromatography paper over the pencil line. You may have to go over the line several times.3. Follow steps 6-10 from above. Results Band Number Distance (mm) Band Color Pigment Rf Values1. 64 Orange Carotene 12. 12 Yellow Xanthophyll .193. 10 Blue Green Chlorophyll a .164. 8 Olive Green Chlorophyll b .13 1. What factors are involved in the separation of the pigments? Factors that are involved in the separation of the pigments are capillary action, the solubility of the pigment in a solvent, and the hydrogen bonds in the pigment. The greater the solubility of the pigment, the farther up the paper it will go. The larger number of hydrogen bonds or the greater the attraction for the paper the pigment has, the lower it will stay on the paper. 2. Would you expect the Rf value of a pigment to be the same if a different solvent were used? Explain. No, I wouldn t expect the Rf value of a pigment to be the same if a different solvent were used. The Rf values depend of how far the pigments travel up the paper, and how far the pigments travel up the paper depends on part by how soluble the pigment is. So, if a pigment weren t as soluble in one solvent as in another then the Rf values for the pigment in the two solvents would be different. 3. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments? The reaction center contains chlorophyll a. Chlorophyll b absorbs red light energy, carotene reflects orange light energy and absorbs blue light energy, and xanthophyll reflects yellow light energy and absorbs blue/purple light energy. These other pigments filter light energy to the chlorophyll a. Conclusion: Do to shortening days, and less energy due to less sunlight, the chlorophyll in plant leaves breaks down, becomes clear and allows the other pigments to begin to show through. The red coloring of the leaves comes from the broken down chlorophyll in the plant vacuole. Sunlight turns the chlorophyll into anthocyanims, a water-soluble pigment. The leaf will be red if in acid water, and blue/purple if in basic water. After the chlorophyll in a leaf is gone, the leaves will loose 99% of their water. The trees then drop their leaves and make an abscission layer of cork to close the xylem tubes which carry water to/from leaves.

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