Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN: 9780134580999
Author: Elaine N. Marieb, Katja N. Hoehn
Publisher: PEARSON
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Question

If the pigments were found in the storma rather than in the thylakoid membrane how would this affect their movement on the tlc plate ?

Background and Procedure: Photosynthetic plants utilize a variety of light-absorbing pigments. Due to the large quantity and intensely colorful nature of the chlorophylls (green), under many circumstances it is not possible to notice the presence of the other pigments. Differences in chemical structure of these pigments result in differing amounts of polarity, allowing us to separate them from one another using Thin Layer Chromatography (TLC). Most of these pigments are found in membranes of the thylakoid and are predominantly hydrophobic. Therefore, we will use organic solvents, such as isopropyl alcohol ("rubbing alcohol"), as the solvent and petroleum ethers for the mobile phase or "developing solution" for the chromatography. 1. Weigh approximately 3-4 grams of fresh, dry spinach leaves with the stems completely removed. 2. Using a pair of scissors cut the leaves into very small pieces into the mortar. Using the pestle grind and pound the leaves into a mash. This will take several minutes and is an essential step, so don't rush it. 3. Add 10 mL of anhydrous isopropyl alcohol to the mortar and carefully, without splashing, continue to mix and crush the slurry for several more minutes. If some of the alcohol has evaporated you can add a couple more milliliters to maintain the original volume. Then place plastic wrap, a beaker, or other object over the top of the mortar to prevent evaporation of the alcohol. Wait five minutes. 4. Stir and crush the mixture for another thirty seconds. The liquid should be a dark green color. Use a transfer pipet to move the isopropyl-pigment solution into a glass test tube. Try to avoid the spinach solids. Keep in mind that this tube of pigment/alcohol solution will also be used for Activity #3. 5. Carefully cut the TLC plate lengthwise, to create two, more narrow strips. One half of the plate will be used per group. Using a ruler draw a thin line 1.5 cm from one end (this will be the bottom) using a standard pencil. 6. Using the glass capillary tube aspirate a small amount of the isopropyl solvent containing the leaf pigments. Carefully make a small dot on the center of the pencil line on the TLC plate. Allow approximately 1 minute to dry. 7. Repeat step 6 at least five times. You want a very dark and concentrated spot of pigments at one place on the thin pencil line. Be certain the spot is dry before continuing to the next step. 8. In a clean glass test tube add 0.75 mL of chromatography developing solution. Caution: this is a mixture of petroleum ethers. This solvent is highly flammable and mildly toxic. Be sure to dispose of this mixture properly (as outlined by your instructor) and take care with handling. 9. Carefully place your TLC strip into the test tube containing the developing solution. The dot containing the of pigments should NOT come in direct contact with the solution. Using a piece of parafilm seal the top the test tube and place in a test tube rack. 10. Observe the TLC plate for the next few minutes to watch the developing solution front move up the plate while the pigments separate. 11. When the developing solution is within 1 cm of the top of the plate remove it from the tube with a pair of forceps and place it on a paper towel to dry. Properly dispose of the remaining developing solution in the organic waste receptacle and dispose of your test tube in the glass waste. Documentation and Retardation Factor (R): Since the developing solution is extremely nonpolar, more hydrophobic pigments will travel easier with the solvent front nearer the top. The more polar (less hydrophobic) pigments will stick to the silica further down on the plate. 4 of 10
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Transcribed Image Text:Background and Procedure: Photosynthetic plants utilize a variety of light-absorbing pigments. Due to the large quantity and intensely colorful nature of the chlorophylls (green), under many circumstances it is not possible to notice the presence of the other pigments. Differences in chemical structure of these pigments result in differing amounts of polarity, allowing us to separate them from one another using Thin Layer Chromatography (TLC). Most of these pigments are found in membranes of the thylakoid and are predominantly hydrophobic. Therefore, we will use organic solvents, such as isopropyl alcohol ("rubbing alcohol"), as the solvent and petroleum ethers for the mobile phase or "developing solution" for the chromatography. 1. Weigh approximately 3-4 grams of fresh, dry spinach leaves with the stems completely removed. 2. Using a pair of scissors cut the leaves into very small pieces into the mortar. Using the pestle grind and pound the leaves into a mash. This will take several minutes and is an essential step, so don't rush it. 3. Add 10 mL of anhydrous isopropyl alcohol to the mortar and carefully, without splashing, continue to mix and crush the slurry for several more minutes. If some of the alcohol has evaporated you can add a couple more milliliters to maintain the original volume. Then place plastic wrap, a beaker, or other object over the top of the mortar to prevent evaporation of the alcohol. Wait five minutes. 4. Stir and crush the mixture for another thirty seconds. The liquid should be a dark green color. Use a transfer pipet to move the isopropyl-pigment solution into a glass test tube. Try to avoid the spinach solids. Keep in mind that this tube of pigment/alcohol solution will also be used for Activity #3. 5. Carefully cut the TLC plate lengthwise, to create two, more narrow strips. One half of the plate will be used per group. Using a ruler draw a thin line 1.5 cm from one end (this will be the bottom) using a standard pencil. 6. Using the glass capillary tube aspirate a small amount of the isopropyl solvent containing the leaf pigments. Carefully make a small dot on the center of the pencil line on the TLC plate. Allow approximately 1 minute to dry. 7. Repeat step 6 at least five times. You want a very dark and concentrated spot of pigments at one place on the thin pencil line. Be certain the spot is dry before continuing to the next step. 8. In a clean glass test tube add 0.75 mL of chromatography developing solution. Caution: this is a mixture of petroleum ethers. This solvent is highly flammable and mildly toxic. Be sure to dispose of this mixture properly (as outlined by your instructor) and take care with handling. 9. Carefully place your TLC strip into the test tube containing the developing solution. The dot containing the of pigments should NOT come in direct contact with the solution. Using a piece of parafilm seal the top the test tube and place in a test tube rack. 10. Observe the TLC plate for the next few minutes to watch the developing solution front move up the plate while the pigments separate. 11. When the developing solution is within 1 cm of the top of the plate remove it from the tube with a pair of forceps and place it on a paper towel to dry. Properly dispose of the remaining developing solution in the organic waste receptacle and dispose of your test tube in the glass waste. Documentation and Retardation Factor (R): Since the developing solution is extremely nonpolar, more hydrophobic pigments will travel easier with the solvent front nearer the top. The more polar (less hydrophobic) pigments will stick to the silica further down on the plate. 4 of 10
updated 7/19 yellow green dark en lighter Finis yellow ravels Light peen d name of pigment
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Transcribed Image Text:updated 7/19 yellow green dark en lighter Finis yellow ravels Light peen d name of pigment
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