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  1. Plant Pigment Chromatography
    • NOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments travel along the paper at different rates and may have different colors. By calculating the relative distance the pigments travel, their resolution factor, and comparing them with literature values, you can identify different pigments. HYPOTHESES: In this exercise the experimental hypothesis is that there will be multiple pigments within the spinach leaves that absorb different wavelengths of sunlight. The null hypothesis is that there is only one type of pigment within the spinach leaf.
    • Use a pencil to make a line two centimeters from one end of the chromatography paper.
    • Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
    • Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
    • Next, wrap the paper around the pipe cleaner so that the bottom edge is barely touching the bottom of the beaker and then secure it with a paperclip.
    • When the paper is secured around the pipe cleaner, remove it from the beaker, and then place a patted-dry spinach leaf over the marked line on the chromatography paper.
    • Roll a coin over the spinach leaf along the pencil line going back and forth multiple times and applying steady pressure. When the leaf is removed, a green line should be clearly present.
    • Next, place 8 mL of chromatography solvent in the beaker.
    • Lower the chromatography strip into the beaker so that the edge of the paper touches the solvent but the green line does not. Adjust the pipe cleaner if needed.
    • Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
    • When the solvent has traveled half way up the chromatography paper, which will take approximately 10 minutes, and the pigments have separated into well-defined bands, remove the paper from the beaker.
    • Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
    • Next, record the number of visible bands and describe their color and relative size.
    • Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1. Click Here to download Table 1
    • Dispose of the chromatography solvent in a waste container under a fume hood. Throw the chromatography strips into the regular trash, and then clean the beakers with soap and water.
  2. Floating Leaf Discs in a Vacuum
    • NOTE: In this experiment you will indirectly observe photosynthesis and cellular respiration using a floating leaf disc in a solution. During photosynthesis, air bubbles will cause the leaves to float, and during respiration, the discs will sink. HYPOTHESES: In this exercise, the experimental hypothesis is that the leaf discs will have a greater rate of photosynthesis in the bicarbonate solution, because bicarbonate provides added CO2 to fuel photosynthesis, causing more leaf discs to float. Additionally, all of the discs will sink in dark conditions as they perform cellular respiration. The null hypothesis is that there will be no difference in the rate of photosynthesis, and therefore the number of floating discs, between the bicarbonate and water, or light and dark treatments.
    • To place leaf discs under vacuum, first remove the plungers from two 20 mL syringes, and then place 10 leaf discs inside each syringe tube. Label one syringe “bicarbonate”, and label the other syringe “water”.
    • Replace the plungers and push the plunger until only a small amount of air remains in the syringe. Take care not to damage the leaf discs.
    • Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
    • Push as much air out as possible without expelling the solution or damaging the leaf discs.
    • Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
    • To create a vacuum, hold one finger over the tip of the syringe while pulling back on the plunger. Hold this for 10 seconds while swirling the syringe to keep the leaf discs in suspension.
    • Then, release the vacuum. NOTE: The discs should have absorbed the solution into the air spaces in their tissues and you should see them sink. If the discs don't sink, you can repeat the vacuum creation up to three times.
    • Next, add 50 mL of bicarbonate solution to a plastic cup or a glass beaker, and then gently add the discs from the bicarbonate vacuum syringe.
    • For the control, add the same amount of water to an identical cup, and then add the leaf discs from the water vacuum syringe. Label the containers appropriately.
    • Place both cups under a light source.
    • Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed. Click Here to download Table 3
    • Next, remove the cups from the light source and then swirl them so that the discs at the surface intermix with any gases also at the surface.
    • Move the cups to a dark place. Every five minutes record the number of leaf discs floating at the surface until 20 minutes have passed. Swirl the cup each time before placing it back in the dark.
    • To clean up, dispose of the leaf discs in the trash, and pour the bicarbonate solution down the drain. Wash the syringes and cups thoroughly.
  3. Results
    • NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
    • Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
    • Calculate the retention factor, or Rf values for the pigments, which is done by dividing the distance the pigment in question moved up the paper from the line by the distance the solvent moved up the paper from the line.
    • Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. Click Here to download Table 2
    • Record these data in Table 1. NOTE: In the second experiment, you observed floating and sinking leaf discs as an indirect measurement of photosynthesis and respiration.
    • Graph the results with time and minutes on the x-axis and number of floating discs on the y-axis. Use two different lines to represent the water control and the bicarbonate treatment.
    • Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
    • Next, starting with the bicarbonate condition, use the graph to determine the point at which 50% of the leaf discs were floating. This is referred to as the effective time, or ET50. NOTE: You will notice that the discs likely hit the 50% floating mark once in the light condition and then again in the dark condition.
    • Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
    • Finally, compare your ET50 values and graphs with the rest of the class.


JoVE Lab Lab: 7 Procedure

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