What Causes Dcip to Lose It's Color?

What Causes DCIP to Lose Its Color?
Chloe Gearhart
2.23.2012
Concepts Lab

Introduction:
Plants consume light for energy by converting carbon dioxide and water into glucose and oxygen, known as a process called photosynthesis. By adding hydrogen ions to carbon dioxide C-H bonds are made that hold energy which is released in mitochondrion, an organelle. This process of the release of energy is known as respiration. Without respiration, photosynthesis would not be able to perform. The process of photosynthesis is able to perform its duties with the help of an organelle called chloroplast as well. Within the chloroplasts lie thylakoids, a membrane system that contains pigments which are used to capture light energy. Photosynthesis is also responsible for reducing carbon dioxide into glucose, because reducing power is present. Reducing power is made after electrons get excited in the chlorophyll molecule. To carry out the process of reducing carbon dioxide into glucose, hydrogen ions must be added to the carbon of carbon dioxide where ATP and NADPH (reducing power) are used to convert carbon dioxide to sugar, all of which takes place in the chloroplast. The part of photosynthesis that converts carbon dioxide to sugar is known as Calvin Cycle of Dark reaction. For this lab, we will test the process of photosynthesis by using Spinach leaves to observe the Hill Reaction. The Hill Reaction tests water splitting in photosynthesis, and can be observed when a color change occurs. This color change will occur when a molecule is split and electrons are removed from molecular oxygen and hydrogen ions. DCIP combined with sucrose or ethanol spinach mixtures, will give either a green color (indicating photosynthesis occurred) or no color at all (indicating no photosynthesis occurred), depending on the factors and environments to which the mixtures will be tested.
In experiment one, membrane integrity of the spinach was tested. If the DCIP was mixed with either the sucrose or ethanol and turned green it means the photosynthesis process worked, but if it stayed blue, photosynthesis was unable to perform. Knowing that sucrose is only a polar molecule because it dissolves in water, and polar molecules are hydrophilic, and ethanol can be both polar and nonpolar, I predict the solution with the sucrose will have a higher photosynthetic success. Sucrose is not hydrophobic; therefore the cells will stay intact rather than blowing up from excess water, making it easier for photosynthesis to work. In experiment two, temperature will be tested by placing one set of two test tubes (one with the ethanol and one with the sucrose solutions) in a cold water bath, and one set in room temperature. If the sucrose and ethanol test tubes were placed in an ice water bath and room temperature, then the test tubes with the solutions at room temperature will undergo the most photosynthesis because most photosynthetic processes occur at a temperature closer to room temperature than extreme cold conditions. The third experiment will test influence of the mixtures exposed to light versus no light on the photosynthetic success of sucrose and ethanol spinach leaf mixtures. If the sucrose and ethanol spinach mixtures are combined with DCIP solution, then the solutions exposed to no light will have a lighter/colorless product because the photosynthesis process functions better in light and the colorless solution proves the greater photosynthetic success. For the fourth experiment, influence of distance to light will be tested for photosynthetic success. Because photosynthesis works better with more exposure to light, the test tubes placed closer to the light will have a higher photosynthetic success.

Methods:
Experiment 1
We collected two test tubes and labeled them as: Ethanol and Sucrose. In the first test tube, Sucrose, we added 4mL of the DCIP solution, and then 4 drops of the ethanol spinach leaf extract. In the second tube, Ethanol, we added 4mL of the DCIP solution, then 4 drops of .5m sucrose spinach leaf extract. We gently swirled each test tube by hand and let sit for ten minutes. After these test tubes sat for the duration of the ten minutes, we drew conclusions and recorded our observations about the change that occurred in the solution.
Experiment 2
We collected four test tubes and labeled them as: Sucrose in Ice Water, Sucrose in Room Temperature, Ethanol in Ice Water, and Ethanol in Room Temperature. For all tubes we gently swirled the contents by hand before letting sit. In the first test tube, Sucrose in Ice Water, we added 4mL of DCIP and 4 drops of sucrose extract, then placed it in an ice water bath. We added the same contents to the second tube, Sucrose in Room Temperature, but let it sit at room temperature in a drying rack. For the third test tube, Ethanol in Ice Water, we added 4mL of DCIP and 4 drops of ethanol extract and placed it in an ice water bath. We added the same contents as tube three to the fourth test tube, Ethanol in Room Temperature, but placed the fourth tube on a drying rack at room temperature. After these test tubes sat for the duration of the ten minutes, we drew conclusions and recorded our observations about the change that occurred in the solution.
Experiment 3
We collected four test tubes and labeled them as: Sucrose Light, Sucrose Dark, Ethanol Light, and Ethanol Dark. For all tubes, we gently swirled the contents by hand before letting sit. For the first test tube, Sucrose Light, we added 4mL of DCIP and 4 drops of sucrose extract then placed that test tube about one inch away from the artificial light. We added the same contents to the second test tube, Sucrose Dark, but placed this tube in a dark cupboard. For the third test tube, Ethanol Light, we added 4mL of DCIP and 4 drops of ethanol extract then placed it about one inch in front of the artificial light. We added the same contents to the fourth test tube, Ethanol Dark, but placed this tube in a dark cupboard. After these test tubes sat for the duration of the ten minutes, we drew conclusions and recorded our observations about the change that occurred in the solution.
Experiment 4
We collected four test tubes and labeled them as: Sucrose 1, Sucrose 5, Ethanol 1, Ethanol 5. For all test tubes we gently swirled by hand before letting sit. In all the test tubes, we added the same contents as we did in experiment 3. For the first test tube, Sucrose 1, we placed it one inch away from the artificial light. For the second test tube, Sucrose 5, we placed it 5 inches away from the artificial light. For test tube three, Ethanol 1, we placed it one inch from the light, and for test tube four, Ethanol 5, we placed it 5 inches from the artificial light. After these test tubes sat for the duration of the ten minutes, we drew conclusions and recorded our observations about the change that occurred in the solution.
Observations/Results:
Experiment 1
Membrane Integrity | Sucrose | Ethanol | Observations | -turned a turquoise color | -remained blue |
This table shows observations of sucrose and ethanol solutions when mixed with the DCIP solution.
Experiment 2
Temperature: Ice Water vs. Room Temperature | Ethanol | Sucrose | Room Temperature | -Medium blue-Clear & transparent | -Light/lime green | Cold Temperature (Ice bath) | -Blue/green-Teal color | -Lime green/clear-Lots of evidence of photosynthesis |
This table shows observations sucrose and ethanol extracts mixed with DCIP solution in room temperature and in ice water.
Experiment 3
Light Exposure: Light vs. Dark

| Sucrose | Ethanol | Light | - more green than ethanol but same color as natural -lime green/clearish color | - stayed blue | Dark | -turned a slight green color*was exposed to light in room for one minute before put in dark | -stayed blue |
This table shows observations of sucrose and ethanol extracts mixed with DCIP solution exposed to light and not exposed to light.
Experiment 4
Light Exposure: One Inch Away vs. Five Inches Away Distance From Light | Sucrose | Ethanol | One Inch | -turned lime green | -stayed blue | Five Inches | -turned lime green | -stayed blue |
This table shows observations of sucrose and ethanol extracts mixed with DCIP solution placed one and five inches away from an artificial light.
Observations for slides under microscope
Sucrose Leaf Extract Ethanol Leaf Extract

This is a picture of the sucrose leaf extract This is a picture of the ethanol leaf extract under the microscope at 10x magnification. under the microscope at 10x magnification.

Discussion:
Throughout this lab, it became very clear the significance of different conditions for the optimal energy production from a photosynthetic reaction. Spinach chloroplasts are isolated in 0.5M sucrose rather than distilled water due to the significance of osmotic pressure. If the spinach were isolated in water, then the salt concentration in the chloroplasts would be higher, therefore cause water to exit the chloroplasts. The spinach solution prepared in the buffer produced a more translucent and clear DCIP solution after being exposed to light and undergoing the photosynthetic process. The spinach solution produced in the ethanol was less successful at reducing the DCIP solution and therefore less successful at undergoing the photosynthetic process. The spinach produced in ethanol produced a clear dark green color, while the spinach solution produced in the buffer, became a clear and light yellow/green color. This proved that electrons are taken from the water from the water during photosynthesis, which causes DCIP to gain electrons and reduce to its colorless form. The significance of SDS and ethanol are important for the success of photosynthetic reactions. SDS is a detergent that causes the cell membrane to further break down and the emulsification of lipid and proteins in the cell. The solution forms complexes within the lipids (cell membrane), which causes them to precipitate out of the solution. Therefore, causing the cell membrane to further break down, further prohibiting photosynthesis to occur. The ethanol solution dehydrates the cell membrane, because it is an alcohol. Therefore, the growth (or photosynthetic success) of the plant is stunted due to the effect of ethanol. This would support the stunted growth, or lack of photosynthetic reaction, when the spinach solutions were introduced to ethanol.