Spinach Lab

The overall experiment consisted of three smaller experiments as a result of the different properties chlorophylls possess and the need to test them individually to receive the most accurate results. Preparation for the lab began with selecting a medium to large spinach leaf and deveining it. Once the spinach was prepared, it was ground in 5 mL of 100% acetone, creating a green acetone extract. The extract was filtered through a funnel with a kimwipe and into a test tube. The volume of the filtered extract was estimated, and one half of the estimated volume of petroleum ether was added to the solution, along with two drops of water. The test tube was then inverted to ensure it was properly mixed, and the phases could separate.
Experiment 1
To prepare the solution, 4 mL of 80% acetone was pipetted into a test tube, and five drops of the top layer of the pigment extract from the first experiment was combined with the acetone. The test tube was inverted until thoroughly mixed. Then a test tube containing 4 mL of 80% acetone was created to blank the spectrophotometer at 380 nm. The absorbance of the tube with the extract and acetone solution was then tested for its absorbency. These tests were repeated with the same solution at 40 nm intervals, until the wavelength reached 740 nm, re-blanking the machine between each test or change in wavelength. All the absorbencies were recorded, and the areas with the highest absorbance were retested at 5 nm intervals. The results were then plotted on Excel to show the relationship between certain wavelengths and the absorbance of the
First, the spectrophotometer was set to a wavelength of 605 nm. Then15 mL of pre-prepared chloroplast membrane suspension was obtained, and kept cold in a beaker containing ice to keep the chloroplast fragments from degrading. Then DCIP was retrieved in a foil wrapped test tube to keep any form of light out. Also, 15 mL of distilled water was obtained along with 7 test tubes; labeled blank and numbers 1-6. A blank was prepared by combining 5 mL of distilled water and 2.5 ml of chloroplast membrane suspension, mixing it well and producing a consistent green color. Tube 1 was set as a control for experiment three by combining .5 mL of DCIP and 2.5 mL of water. The absorbance of the first test tube was recorded initially at 0 minutes, and then placed under a light for one minute. Then the absorbance was tested again. and this process was repeated until there were five total readings. The second tube was used as a positive control against white light. The solution was produced by combining .5 mL of DCIP with 2.5 mL of CMS. Like the blank, this tube was screened for its original absorbance, and then exposed to white light for one minute until five readings were obtained. The third test tube was a negative control with no exposure to light. The tube was wrapped in a foiling casing and cap, and contained the same solution as test tube 2, .5 mL of DCIP and 2.5 mL of CMS. The