Before the spectrophotometric experiment began, the spectrophotometer was warmed up for about 15 minutes. A pH 6.5 buffer solution was also prepared from phosphoric acid and sodium phosphate. Powdered 2,6-dichlorophenol-indolphenol (DCPIP) was dissolved in this solution, and a 0.2mM DCPIP stock solution was produced. This stock solution was made available at the beginning of the procedure as well as a pure pH 6.5 phosphate buffer solution. Additionally, microfuge tubes (1.5mL volume) with spinach leaf extract and acetone were prepared during the same lab day and supplied to researchers for the second part of the experiment.
The DCPIP solution (approximately 20 mL) and the pH 6.5 buffer solution (approximately 20 mL) were transferred into two separate 50 mL beakers. The DCPIP solution (0.5 mL) and the pH buffer (4.5 mL) was volumetrically pipetted into a dry test tube, then transferred into a spectrophotometric cuvette. This cuvette solution was used as the first sample solution for the experiment. A blank solution was then prepared with 5.0 mL of pH 6.5 phosphate buffer and was set aside.
The spectrophotometer was reset and set to Absorbance mode with a wavelength of 625 nm and a blank transmittance. The blank solution was then cleaned with a Kim Wipe and inserted while the vertical line on the cuvette was aligned with the painted line on the sample holder. The 0A/100%T button was pressed and the spectrophotometer was blanked for the experiment. The first cuvette solution prepared was then cleaned with a Kim Wipe and inserted identically to the blank solution, and the Absorbance value was recorded from the reading on the spectrophotometer.
This procedure was performed iteratively with four more samples of varied compositions. The solutions were first pipetted into test tubes. Then, they were poured into spectrophotometric cuvettes. The solutions prepared were, in order: 1.0 mL of 0.2mM DCPIP with 4.0 mL of pH 6.5 buffer; 1.5 mL of 0.2mM DCPIP with 3.5 mL of pH 6.5 buffer; 2.0 mL of 0.2mM DCPIP with 4.0 mL of pH 6.5 buffer; and 2.5 mL of 0.2mM DCPIP with 2.5 mL of pH 6.5 buffer. After the spectrophotometer was prepared when the blank solution was inserted into the sample holder, each cuvette was cleaned and placed in order into the spectrophotometer, and the Absorbance value on the spectrophotometer was read and recorded. A standard curve for DCPIP in pH 6.5 phosphate buffer was created with the Absorbance values collected in the procedure and was reserved for the analysis of later experiments.
During the same day, the spinach leaf extract and acetone was poured from the tubes into one cuvette and was sealed with Parafilm. A blank solution was prepared with approximately 3 mL of acetone pipetted into a cuvette. This cuvette was sealed with Parafilm in between uses. The spectrophotometer wavelength was first adjusted to 375 nm. The blank solution of acetone was cleaned and inserted identically to the blank solution of DCPIP. After the machine was blanked, the blank solution was taken out and the solution of spinach leaf extract and acetone was cleaned and inserted into the sample holder. The Absorbance value was read from the spectrophotometer before the solution of spinach leaf extract and acetone was removed from the sample holder.
This process, including blanking the spectrophotometer, was performed iteratively at 25nm intervals, beginning at 400nm and increasing to 750 nm. An absorption spectrum for the spinach extract was graphed using the Absorbance values from the experiment before the equipment and reagents were cleaned and disposed.
Before the photosynthetic experiment was performed, the spectrophotometer was warmed up for about 15 minutes. Spinach leaves (15 g) were torn and blended with 100 mL of cold 0.2 M sucrose solution for one minute. The homogenate mixture was filtered through layers of cheesecloth into a cold beaker and distributed into two 50 mL cold centrifuge tubes. The tubes were placed in plastic holders and balanced in the centrifuge, which was centrifuged for 5 minutes at 3500 rpm. The resulting supernatant was decanted into a cold 50 mL beaker and pipetted into several 1.5 mL microfuge tubes. These microfuge tubes were sealed and centrifuged in a refrigerated microcentrifuge for 3 minutes at 12,000 rpm. While the centrifuge was performed, an experimental site was made with a gooseneck desk lamp that was plugged in and positioned approximately 30 cm away from an empty test tube rack. A black trifold board was also wrapped around the test tube rack.
After the centrifuge was completed, the microfuge tubes were extracted and kept in ice buckets with dark wrapping. The supernatant of the extract was decanted and discarded and the pellet was resuspended as 1.5 mL of cold 0.2 M sucrose was continuously pipetted into the tube with a Pasteur pipette. The resulting 1.5 mL suspension was transferred into a test tube. Then, 5.5 mL cold 0.2 M sucrose was added to the test tube, and the tube was stored in ice. Once the extract and spectrophotometer were ready for use, a blank solution was made from 2.0 mL pH 6.5 phosphate buffer and 1.0 mL of the suspended chloroplast. The blank solution was cleaned and inserted into the spectrophotometer, blanking the spectrophotometer, then was set aside.
Five cuvettes were filled with identical solutions of 2.0 mL pH 6.5 phosphate buffer, 2.0 mL 0.2 M DCPIP in pH 6.5 buffer solution, and 1.0 mL suspended chloroplast. The cuvettes were immediately stored in a dark chamber. Each cuvette was procedurally removed from the chamber, cleaned, and placed into the sample holder to take an initial Absorbance value. Then, the gooseneck desk lamp was turned on, and the cuvettes were either placed in a cylindrical filter made from different acetate filters and tape, black construction paper and tape, or no filter, and placed into the test tube rack. These cuvettes were removed, tested, and placed into cylindrical filters at 30-second intervals. In order, the initial Absorbance reading of the cuvette with no light filter was recorded; then the cuvette with a blue acetate filter; then the cuvette with a red acetate filter; then the cuvette with a green acetate filter; lastly, the cuvette with a cylindrical filter of black construction paper was recorded. Every cuvette was extracted from the test tube under the gooseneck light every 2.5 minutes, removed from a filter if it had one, cleaned, and placed into the spectrophotometer sample holder. The Absorbance reading was recorded before the cuvette was placed back into a filter if it had one and placed in the test tube rack. This process was repeated for each cuvette seven times, and each cuvette was studied over the course of 20 minutes.
Afterwards, the Absorbance values were converted to values for the current concentration of DCPIP (mM) using the standard curve of DCPIP in pH 6.5 phosphate buffer solution. The concentration values were plotted against the time elapsed in the experiment in a series of progress curves. The slope of the initial reaction rate was derived from each progress curve, which was plotted on a bar graph depicting the effect of different light treatments on the studied chloroplast suspensions.