How Does Blackman Uses Direct Light To Trap Radiant Energy

In 1905, Frederick Blackman, a British physiologist, concluded that photosynthesis, the capture of light and conversion of it into chemical energy is a two-way process, of which only one uses direct light to trap radiant energy. In the Hill Reaction activity, our group will be focusing directly on the light dependent reaction. The overall purpose of this experiment is to try to prove the hypothesis that this reaction does produce compounds that have the energy of light. The first of the two major reactions is referred to as the light reactions because they require light and are light dependent. These reactions are responsible for trapping radiant energy. The second reaction is concerned with using energy (light not required) captured by light
Isolation of chloroplasts
• Blend 50g of spinach with 150ml 0.4M NaCL solution
• Filter, then divide cell suspension into 4 centrifuge tubes
• Centrifuge at 100 X g for 3 minutes
• Decant into additional centrifuge tubes and discard the pellet at the bottom
• Centrifuge for 10 minutes at 1400 X g
• Discard supernatant, you now have chloroplast
• Resuspend chloroplasts pellet in 20mL 0.05M NaCl solution
• Centrifuge again with same parameters
• Discard liquid and resuspend pellet in 20mL of 0.5M sodium phosphate buffer at pH 6.4
Part B. The reducing power of chloroplasts
• Label 9 cuvettes. Pipette 4mL and 1mL DCPIP in each.
• Making blank, pipette 1mL chloroplast and add ascorbic acid
• Set spectrophotometer to 595nm
• Pipette 1mL chloroplast into each cuvette, make sure light is maximized
• Remove foil, determine absorbance at time zero. Place in front of light at lower intensity.
• Take absorbance for same tube at 2min intervals for 8min or until 0.5 ABS
• Repeat at each light intensity, 100. Expose each cuvette for 4 min or until 0.5ABS
• Using molar extinction coefficient 0.014, calculate rate of reduction DCPIP per chloroplast. Plot (electron transfer) per minute vs light intensity.

Results:
This resulted in light intensity to increase as the distance from the light source decreased. The purpose of placing them at different distances was to investigate the effect of light intensity on the rate of the Hill reaction. It is expected that the DCPIP at each light intensity will reduce at different rates (Figure 1). (Figure 2) shows the rate of electrons being transferred vs. the rate of photosynthesis. It is expected that the greater the light intensity, the higher the rate of electrons being transferred, and the higher the rate of photosynthesis. This higher rate of electrons transfers results in a much faster reduction of DCPIP which makes the darker green solution turn more clear, while also decreasing its absorbance. As light intensity goes up, the metabolic activity of the chloroplast goes up and the more electrons its gives up. DCPIP absorbs these electrons and the absorbance of the DCPIP decreases. The number of electrons absorbed is measured by taking the absorbance of the solutions. Chlorophyll electrons are going to absorb light energy and than transfer the energy through redox reactions until it gets to NADP which is the final electron acceptor. Water will then split and produce oxygen electrons as well as hydrogen ions. This DCPIP is supposed to be a replacement for the NAD+ and is the artificial electron acceptor we use. As DCPIP