AP BIOLOGYLAB 4B
Photosynthesis
DPIP
When chloroplasts are exposed to light, electrons reduce NADP+ to NADPH
ELECTRON ACCEPTOR
• DPIP= substitute for NADP+
• DPIPox = BLUE
• DPIPre = colorless
SPECTROPHOTOMETER
• Measures % light transmitted
• If DPIP is oxidized (blue) % transmittance will be low
• If electrons are excited and move to reduce DPIP(colorless) % transmittance will increase
SET UP LIGHT SINK
Image modified from Holtzclaw & Holtzclaw Study Guide
Cover tube # 2 completely with foilMake a removable foil top
SET UP TUBES
Add Phosphate buffer, distilled water, & DPIP totubes as shown
WAIT TO ADD CHLOROPLASTS!
SPECTROPHOTOMETER
• BLANK has all ingredients except DPIP
• Use Blank to calibrate machine
• Spectrophotometer is not digital
• Slow to respond
• Adjust dial and WAIT!BE PATIENT!
ZERO MACHINE
• Place BLANK in chamber and close lid
• Slowly turn dial on front until window reads 100% (Wait for lag time!)
• 100% with blank
• 0% with no tube/door closed
• Add 3 drops of UNBOILED CHLOROPLASTS to tube #2
• Put Parafilm on top and gently mix
• Remove from foil sleeve and immediately put sample in chamber, read % transmittance, and record in lab book
• Remove sample from chamber and return to foil sleeve, cover and place in test tube rack under light
• Start timer
• Take and record readings at 5, 10, & 15 min
REPEAT WITH EACH TUBE
• You can run multiple timers
• Just make sure you stagger time points so you are not taking readings of multiple tubes at same time
• Use cuvette #1 (blank) occasionally to check and adjust the spectrophotometer to 100% transmittance
Image modified from Holtzclaw & Holtzclaw Study Guide