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