LabBench Activity

Design of the Experiment II

Pigments absorb in the visible range of the spectrum (380–760 nm). After illuminating the reaction tubes as shown below, we will use the spectrophotometer to measure the percentage of transmittance at wavelength 605 nm.

Flowchart of the experiment

If DPIP is in an oxidized state, it will be blue, and the percentage of light transmitted will be low. If, on the other hand, chlorophyll's electrons have been excited and move on to reduce the DPIP, the sample will become progressively paler, allowing more light energy to pass through the sample. We can measure this change over time until the sample has been completely reduced, is almost colorless, and the percentage of transmittance is very high.

For this experiment, one tube (the blank) will contain all the solutions used in the reaction except the DPIP. Since the blank contains chloroplasts, it will be green; you will use this tube to calibrate the machine. The other tubes will be experimental, and will contain either boiled or unboiled chloroplasts.

pipette Helpful Hints

  1. The first reading provides a baseline. Be sure you read the % transmittance immediately after adding chloroplasts to each experimental tube.
  2. Keep the test tube glass clean! Use a Kimwipe or lens paper to remove your fingerprints.
  3. Thoroughly mix each sample before you measure it.
  4. Be sure to place all the tubes into the spectrophotometer in the same orientation each time so variations in the glass don't mask your results.
  5. Maintain a constant distance from the light source to the sample for all the tubes.