Title

An Investigation of Light-Dependent Electron Transport using DCPIP (2,6 dichlorophenol-

indophenol).

Introduction
Photosynthesis is a chemical process whereby plants take in carbon dioxide and water
from their surroundings and use light energy from Sun to convert these into carbohydrates,
sugars and etc. Photosynthesis takes place inside the chloroplast and is categorised into two
processes; in light dependent reactions, light energy is necessary, in light independent
reactions, light energy is not needed.

Light dependent reactions occur when chlorophyll assimilate sunlight and convert it
to chemical energy which is utilized to separate water to give out oxygen, protons and a
reduced electron acceptor. The protons are utilized to make ATP from ADP during
chemiosmosis, the reduced electron acceptor is used to produce NADPH through the electron
transport chain.

The aim of this experiment is to investigate photosynthetic electron transport, using

isolated chloroplasts from the silverbeet leaves. DCPIP is used to determine if photosynthesis
occurs. Electrons produced during light reactions will be accepted by DCPIP, DCPIP is
reduced and changes colour from blue to colourless. The hypothesis of this experiment is the
faster the rate of photosynthesis, the faster the DCPIP will change to colourless and the more
rapid is the decrease in absorbance when measured with spectrophotometer.

Methods

“Seven spectrophotometer tubes were numbered and solutions A-D were added
according to the volumes shown in Table 1. Tube 1 was capped and inverted several times.
The spectrophotometer was calibrated using Tube 1, which contained chloroplasts and
sucrose only, as the blank, to ensure that any changes in colour for the other treatments could
be attributed to the reduction of the dye DCPIP. At time zero (mins), absorbance was
recorded for all treatments immediately after addition of DCPIP and mixing of contents.
Immediately following the time zero reading, all tubes (1-7) were placed in larger plastic
tubes: tube 2 in a light-proof (black) tube, and tubes 6 and 7 in tubes covered in red and green
cellophane respectively. All tubes were then placed horizontally on ice, under lights. At
fifteen minutes intervals, readings of absorbance were taken for all treatments, except for the
dark tube which was kept in the light-proof tube for 60 mins, after which its absorbance was
measured.

Results
Table 1. Absorbance readings taken at 15 minutes intervals for each tube.

  ABSORBANCE
Time (mins) DARK LIGHT BOILED DCMU RED GREEN
Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7
0 1.40 1.47 1.29 1.18 1.26 1.31
15   1.20 1.27 1.18 1.22 1.32
30   1.14 1.26 1.16 1.19 1.28
45   1.08 1.25 1.14 1.17 1.25
60 1.85 1.02 1.22 1.11 1.14 1.22

Figure 1. Graph of Absorbance against Time for each tube

Graph of Absorbance against Time

1.80

1.60
Absorbance

1.40

1.20

1.00

0.80

0.60
0 15 30 45 60

Time (mins)

Tube 3 Tube 4 Tube 5 Tube 6 Tube 2 Tube 7

Description of the outcome for each tube based on Figure 1:

 Tube 2 has an increase in absorbance value, it was covered with aluminium foil.
 Tube 3 has a significant drop from 0-15mins, then a continuous decrease under light
condition.
 Tube 4 has a very slight drop in absorbance value throughout the experiment.
  Tube 5 has the same absorbance value from 0-15mins and a very slight drop from 30-
60mins
 Tube 6 has a steady drop in absorbance value
 Tube 7 has an increase in 0.01 absorbance value from 0-30mins then it drops
considerably until the end of the experiment

Discussion

Tube 1 acted as a control (blank solution), this was to calibrate the absorbance value
of DCPIP in the spectrophotometer and to minimize the errors when DCPIP is measured.

Tube 2 which was covered with aluminium foil prevents light energy to reach the
chloroplast. However, light can still penetrate the chloroplast when the solution was poured
out of the test tube to measure its absorbance then poured back into it again. Light reaction
occurred indicates that electrons were accepted by the chlorophyll pigments, as confirmed by
DCPIP test (changed to colourless), this caused the increase in absorbance value.

Tube 3 was placed under light allows chlorophyll pigments to absorb light for light
reaction to occur. This caused the significant drop in absorbance value as many water
molecules broke down to produce many electrons. The colour change of DCPIP from blue to
colourless confirmed that photosynthesis had occurred. Hence, the rate of photosynthesis in
tube 3 is proportional to the amount of light absorbed and inversely proportional to the
absorbance.

The chlorophyll pigments in Tube 4 has denatured as the chloroplast was boiled. The
slight drop in absorbance value shows that very little light reaction has taken place. It was
expected to achieve a constant absorbance value as the chlorophyll pigments have been
denatured at high temperatures, hence, no light can be absorbed, photosynthesis cannot take
place. Therefore, there will not be any colour changes on DCPIP. The results obtained
showed that photosynthesis had occurred, this is due to some chloroplast pigments were not
completely denatured and still active.

Tube 5 contained DCMU which acts an electron transport chain (ETC) inhibitor
shows a very slight drop in absorbance value as DCMU prevents the release of electron to the
ETC. Therefore, the light reaction happened at a very slow rate resulting in a very slow rate
of photosynthesis.
 Tube 6 demonstrated a steady drop in absorbance value due to other lights from the
visible spectrum had penetrated when the solution was poured out of the tube to measure its
absorbance. This is a source of error that had occurred. It was expected to have a drastic drop
in absorbance value and a speedy rate of photosynthesis as photosynthesis occur best under
visible red light, more electrons would have been released through the water splitting process
to restore oxidised PS(II).

Tube 7 which was covered with green plastic has a steady considerable and steady
drop in absorbance value as chlorophyll pigments prefer red or blue light. Therefore, green
light was not fully absorbed.