Title

A study of Light-Dependent Electron Transport using DCPIP (2,6 dichlorophenol-

indophenol).

Introduction
Photosynthesis is a chemical change and process whereby plants absorb carbon
dioxide, water from the environment and utilize light energy from Sun to convert to
carbohydrates, sugars and etc. (Atkins. T. and Escudier. M. (2013)) Photosynthesis occur
inside the chloroplast and is categorised into two processes; light reactions and dark
reactions. Light dependent reactions (light reactions) require light energy while light
independent reactions (dark reactions) do not require light. (Johnson. M. P. (2016)).

Light dependent reactions occur when chlorophyll absorb sunlight and change it to
chemical energy, hydrolysis of water occurs and oxygen, protons, a reduced electron acceptor
are produced. During chemiosmosis, the protons are used to make ATP from ADP, the
reduced electron acceptor is used to produce NADPH through electron transport chain.

The aim of this experiment is to study the photosynthetic electron transport using
chloroplast from silverbeet leaves that has been kept in the dark by wrapping the tube
containing chloroplast suspension with aluminium foil. 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 speedier the rate of photosynthesis, the quicker the DCPIP will change to
colourless and more rapid is the declining in absorbance value 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 containing chloroplast
suspensions.

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 was a blank solution which acted as a control, this was to calibrate the
absorbance value of DCPIP and to minimize errors of measurements of DCPIP with
spectrophotometer.

Tube 2 which was wrapped with aluminium foil prevents light energy from reaching
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 has broken down and gave out many electrons. The colour change of DCPIP from
blue to colourless confirmed that photosynthesis had occurred. In tube 3, the rate of
photosynthesis corresponds to the quantity of light absorbed and contrarily to the absorbance.

In Tube 4, the chlorophyll pigments has denatured as the chloroplast suspension was
boiled. There was a slight drop in absorbance value which 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. Therefore, in the water splitting process which restores oxidised PS(II),
more electrons will be released.

Tube 7 which was covered with green plastic only allowed green light to enter the
tube. It steady drop in absorbance value as chlorophyll pigments prefer red or blue light.
Therefore, green light was not fully absorbed.

References

1. Johnson. M. P. (2016). Photosynthesis. Sheffield: University of Sheffield, 255-273.

2. Atkins. T. and Escudier. M. (2013). A dictionary of Mechanical Engineering. Oxford:

Oxford University Press, 452.