HILLS REACTION CPAC OLIVIA BENNY 12E1
Scientific introduction:
This investigation is based around the investigation carried out by Robert hill in 1938 [5] to which he concluded that the
water had been split into hydrogen and oxygen which is now called the hills reaction. The conclusion came from the fact
the NADP is the electron acceptor which will be reduced in the light dependent reactions and provides electrons and
hydrogens for the light-independent reactions. In this practical, I used the DCPIP as the reducing agent as it acts as an
electron acceptor and will become colourless when it becomes reduced. In plants for light-dependant photosynthesis to
occur, the presence of lights and chloroplasts must be present as seen in the results of this investigation. The light energy is
used various ways e.g., in the process of photolysis which is the splitting of water using light in order to produce H+ ions
which acts as a reducing agent and reduces NADP. Chloroplasts are the site of photosynthesis. The DCPIP is a blue dye and
gets oxidised at pH 7.0 and becomes colourless when reduced so is used to monitor the colour change as an indication that
the DCPIP has accepted electrons [6]. DCPIP is used as a substitute for NADP which allows photolysis to take place as DCPIP
can accept the electrons from the electron transport chain.
Hypothesis:
Tube Contains Colour change YES/NO Predictions to why?
1 Leaf extract + DCPIP (light YES There is a source of light as
source as well) well as chloroplasts
2 Isolation medium + DCPIP NO The DCPIP will not
(light) decolourise not exposed to
light, no chloroplasts
present
3 Leaf extract + DCPIP (dark) NO No light source, light source
is necessary for
photosynthesis
4 Leaf extract + distilled NO Photosynthesis is actually
water (light) occurring but we have no
indication of it occurring as
DCPIP isn’t present
5 Supernatant + DCPIP NO (if its clear) No colour change if its clear
however if its not clear it
means that some
chloroplasts are present
which may photosynthesise
Variables:
Independent variable - The independent variable is the variable you
change, I changed the light intensity to
investigate the effect of light intensity on the
photosynthesis, whether it was a necessary
condition. The light source I used was a lamp
and in one test tube I used the absence of light,
the others I included the source of light.
- Another variable I changed was the absence of
the isolated chloroplasts, this was to investigate
whether chloroplasts was a necessary condition
in the light dependant reaction, I did this by in
, some test tubes only using DCPIP and
supernatant and using distilled water in one test
tube as a control
Dependant variable - The dependant variable is the variable you
measure, and I measured the colour change of
the DCPIP, or if there was any colour change at
all as colour change was an indication of
photosynthesis/ somewhat of a reaction
occurring. The DCPIP will turn colourless and
therefore a green colour will be seen.
Control variable - The amount of time, I made sure all tubes were
left for the same amount of time before
comparing the different colours of the mixture, I
used a stop watch to ensure accuracy of time.
Time is important as different observations may
be made with more or less time.
- The amount of DCPIP added to the test tube- I
added 5cm^3 of DCPIP in each test tube using a
syringe to ensure accuracy, this is important as a
higher concentration of DCPIP may affect the
results of the colour change e.g., may take
longer for a larger volume of DCPIP to
decolourise.
- Using chloroplasts from the same species of
plant, I have controlled this by using spinach
only from the same habitat and environment to
ensure minimal genetic variation and genetic
variation can affect the results.
- Amount of chloroplast/ if any chloroplast added
to each test tube, I controlled this using a
syringe and using 0.5cm^3 of the chloroplast
solution with the isolation medium, not using
the same volume of solution made can affect
the results as a higher concentration of
chloroplasts may reduce the time overall taken
to see an overall colour change in the test tube.
Method:
1. Cut three small green spinach, lettuce or cabbage leaves into small pieces with scissors, but discard the tough
midribs and leaf stalks. Place in a cold mortar of cold isolation medium (scale up the quantities)
2. Grind vigorously and rapidly for about 10 seconds
3. Place four layers of muslin in a funnel and wet with cold isolation medium
4. Filter the mixture through the funnel into the beaker and pour the filtrate into pre cooled centrifuge supported in
an ice water salt bath. Gather the edges of muslin, wring thoroughly into the beaker and add filtrate to the tubes
5. Check that each centrifuge tube contains about the same volume of filtrate
6. Centrifuge the tubes for sufficient time to get a small pellet of chloroplasts (10 mins at high speed Is sufficient)
7. Pour off the liquid (supernatant) into a boiling tube being careful not to lose the pellet. Resuspend the pellet with
2cm^3 of isolation medium, using a glass rod. Squirting in and out of. A Pasteur pipette five or six times giving a
uniform suspension.
8. Store this leaf extract in an ice water salt bath and use as soon as possible
9. Set up 5 labelled tubes with alternating DCPIP, light source, leaf extract and the supernatant/ distilled water
10. Set them according to this table:
Tube Leaf extract/ Supernatant/ Isolation Distilled DCPIP
cm^3 cm^3 medium/ water/cm^3 solution/cm^3
cm^3
1 0.5 - - - 5
2 - - 0.5 - 5
3 0.5 - - - 5
4 0.5 - - 5 -
Scientific introduction:
This investigation is based around the investigation carried out by Robert hill in 1938 [5] to which he concluded that the
water had been split into hydrogen and oxygen which is now called the hills reaction. The conclusion came from the fact
the NADP is the electron acceptor which will be reduced in the light dependent reactions and provides electrons and
hydrogens for the light-independent reactions. In this practical, I used the DCPIP as the reducing agent as it acts as an
electron acceptor and will become colourless when it becomes reduced. In plants for light-dependant photosynthesis to
occur, the presence of lights and chloroplasts must be present as seen in the results of this investigation. The light energy is
used various ways e.g., in the process of photolysis which is the splitting of water using light in order to produce H+ ions
which acts as a reducing agent and reduces NADP. Chloroplasts are the site of photosynthesis. The DCPIP is a blue dye and
gets oxidised at pH 7.0 and becomes colourless when reduced so is used to monitor the colour change as an indication that
the DCPIP has accepted electrons [6]. DCPIP is used as a substitute for NADP which allows photolysis to take place as DCPIP
can accept the electrons from the electron transport chain.
Hypothesis:
Tube Contains Colour change YES/NO Predictions to why?
1 Leaf extract + DCPIP (light YES There is a source of light as
source as well) well as chloroplasts
2 Isolation medium + DCPIP NO The DCPIP will not
(light) decolourise not exposed to
light, no chloroplasts
present
3 Leaf extract + DCPIP (dark) NO No light source, light source
is necessary for
photosynthesis
4 Leaf extract + distilled NO Photosynthesis is actually
water (light) occurring but we have no
indication of it occurring as
DCPIP isn’t present
5 Supernatant + DCPIP NO (if its clear) No colour change if its clear
however if its not clear it
means that some
chloroplasts are present
which may photosynthesise
Variables:
Independent variable - The independent variable is the variable you
change, I changed the light intensity to
investigate the effect of light intensity on the
photosynthesis, whether it was a necessary
condition. The light source I used was a lamp
and in one test tube I used the absence of light,
the others I included the source of light.
- Another variable I changed was the absence of
the isolated chloroplasts, this was to investigate
whether chloroplasts was a necessary condition
in the light dependant reaction, I did this by in
, some test tubes only using DCPIP and
supernatant and using distilled water in one test
tube as a control
Dependant variable - The dependant variable is the variable you
measure, and I measured the colour change of
the DCPIP, or if there was any colour change at
all as colour change was an indication of
photosynthesis/ somewhat of a reaction
occurring. The DCPIP will turn colourless and
therefore a green colour will be seen.
Control variable - The amount of time, I made sure all tubes were
left for the same amount of time before
comparing the different colours of the mixture, I
used a stop watch to ensure accuracy of time.
Time is important as different observations may
be made with more or less time.
- The amount of DCPIP added to the test tube- I
added 5cm^3 of DCPIP in each test tube using a
syringe to ensure accuracy, this is important as a
higher concentration of DCPIP may affect the
results of the colour change e.g., may take
longer for a larger volume of DCPIP to
decolourise.
- Using chloroplasts from the same species of
plant, I have controlled this by using spinach
only from the same habitat and environment to
ensure minimal genetic variation and genetic
variation can affect the results.
- Amount of chloroplast/ if any chloroplast added
to each test tube, I controlled this using a
syringe and using 0.5cm^3 of the chloroplast
solution with the isolation medium, not using
the same volume of solution made can affect
the results as a higher concentration of
chloroplasts may reduce the time overall taken
to see an overall colour change in the test tube.
Method:
1. Cut three small green spinach, lettuce or cabbage leaves into small pieces with scissors, but discard the tough
midribs and leaf stalks. Place in a cold mortar of cold isolation medium (scale up the quantities)
2. Grind vigorously and rapidly for about 10 seconds
3. Place four layers of muslin in a funnel and wet with cold isolation medium
4. Filter the mixture through the funnel into the beaker and pour the filtrate into pre cooled centrifuge supported in
an ice water salt bath. Gather the edges of muslin, wring thoroughly into the beaker and add filtrate to the tubes
5. Check that each centrifuge tube contains about the same volume of filtrate
6. Centrifuge the tubes for sufficient time to get a small pellet of chloroplasts (10 mins at high speed Is sufficient)
7. Pour off the liquid (supernatant) into a boiling tube being careful not to lose the pellet. Resuspend the pellet with
2cm^3 of isolation medium, using a glass rod. Squirting in and out of. A Pasteur pipette five or six times giving a
uniform suspension.
8. Store this leaf extract in an ice water salt bath and use as soon as possible
9. Set up 5 labelled tubes with alternating DCPIP, light source, leaf extract and the supernatant/ distilled water
10. Set them according to this table:
Tube Leaf extract/ Supernatant/ Isolation Distilled DCPIP
cm^3 cm^3 medium/ water/cm^3 solution/cm^3
cm^3
1 0.5 - - - 5
2 - - 0.5 - 5
3 0.5 - - - 5
4 0.5 - - 5 -
