Core Practical 11 - 22/03/23
Core practical 11 - Investigate photosynthesis using isolated chloroplasts (the Hill reaction)
Aim: To investigate experimentally the link between the light-dependent and light
independent reactions
Introduction
The process of photosynthesis is based on 2 reactions that are carried out by separate parts
of the chloroplast. The light dependent reaction occurs in the chloroplast thylakoid
membrane and involves the splitting of water into oxygen, protons and electrons. The
protons and electrons are then transferred through the thylakoid membrane to create the
energy storage molecules adenosine triphosphate (ATP) and nicotinamide-adenine
dinucleotide phosphate (NADPH). (Johnson, 2016)
DCPIP (dichlorophenol-indophenol) is a blue dye that works like NADP+ in the light reaction
of photosynthesis. In the presence of light, chloroplasts can convert DCPIP to DCPIPre just
as NADP+ is converted to NADPH. AS this reaction progresses, there is less blue pigment -
this colour change can be measured spectrophotometrically, thus making DCPIP a good
indicator of photosynthetic activity (www.proquest.com, n.d.)
In this experiment, chloroplasts are isolated from plant cells and exposed to light showing
how isolated chloroplasts would continue to perform some of the reactions of photosynthesis
if provided with the correct condition (Lesson Element The Hill Reaction Instructions and
answers for teachers, n.d.).
Hypothesis : The light dependent reaction is dependent on both light and chloroplast
Risk assessment
- DCPIP is an irritant so wash hands after use - Low risk
- Fasten the centrifuge before turning it on
- The bulb of lamp may be hot as it would have been on for a while, so care should be
taken not to make contact with it
- Place the bench lamp away from the ice bath and be cautious of any spillages to
prevent electrocution
- Glasswares such as test tubes are fragile causing them to break easily. They have
very sharp edges when they’re broken, this may cause skin injury - Handle glass with
care and put away safely and keep away from edges of surfaces where it can fall
Variables
Independent variable : The different solutions made and the conditions in which the test
tube were placed
Dependent Variable : The colour change of the sample and whether DCPIP was reduced or
not
Control variables :
- Use chloroplast from the same species of plant - chopped spinach
- Amount of DCPIP solution - 5 cm3 measured using 5 cm3 pipette
- Time - All tubes will be left for the same amount of time before comparing the colour
of the mixture
- Same pH used - buffer solution will be added to maintain ph of 7.0
, Core Practical 11 - 22/03/23
Equipment
Equipment Why is it needed?
Fresh green spinach, lettuce or cabbage Source of chloroplast
leaves
Cold pestle and mortar To crush the spinach to make a solution
that can be centrifuged
Scissors To cut up the leaves into smaller pieces to
be grinded
Centrifuge and centrifuge tubes Used to separate organelles and other
subcellular particles based on their weight.
Heavy organelles such as chloroplast settle
at the bottom forming a thick sediment
known as a pellet
Ice-water-salt bath Solution must be cold to reduce the activity
of enzymes breaking down organelles
Pasteur pipette Squirting in and out of a pasteur pipette
gives a uniform suspension
Isolation medium (sucrose and KCl in 0.05 The isolation medium will prevent damage
M phosphate buffer, pH 7.0) due to osmosis or extreme pH
Measuring cylinder, 20 cm3 To measure accurately the volume of
isolation medium
Beaker and funnel, 100 cm3 To filter the crushed leaves and isolation
medium mixture
Pipettes, 5 cm3 and 1 cm3 To measure the volume of different
solutions and DCPIP to put into the 5
different test tubes
Bench lamp with 100 W bulb Source of light for light dependent reaction
to occur
DCPIP solution Indicator used to study the rate of the Hill
reaction
Colorimeter and data logger To quantify the colour change
Method
PART A
1. Cut a couple of green spinach leaves into small pieces - ensuring stalks and other
components as removed
Core practical 11 - Investigate photosynthesis using isolated chloroplasts (the Hill reaction)
Aim: To investigate experimentally the link between the light-dependent and light
independent reactions
Introduction
The process of photosynthesis is based on 2 reactions that are carried out by separate parts
of the chloroplast. The light dependent reaction occurs in the chloroplast thylakoid
membrane and involves the splitting of water into oxygen, protons and electrons. The
protons and electrons are then transferred through the thylakoid membrane to create the
energy storage molecules adenosine triphosphate (ATP) and nicotinamide-adenine
dinucleotide phosphate (NADPH). (Johnson, 2016)
DCPIP (dichlorophenol-indophenol) is a blue dye that works like NADP+ in the light reaction
of photosynthesis. In the presence of light, chloroplasts can convert DCPIP to DCPIPre just
as NADP+ is converted to NADPH. AS this reaction progresses, there is less blue pigment -
this colour change can be measured spectrophotometrically, thus making DCPIP a good
indicator of photosynthetic activity (www.proquest.com, n.d.)
In this experiment, chloroplasts are isolated from plant cells and exposed to light showing
how isolated chloroplasts would continue to perform some of the reactions of photosynthesis
if provided with the correct condition (Lesson Element The Hill Reaction Instructions and
answers for teachers, n.d.).
Hypothesis : The light dependent reaction is dependent on both light and chloroplast
Risk assessment
- DCPIP is an irritant so wash hands after use - Low risk
- Fasten the centrifuge before turning it on
- The bulb of lamp may be hot as it would have been on for a while, so care should be
taken not to make contact with it
- Place the bench lamp away from the ice bath and be cautious of any spillages to
prevent electrocution
- Glasswares such as test tubes are fragile causing them to break easily. They have
very sharp edges when they’re broken, this may cause skin injury - Handle glass with
care and put away safely and keep away from edges of surfaces where it can fall
Variables
Independent variable : The different solutions made and the conditions in which the test
tube were placed
Dependent Variable : The colour change of the sample and whether DCPIP was reduced or
not
Control variables :
- Use chloroplast from the same species of plant - chopped spinach
- Amount of DCPIP solution - 5 cm3 measured using 5 cm3 pipette
- Time - All tubes will be left for the same amount of time before comparing the colour
of the mixture
- Same pH used - buffer solution will be added to maintain ph of 7.0
, Core Practical 11 - 22/03/23
Equipment
Equipment Why is it needed?
Fresh green spinach, lettuce or cabbage Source of chloroplast
leaves
Cold pestle and mortar To crush the spinach to make a solution
that can be centrifuged
Scissors To cut up the leaves into smaller pieces to
be grinded
Centrifuge and centrifuge tubes Used to separate organelles and other
subcellular particles based on their weight.
Heavy organelles such as chloroplast settle
at the bottom forming a thick sediment
known as a pellet
Ice-water-salt bath Solution must be cold to reduce the activity
of enzymes breaking down organelles
Pasteur pipette Squirting in and out of a pasteur pipette
gives a uniform suspension
Isolation medium (sucrose and KCl in 0.05 The isolation medium will prevent damage
M phosphate buffer, pH 7.0) due to osmosis or extreme pH
Measuring cylinder, 20 cm3 To measure accurately the volume of
isolation medium
Beaker and funnel, 100 cm3 To filter the crushed leaves and isolation
medium mixture
Pipettes, 5 cm3 and 1 cm3 To measure the volume of different
solutions and DCPIP to put into the 5
different test tubes
Bench lamp with 100 W bulb Source of light for light dependent reaction
to occur
DCPIP solution Indicator used to study the rate of the Hill
reaction
Colorimeter and data logger To quantify the colour change
Method
PART A
1. Cut a couple of green spinach leaves into small pieces - ensuring stalks and other
components as removed
