Cambridge International

AS and A Level Biology (9700)

Practical booklet 9

Measuring the effect of wavelength of light

on photosynthesis

Candidate Name: Date:

AS Level skills How learners develop the skills
MMO collection Using different methods to measure the size of cells
PDO recording Recording quantitative data in a table
ACE analysis Calculating a mean
PDO display Showing all the steps in their calculations
Deciding which method provides the most accurate results and therefore
ACE evaluation develop an understanding of how modifying a procedure can increase
accuracy
C = corrosive substance F = highly flammable substance
Hazard symbols H = harmful or irritating substance O = oxidising substance
N = harmful to the environment T = toxic substance

 Independent variable: Variable that is changed by investigator

 Dependent variable: Variable that is measured, but not
processed
 Variables affecting Dependent Variable must be standardised
(controlled).
 Practical 9 – Worksheet

Measuring the effect of wavelength of light on photosynthesis

Aim
To using redox dye, DCPIP, to measure the effect of light on photosynthesis by varying the
wavelength of light. This practical is intended to focus on planning, in particular, defining the
problem and identification of variables.

Null Hypothesis: ………………………………………………………………………………………

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Alternative Hypothesis: …………………………………………………………………………….
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Principle
In the light dependent reaction of photosynthesis, electrons are excited using energy from
light. These high-energy electrons are passed through chains of electron carriers into NADP,
which becomes “reduced NADP” [NADPH] by accepting the electrons and hydrogen ions
from the photolysis of water.
Chloroplasts contain a variety of pigments that can absorb light of different wavelengths, but
some wavelengths of light are more effectively absorbed than others. Apart from pigments,
chloroplast has electron carriers and enzymes to reduce NADP and synthesise ATP. If
chloroplasts are extracted from leaves, the same reactions occur and other oxidised
materials can accept the electrons and hydrogen ions, releasing oxygen. This is called
the Hill reaction after its discoverer, Robert Hill.
Some coloured chemicals can act as electron and hydrogen ion acceptors and change
colour as they are reduced. Oxidised 2, 6-dichlorophenolindophenol (DCPIP) is bright
blue, and when reduced it becomes colourless. Methylene blue can be used but DCPIP
works best as it is very sensitive. DCPIP provides a way of measuring how fast the light
dependent reaction is happening as the time taken for the colour to change from blue to
colourless can be timed. Understanding the light dependent process of photosynthesis, the
visible light spectrum, the absorption spectrum and the information about how DCPIP can be
used to demonstrate the release of electrons and hydrogen ions are necessary to
understand this experiment.

The extraction medium [Do not keep more than 48 hours in a refrigerator and must be very
cold when used]. This consists of Phosphate buffer [ph7.2], sucrose and potassium chloride
(KCl). The best concentration to use is 34.23 g sucrose and 0.19 g KCl in 250 cm3 of
phosphate buffer solution.

To prepare 125ml of extraction medium

17.115 g sucrose
0.095 g (95mg) KCl
125ml phosphate buffer solution

The DCPIP can be bought as a ready-made solution or made from the powder and
phosphate buffer or made from the powder, KCl and phosphate buffer. A concentration of
DCPIP that work well is 0.4 g DCPIP and 0.93 g KCl in 250 cm3 phosphate buffer
solution at room temperature. It does not keep more than 48 hours in a refrigerator.
To prepare 125ml of of DCPIP
0.2g (200mg) of DCPIP
0.465g (465mg) of Kcl

1 Cambridge International AS and A Level Biology 9700

Biology Practical 9 – Worksheet

125 ml of phosphate buffer solution at RT

Phosphate buffers consist of 4.48 g disodium hydrogen phosphate (Na2HPO4.12H2O) and

1.7 g potassium dihydrogen phosphate (KH2PO4) in 500 cm3 of distilled water. This keeps for
several weeks if stored in a refrigerator.

[Buy prepared Phosphate buffer, pH 7.2]

Method
Safety glasses must be worn.
Preparation of leaf extract.
1. Placed the leaf onto a tile.
2. Chopped the leaf into small pieces after discarding any large veins.
3. Took the pieces into a plastic tube and added 2 cm3 of very cold extraction medium.
4. Ground the contents with a glass rod for one minute to give a green liquid of the leaf
extract.
5. Decanted the leaf extract slowly into a Petri dish by filtering through a muslin cloth to
remove leaf debris.
6. Covered the Petri dish with a metal foil to keep light out. Steps 4 and 5 are done fast.
7. Folded different coloured filters [wavelength] along their length to make little tents, and
put them on the white tile as shown in the drawing.

colour wavelength / nm
purple 425
blue 450
green 525
orange 625
red 675

8. Dipped the end of one of the capillary tubes into the leaf extract in the Petri dish so that
some extract rises up the tube. This is the control. Laid this tube on the white tile.
9. Added required number of drops of DCPIP solution to the leaf extract in the Petri dish
and mixed so that the green leaf extract is a blue-green colour. Covered the extract with
the metal foil immediately. This is done as fast as possible.
10. Lifted the edge of the metal cover and dipped the end of another capillary tube in the leaf
extract and DCPIP mixture. Covered the dish again. This is a test extract. Laid this on the
tile next to the control. Covered the two capillary tubes with a tent of a purple filter.
11. Switched the lamp so that the light falls evenly onto the filter and started the stop clock.
At one minute intervals lifted the filter on the side opposite to the lamp and recorded
the colour of the test extract.
12. Recorded the time taken for colour change. Recorded as ‘>10 minutes’, when the extract
was still blue even after 10 minutes.
13. Discarded the test capillary as soon as it has changed back to green.
14. Left the control on the white tile.
15. Repeated the investigation from step 10, but with a different coloured filter.
[Expected time: Purple and blue (1-2 min); Red (2-3 min); Green (8-10 min)]

Results
1. Prepared a table to record the colour of each tube at 1 minute intervals.

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 Biology Practical 9 – Worksheet

Colour of the Minute wise observation of colour change

filter Min Min Min Min Min Min Min Min Min Min
(wavelength) 1 2 3 4 5 6 7 8 9 10

Purple 425nm
[R1]
Purple 425nm
[R2]
Purple 425nm
[R2]
Blue 450nm
[R1]
Blue 450nm
[R2]
Blue 450nm
[R3]
Green 525nm
[R1]
Green 525nm
[R2]
Green 525nm
[R3]
Orange 625nm
[R1]
Orange 625nm
[R2]
Orange 625nm
[R3]
Red 675nm
[R1]
Red 675nm
[R1]
Red 675nm
[R1]

Colour of the Time taken for colour change Mean Rate of

filter colour change / s-1
(wavelength) R1 R2 R3 Mean Mean
1
/ min / min / min / min / sec
time / s
Purple
Blue
Green
Orange
2. Identified
Red anomalies in
data, if any.
3. Calculated the mean rate of reaction using “1 / time” taken for colour to disappear.
When, the time is recorded as >10 minutes, then recorded 1 / time taken for colour to
disappear as 0.
4. Used the formulae below to calculate the standard deviation (s) and standard error (SM)
for each wavelength of light.
Biology Practical 9 – Worksheet

√
∑ ( x−x )
2 and s
SM=
s= √n
n−1

where n = sample size (number of observations), x = mean, Ʃ= ‘sum of’

5. Plotted a graph showing the effect of light intensity on the rate of photosynthesis. Added
error bars using calculated standard error.

Interpretation and evaluation

1. Use the length of the error bars to assess the reliability of the data collected.

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2. Describe the effect of the wavelengths of light on the rate of photosynthesis.

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3. Compare your graph to the shape of the graph of the absorption spectrum and draw
conclusions about the relationship between the wavelengths of light absorbed and the
rate of photosynthesis.

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 Biology Practical 9 – Worksheet

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Extension
Write a procedure, to test the effect of different light intensity on the rate of hotosynthesis.
Use the following information about the filters used.
description % of light transmitted
pale grey 70
mid-grey 50
dark grey 25

Your method should have practical details that would allow another person to use it
without any further information about the procedure.

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Biology Practical 9 – Worksheet

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