Biology 11–12 · Year 11

The light-dependent reaction: isolated chloroplasts reducing DCPIP (the Hill reaction)

Module 1: Cells as the Basis of Life (Cell Function)

PracticalLow risk

The idea

Chloroplasts in light release electrons from water, and a blue dye that accepts those electrons loses its colour, showing the light-dependent stage of photosynthesis happening outside the living cell.

Safety card

Low riskAn adult supervises

Hazards

Centrifuge rotor
Hot lamp and mains electricity near water
DCPIP may cause skin sensitisation with prolonged contact
Ice-salt bath is very cold

Controls

Balance the centrifuge tubes and keep the lid closed until the rotor stops
Keep the lamp away from water; do not handle it with wet hands
Avoid skin contact with DCPIP; wash splashes off
Handle the ice bath with dry hands for short periods

Note

All solutions are low hazard per the source; RiskAssess (https://www.riskassess.com.au/) and the NSW Department of Education Chemical Safety in Schools package (https://education.nsw.gov.au/content/dam/main-education/asset-management/chemical-safety/1._Section_1_-_General_information_for_all_staff.pdf).

What you need

Three small fresh spinach, lettuce or cabbage leaves with midribs removed; scissors
Cold isolation medium (0.4 mol/L sucrose and 0.01 mol/L potassium chloride in 0.05 mol/L phosphate buffer, pH 7.0), 25 mL (20 mL for the grind, about 2 mL to resuspend the pellet, 0.5 mL for the control tube and a reserve for the 1 in 5 dilution), and the phosphate buffer, both prepared by the technician to the source recipes and kept at 0 to 4 degrees Celsius
DCPIP solution, 0.007 to 0.01 g made up to 100 mL with the phosphate buffer (the source recipe; this is about 2.4 to 3.4 x 10^-4 mol/L computed from the molar mass of the sodium salt, higher than the approximate 1 x 10^-4 mol/L the source also states)
Pestle and mortar chilled in a freezer for 15 to 30 minutes, or a blender; muslin; funnel; ice-water-salt bath; bench centrifuge and tubes
Five test tubes; 5 mL and 0.5 mL pipettes with fillers; bright bench lamp; foil; colorimeter with a red filter, or a light sensor and data logger

How to do it

Keeping everything cold, grind the leaf pieces rapidly in 20 mL of cold isolation medium, filter through four layers of muslin into chilled centrifuge tubes, and centrifuge about 10 minutes to obtain a small green pellet.
Pour the liquid (supernatant) into a tube and keep it; resuspend the pellet in about 2 mL of cold isolation medium to make the chloroplast extract and keep it on ice.
Set up the source's five tubes: 1, 0.5 mL extract with 5 mL DCPIP in light; 2, 0.5 mL isolation medium with 5 mL DCPIP in light; 3, 0.5 mL extract with 5 mL DCPIP in the dark (foil); 4, 0.5 mL extract with 5 mL distilled water in light; 5, 0.5 mL supernatant with 5 mL DCPIP in light.
Place the lit tubes 12 to 15 cm from the lamp and time how long each takes to lose its blue colour, or read the absorbance every minute for 20 minutes in the colorimeter.
If the extract decolourises within seconds, dilute it 1 in 5 with isolation medium and repeat.
Plot absorbance against time for each tube and explain each control.

What you should see

The extract in light decolourises DCPIP steadily while the dark tube and the tube without extract stay blue; the supernatant, with fewer chloroplasts, decolourises more slowly, and tube 4 shows the green colour of the extract alone for comparison. In the source's bench-centrifuge sample run, tube 1 fell from 5.0 absorption units at 2 minutes to 0.3 at 20 minutes, the dark tube still read 5.4 after 20 minutes and DCPIP without extract read 6.2. The learner knows it worked when only the illuminated tubes containing chloroplast material lose their colour.

What changes

What you change: light or dark, and chloroplast content (pellet, supernatant, none)
What you measure: time to decolourise DCPIP or absorbance against time
What you keep the same: volumes of extract and DCPIP
lamp distance
temperature of the solutions
time from extraction to test

Common misconceptions

Each of these ideas is wrong, and the activity is a chance to test it.

Photosynthesis only happens inside a living leaf; isolated chloroplasts carry out the light-dependent stage.
The oxygen from photosynthesis comes from carbon dioxide; the electrons that reduce the dye come from splitting water.
Light alone makes the dye lose colour; the tube without chloroplasts stays blue in the same light.

Curriculum references

The NSW syllabus outcomes and Australian Curriculum v9 codes this activity supports. They are references, not a verified or complete curriculum alignment.

Biology Stage 6 Syllabus (2017), current: Year 11 taught to the end of 2026 and Year 12 to Term 3 2027BIO11-8 BIO11/12-3 BIO11/12-4
Biology 11–12 Syllabus (2025), Year 11 focus area Cells as the basis of life; new syllabus not yet taught: Year 11 from Term 1 2027, Year 12 from Term 4 2027BI-11-01
Biology 11–12 Syllabus (2025), Year 11 Working scientifically; new syllabus not yet taught: Year 11 from Term 1 2027, Year 12 from Term 4 2027BI-11WS-02 BI-11WS-03 BI-11WS-04
Australian Curriculum v9No Australian Curriculum v9 code is listed.

Sources

The pages the author read to write this activity.

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