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Practical work is a core component of the Edexcel A-Level Biology (9BI0) specification. This lesson consolidates the required practicals for photosynthesis and respiration, focusing on experimental design, data analysis, controls, and common sources of error. You must be able to describe, evaluate, and improve these methods.
This is the most commonly examined practical for measuring photosynthetic rate by collecting oxygen gas produced by an aquatic plant.
| Item | Purpose |
|---|---|
| Elodea or Cabomba (aquatic plant) | Photosynthesising organism |
| Sodium hydrogencarbonate (NaHCO₃) solution | Provides constant supply of dissolved CO₂ |
| Bench lamp (with known wattage) | Light source; distance varied to change intensity |
| Ruler / metre rule | Measure distance from lamp to plant |
| Stopwatch | Time the counting period |
| Beaker of water | Acts as heat shield (absorbs infrared) |
| Thermometer | Monitor temperature |
| Audus microburette / photosynthometer | More accurate measurement of O₂ volume than bubble counting |
| Variable type | Variable | How controlled |
|---|---|---|
| Independent | Light intensity (varied by distance; calculated as 1/d²) | Measured with ruler |
| Dependent | Rate of photosynthesis (bubbles per minute or volume of O₂) | Counted or measured |
| Control | Temperature | Water bath or beaker of water between lamp and plant as heat shield |
| Control | CO₂ concentration | Constant concentration of NaHCO₃ |
| Control | Species and size of plant | Same species and similar-length pieces |
| Control | Wavelength of light | Same lamp; or use colour filters to investigate specific wavelengths |
| Error/Limitation | Improvement |
|---|---|
| Bubbles are unequal in size → inaccurate count | Use a photosynthometer to measure volume of gas collected |
| Lamp heats the water → temperature changes | Place a water-filled beaker between the lamp and the plant as a heat shield |
| Air bubbles may not all be O₂ | Collect the gas and test with a glowing splint (relights if O₂) |
| Plant not acclimatised | Allow 5 minutes at each new distance before recording |
| Bubbles may dissolve in water | Use water that has been equilibrated with air at the experimental temperature |
Exam Tip: When asked to describe improvements, always give both the problem and the solution. "The temperature may increase due to the lamp" is the problem; "Place a water-filled beaker between the lamp and the plant to absorb infrared radiation" is the improvement.
An alternative method using leaf discs floating in NaHCO₃ solution.
| Advantage | Limitation |
|---|---|
| Simple equipment | Leaf disc size must be consistent |
| Can investigate multiple factors | Some discs may not fully de-gas |
| Quantitative (time or number floating) | Temperature must be controlled |
The respirometer has been described in detail in the previous lesson. Here we focus on the practical skills assessed in the exam.
Plot a graph of:
| Graph feature | Interpretation |
|---|---|
| Straight line (volume vs time) | Constant rate of respiration |
| Increasing gradient | Rate is accelerating (unusual in a controlled experiment) |
| Plateau | O₂ or substrate exhausted |
| Rate increases with temperature | Enzyme kinetics (more collisions) |
| Rate drops at high temperature | Enzyme denaturation |
Dehydrogenase enzymes in respiration remove hydrogen atoms from substrates and transfer them to coenzymes (NAD⁺ → reduced NAD). The activity of dehydrogenases can be measured using a redox indicator such as DCPIP (dichlorophenolindophenol) or methylene blue.
| Variable | Detail |
|---|---|
| Independent | Temperature (or substrate concentration, or inhibitor concentration) |
| Dependent | Rate of colour change (absorbance change per minute) |
| Controlled | Volume and concentration of DCPIP, volume and concentration of yeast suspension, temperature |
Exam Tip: DCPIP acts as an artificial hydrogen acceptor, replacing NAD⁺. The faster the colour fades, the greater the rate of dehydrogenase activity (and therefore respiration).
Photosynthetic pigments can be separated using thin-layer chromatography (TLC) or paper chromatography.
Rf = distance moved by pigment / distance moved by solvent front
| Pigment | Typical colour on chromatogram | Relative Rf |
|---|---|---|
| Carotene | Yellow/orange | Highest (most soluble in solvent) |
| Xanthophyll | Yellow | Intermediate |
| Chlorophyll a | Blue-green | Intermediate |
| Chlorophyll b | Yellow-green | Lowest (least soluble in solvent) |
Exam Tip: Rf values depend on the solvent used, so always state the solvent system when reporting Rf values. Carotene has the highest Rf because it is the most non-polar pigment and dissolves most readily in the non-polar solvent.
You may need to apply statistical analysis to your practical data:
| Test | When to use | What it tests |
|---|---|---|
| Mean and standard deviation | Always | Central tendency and spread |
| t-test | Comparing two means | Whether two means are significantly different |
| Chi-squared (χ²) | Categorical data | Whether observed data differ significantly from expected |
| Spearman's rank | Correlation between two variables | Whether there is a significant correlation |
When evaluating practical data:
| Term | Definition |
|---|---|
| Photosynthometer | Apparatus that accurately measures the volume of O₂ produced by an aquatic plant |
| Respirometer | Apparatus that measures the rate of O₂ consumption by an organism |
| DCPIP | A redox indicator: blue when oxidised, colourless when reduced; used to measure dehydrogenase activity |
| Rf value | The ratio of the distance moved by a substance to the distance moved by the solvent front in chromatography |
| Acclimatisation | Allowing an organism or apparatus to stabilise at new conditions before taking measurements |
This material sits in Edexcel 9BI0 Topic 5 (On the Wild Side — Photosynthesis, Energy and Ecosystems) with a strong overlap into Paper 3 (General and Practical Principles in Biology), which assesses experimental design, controls, calculations and evaluative reasoning across the whole course. The lesson centralises Core Practical 7 (factors affecting the rate of photosynthesis — Elodea / pondweed and leaf-disc methods) and Core Practical 12 (rate of yeast respiration — methylene-blue redox indicator and respirometer). It is therefore the practical "spine" connecting the conceptual chapters: lessons 1–2 (light reactions and Calvin cycle) describe what photosynthesis CPs measure; lessons 4–7 (glycolysis, link reaction, Krebs cycle, oxidative phosphorylation, anaerobic respiration) describe what respiration CPs measure; lesson 3 / lesson 8 (factors affecting photosynthesis / respiration) sets up the limiting-factor framework. Wider synoptic ties: Topic 1 (enzymes and rate measurement) — Michaelis-Menten and rate-vs-temperature curves underpin every practical; Topic 7 (transpiration potometer) — the apparatus is structurally analogous to a respirometer (capillary, fluid displacement, control tube, equilibration time). Refer to the official Pearson Edexcel 9BI0 specification document for exact wording.
Question (8 marks): A student investigates the rate of photosynthesis using the leaf-disc floating assay. Ten 6 mm diameter discs cut from spinach leaves are vacuum-infiltrated in 0.2 mol dm−3 NaHCO3 solution and transferred to a beaker of NaHCO3 at 25 °C, 30 cm from a lamp. The mean time for half the discs to float is recorded. A parallel beaker is prepared with the same discs in NaHCO3 kept in the dark.
| Condition | Mean time for 50% of discs to float (min) |
|---|---|
| Light, 30 cm from lamp | 8.0 |
| Dark | Discs do not float in 30 min |
(a) Calculate the rate of photosynthesis under the lit condition, expressed as discs per minute. (2)
(b) Explain why the dark control does not produce floating discs, with reference to net versus gross O2 evolution. (3)
(c) Suggest, with reasoning, one modification that would convert the lit measurement from net to gross O2 evolution. (3)
Solution with mark scheme:
(a) M1 (AO2) — 5 discs floated in 8.0 minutes; rate = 5 / 8.0.
A1 (AO2) — Rate = 0.625 discs per minute (accept 0.63).
(b) M1 (AO1) — Photosynthesis evolves O2 via the light reactions (lessons 1–2); respiration consumes O2 via Complex IV in the mitochondrion (lesson 6). What the discs accumulate in their air spaces is net O2 = photosynthesis O2 — respiration O2.
M1 (AO2) — In the dark, the light reactions cannot run (no excitation of chlorophyll, no electron flow, no O2 evolution from photolysis), but mitochondrial respiration continues and consumes any residual O2 in the air spaces.
A1 (AO3) — Net O2 evolution is therefore negative in the dark — the discs lose buoyancy or stay sunken. The discs in the lit beaker float because gross photosynthesis exceeds respiration at this irradiance.
(c) M1 (AO3) — Measure the dark respiration rate of an identical leaf-disc batch separately (e.g. by O2-electrode in a dark chamber, or by methylene-blue decolourisation rate, if respiration data are convertible to disc-buoyancy units).
M1 (AO2) — Then add the dark respiration rate to the lit (net) rate to recover gross O2 evolution = net + respiration.
A1 (AO3) — This assumes respiration rate is the same in light and dark — a standard simplifying assumption that is actually only approximately true (mitochondrial respiration in illuminated photosynthetic tissue is partially suppressed; photorespiration adds another correction). For Edexcel A-level purposes, the assumption is acceptable provided it is stated. (Total: 8 marks; M5 A3.)
Question (6 marks): A student investigates the Hill reaction by adding a chloroplast suspension to DCPIP solution and measuring the time taken for the blue colour to fade in the light at 25 °C. The result is compared with a parallel tube kept in the dark, where DCPIP remains blue. Explain the result with reference to the stage of photosynthesis being measured, the role of DCPIP, and the appropriate controls.
Mark scheme decomposition by AO:
| Mark | AO | Earned by |
|---|---|---|
| 1 | AO1.1 | Identifying that DCPIP is a redox indicator — blue when oxidised, colourless when reduced — and that it accepts electrons in place of NADP+ at the end of the light-reaction electron transport chain |
| 2 | AO1.2 | Stating that the assay therefore measures the light reactions only (photolysis of water, electron transport from PSII to PSI), not the Calvin cycle |
| 3 | AO2.1 | Reasoning that decolourisation rate is proportional to the rate of electron flow through the thylakoid membrane, which is proportional to light intensity (within the linear range) |
| 4 | AO2.2 | Identifying the dark control as essential — it confirms that DCPIP is not reduced by any non-photosynthetic process (e.g. ascorbate in the chloroplast extract); the persistence of blue colour in the dark validates the assay |
| 5 | AO3.1 | Recognising that a boiled-chloroplast control would also be needed to confirm the reduction is enzyme-catalysed (denatured thylakoids cannot transfer electrons, so DCPIP remains blue even in the light) |
| 6 | AO3.2 | Concluding that the Hill reaction isolates light reactions experimentally — the Calvin cycle is bypassed because DCPIP intercepts electrons before NADP+ reduction, so the assay reports light-reaction flux even when CO2 fixation is impossible |
Total: 6 marks (UMS-band-anchored at A; AO1 = 2, AO2 = 2, AO3 = 2). Specimen question modelled on the Edexcel 9BI0 paper format. The structure mirrors Edexcel's preference for combining a familiar practical procedure with mechanistic reasoning that connects the measurement to a specific stage of metabolism, while demanding articulate control logic.
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