Biology 9700/51 — October/November 2023

Background Concept

Ripening of a fruit such as an apple is a coordinated set of molecular and physiological changes: starches are converted into sugars, chlorophyll is broken down to reveal underlying pigments, and cell walls are softened by enzymes such as pectinase. These changes occur over a continuous timescale, but they are not strictly synchronised with growth in size. A fruit can be expanding rapidly and yet still be biochemically unripe.

When a variable (here, diameter) is used to select experimental material, it must reflect the property being studied (ripeness). Diameter is a cumulative measure of all previous growth, whereas ripeness is a current biochemical state; the two need not correspond.

Understanding the Question

The student removes apples of exactly $5\ \text{cm}$ in diameter and assumes they are all at the same, early, ripening stage. The question asks why this assumption may be inaccurate.

Approach

Think about what could decouple diameter from ripeness: variation between individual fruits on a tree, the influence of environmental conditions on growth, and the fact that size is a cumulative measure while ripeness is a current-state measure.

Step-by-Step Reasoning

The mark scheme accepts any one of these points (1 mark):

1. Size is not proportional to ripeness. Two apples of the same diameter may have been on the tree for different lengths of time or grown under different conditions, so they may be biochemically at different points along the ripening pathway even though they look similar in size.
2. Apples ripen at different rates. Genetic and microenvironmental differences between individual apples mean two apples of the same size need not be at the same stage of ripening.
3. Other factors affect ripeness and/or diameter. Position on the tree, light exposure, water availability and pest damage can all change either how big an apple grows or how quickly it ripens, so a single diameter value cannot capture this variation.

Any of these is sufficient. The strongest answer ties the two ideas together: diameter sums all previous growth, while ripeness reflects recent biochemistry, so they need not match.

Key Takeaways

• Selecting experimental material requires choosing a variable that reflects the property you actually want to study.
• A convenient, easy-to-measure variable is not necessarily a good proxy for the underlying biology.
• Variation between individuals of the same species and developmental stage can introduce uncontrolled variables.

Common Mistakes

• Saying that diameter is "not accurate" without giving a reason. The mark scheme requires the reason (size may not be related to ripeness, or apples may ripen at different rates).
• Confusing this with measurement error; the issue here is the validity of the criterion, not the precision of the measurement.

Things to Be Careful About

• Stay close to the mark scheme wording: "size may not be related to ripeness" or "apples may ripen at different rates" are accepted; do not invent unrelated criticisms such as "the ruler was inaccurate".

Background Concept

The stem of the question defines ripeness by three changes: an increase in sugar content, a change in colour, and a softening of the fruit. These are the direct indicators of ripeness, in contrast to diameter which is an indirect and unreliable proxy. A good selection method uses one of these direct indicators.

Understanding the Question

Following the criticism in (a)(i), the student is asked to describe a more accurate way of selecting apples that are all at the same stage of ripening. The improvement must give a more accurate estimate of ripeness than diameter does.

Approach

Replace the diameter criterion with one of the actual ripening indicators mentioned in the stem: sugar content, colour, or softness. The improvement should be both measurable (so it can be standardised across apples) and biologically meaningful (so it really tracks ripeness).

Step-by-Step Reasoning

The mark scheme accepts any one of these (1 mark):

1. Select apples with the same / similar sugar content, colour, softness, or age. Choosing apples that match on one of these direct ripeness indicators ensures the sample starts the experiment at a comparable biological state.
2. Test / measure the sugar content, colour, softness, or age of the apples. This makes the criterion operational — instead of just describing what would be a better criterion, the student specifies the measurement that will be used to apply it (e.g. a refractometer for sugar content, a colour chart for colour, a penetrometer for firmness, or a known picking date for age).

A complete answer often combines the two ideas: "Measure the sugar content of each apple and only use those with similar sugar contents." That scores as one mark for either half, with the other half being bonus detail.

Key Takeaways

• Whenever possible, the criterion used to standardise a variable should be the direct measure of the property of interest.
• A measurable criterion (e.g. Brix refractometer reading, colour score, penetrometer reading) is stronger than an unmeasurable one (e.g. "similar appearance").
• The improvement should be specific to the question, not a generic method-improvement such as "use more samples".

Common Mistakes

• Saying "use apples of the same mass" — this is another proxy for growth, not for ripeness, and is not credited.
• Saying "use a colourimeter to measure the colour" is acceptable; the mark scheme accepts "measure colour", and a specific instrument is bonus detail.

Things to Be Careful About

• Stay within the biological indicators given in the stem: sugar content, colour, softness. Do not invent new criteria like "use the same cultivar" or "pick from the same branch" — these are controls, not ripeness measures.

Background Concept

A serial dilution reduces the concentration of a solution in controlled steps using the relationship $C_1V_1 = C_2V_2$, where $C_1$ and $V_1$ are the concentration and volume of the starting solution, and $C_2$ and $V_2$ are the concentration and volume of the diluted solution. The total volume of the diluted solution is the volume of stock plus the volume of diluent (here, distilled water).

In a two-step serial dilution, the output of the first step becomes the input to the second step. The mathematics is the same for each step, but the working out must be kept separate so that the volumes in step 2 are not confused with the volumes in step 1.

Understanding the Question

The student has a $0.01\ \text{mol dm}^{-3}$ stock of DCPIP and needs a $0.0005\ \text{mol dm}^{-3}$ working solution. They do this in two stages, producing an intermediate solution A of $0.001\ \text{mol dm}^{-3}$. The question gives the volumes of stock (10 cm³) and final water (50 cm³) for one of the two stages, and asks for the missing volumes in each stage.

Approach

For each step, apply $C_1V_1 = C_2V_2$, solve for the missing total volume $V_2$, and then subtract the known volume of stock/solution A to find the volume of distilled water required.

Step-by-Step Reasoning

Step 1

• $C_1 = 0.01\ \text{mol dm}^{-3}$ (stock)
• $V_1 = 10\ \text{cm}^3$ (volume of stock used)
• $C_2 = 0.001\ \text{mol dm}^{-3}$ (target concentration of solution A)
• Substitute into $C_1V_1 = C_2V_2$:
  $0.01 \times 10 = 0.001 \times V_2$, giving $V_2 = 100\ \text{cm}^3$.
• Distilled water to add = $V_2 - V_1 = 100 - 10 = 90\ \text{cm}^3$.

Step 2

• $C_1 = 0.001\ \text{mol dm}^{-3}$ (solution A)
• $C_2 = 0.0005\ \text{mol dm}^{-3}$ (final working solution)
• $V_2 = 50\ \text{cm}^3$ (solution A) $+ 50\ \text{cm}^3$ (distilled water) $= 100\ \text{cm}^3$.
• Substitute into $C_1V_1 = C_2V_2$:
  $0.001 \times V_1 = 0.0005 \times 100$, giving $V_1 = 50\ \text{cm}^3$.
• Volume of solution A required = $50\ \text{cm}^3$.

So the missing values are $90\ \text{cm}^3$ (in step 1) and $50\ \text{cm}^3$ (in step 2). Each is worth 1 mark.

Key Takeaways

• The dilution equation $C_1V_1 = C_2V_2$ requires the total volume of the diluted solution ($V_2$) to be used, not just the volume of diluent added.
• In a two-step dilution, the output of step 1 is the input to step 2 — keep the working separate and clearly labelled so the two stages are not muddled.
• A useful sanity check: in step 1, the stock is diluted by a factor of 10 (from 0.01 to 0.001), so the total volume must be 10× the stock volume (10 cm³ → 100 cm³), giving 90 cm³ of water. In step 2, the dilution factor is 2 (0.001 to 0.0005), so equal volumes of solution A and water are needed.

Common Mistakes

• Using $V_2$ as the volume of diluent (e.g. writing $V_2 = 100$ and giving the answer as "100 cm³ of water" instead of subtracting the 10 cm³ of stock).
• Dividing by the wrong concentration, e.g. using 0.01 instead of 0.001 in step 2, which would give 5 cm³ instead of 50 cm³.
• Forgetting that the units on both sides of the equation must be the same — the cm³ cancel cleanly as long as the same unit is used throughout.

Things to Be Careful About

• The $V_2$ in the equation refers to the total volume of the new solution, which equals (volume of stock + volume of water added). Always subtract the stock volume to get the volume of water.
• Double-check the direction of the dilution: if $C_2 < C_1$ then $V_2 > V_1$, and the answer must be a positive volume of water.

Background Concept

DCPIP titration. DCPIP (2,6-dichlorophenolindophenol) is a blue redox dye that is reduced by ascorbic acid to a colourless form. In a titration, a standardised DCPIP solution is added to a solution containing ascorbic acid; the end-point is the first drop of DCPIP that remains blue, because at that point all of the ascorbic acid has been oxidised and there is nothing left to reduce the next drop. The volume of DCPIP used is directly proportional to the concentration (or amount) of ascorbic acid in the sample.

Why standardise everything. Because the student wants to track how ascorbic acid concentration changes with ripeness, the only thing that should change between samples is ripeness. Anything else that varies (apple variety, storage temperature, mass of apple used, volume of extract pipetted, etc.) will introduce noise that masks the effect of ripeness, so each of these must be controlled.

Understanding the Question

The student has prepared a $0.0005\ \text{mol dm}^{-3}$ DCPIP solution in (c)(i). Now they need (i) a way to get the ascorbic acid out of the apple tissue in a form that can be titrated, and (ii) a titration procedure that gives reliable, comparable results across the ripening period. The method must be detailed enough for another person to follow, and it must include safety.

Approach

Structure the method in three blocks:

1. Sampling and standardisation — how often the apples are tested and what is held constant.
2. Extraction — how the apple is converted into a clear, measurable solution.
3. Titration — how the DCPIP is added to the extract and the result read off.

Add a safety section covering DCPIP and any sharp tools used to cut the apple.

Step-by-Step Reasoning

The mark scheme offers 14 possible points; any 9 score 9 marks. The points group naturally into the four blocks above.

Sampling and standardisation (mark-scheme points 1–3):

1. Test on at least 5 days. Ripening is a slow process; a single comparison between day 0 and day 8 would not capture the rise-peak-fall pattern. Five or more time points spread across the ripening period are needed.
2. Use apples from the same tree / variety. Different apple cultivars have very different ascorbic acid contents, and even within one cultivar, trees grown in different soils / climates differ. Mixing sources would add uncontrolled variation.
3. Same storage conditions (temperature, humidity, light) between tests. Storage conditions affect how quickly the apple ripens and how much ascorbic acid is lost to oxidation. Holding these constant removes them as confounders.

Extract preparation (mark-scheme points 4–7):

1. Remove the skin and seeds. The skin is low in ascorbic acid and the seeds contain compounds that could interfere with the titration; the flesh is the part actually being studied.
2. Use a set / stated mass of apple. Without a fixed mass, a $1\ \text{cm}^3$ sample of extract from a 5 g piece of apple contains a different amount of ascorbic acid than the same volume from a 20 g piece, so the titration result would not be comparable between days.
3. Grind the apple. Cells must be broken open to release the ascorbic acid; a mortar and pestle (with a small volume of water to help break the cells) is the standard method.
4. Centrifuge or filter the homogenate. The result must be a clear solution, not a suspension; otherwise the suspended solids would obscure the colour change of the DCPIP.

DCPIP titration (mark-scheme points 8–13):

1. Place a set / stated volume of the extract in a conical flask. The volume of extract titrated must be the same on every day, otherwise the volume of DCPIP used is not directly comparable.
2. Add DCPIP dropwise to the extract solution until the blue colour remains. This is the end-point: at this point, all the ascorbic acid has been oxidised and any further DCPIP stays blue because nothing in the extract can reduce it.
3. Shake / mix after each drop. The DCPIP must contact the ascorbic acid in the extract before the next drop is added; without mixing, locally the extract becomes saturated and the colour change is slow / uneven.
4. Measure and record the volume of DCPIP used. This is the dependent variable; record the difference between the final and initial burette readings.
5. Read the volume from the bottom of the meniscus (at eye level). Standard practice for reading a burette; avoids parallax error.
6. At least 3 replicates per day, then take a mean. Random variation between apples (and between extractions) means a single titration per day is unreliable. Three or more replicates allow a mean to be calculated and anomalies to be identified.

Safety (mark-scheme point 14):

1. State a hazard, the risk it poses, and the precaution to take. The mark scheme table gives credit for hazard + risk + precaution. Examples:
   • DCPIP is an irritant / toxin → wear gloves and goggles, wash hands after use.
   • Knife / scalpel used to cut the apple → cut on a board, away from the hand.
   • Some people are allergic to apples → do not eat the apple during the practical; allergy sufferers should not handle the extract.

A full 9-mark method covers all four blocks; usually a student will write 9 of the 14 possible points.

Key Takeaways

• A titration method needs (i) a defined end-point, (ii) a way to detect it, and (iii) a way to record the amount of titrant used.
• Standardising the variables that are not being studied is essential to isolate the effect of the variable that is.
• Replication (≥3) and a calculated mean are required for a reliable result.
• Safety points need to link a specific hazard to a specific risk and a specific precaution; vague answers like "be careful" are not credited.

Common Mistakes

• Saying "add DCPIP until the solution turns blue" without saying dropwise — this misses the pointwise addition needed to detect the end-point precisely.
• Omitting the meniscus reading point — a common error because it is assumed rather than stated.
• Replicating the dilution step from (c)(i) — the question explicitly says not to repeat the dilution details.
• Vague safety ("be careful with the chemicals") — the mark scheme requires hazard + risk + precaution, all three linked.
• Forgetting the control of variables: testing apples from different trees, or at different storage temperatures, makes the day-to-day comparison invalid.

Things to Be Careful About

• "DCPIP changes colour from blue to colourless when it is reduced by ascorbic acid" — so during the titration the blue disappears as the DCPIP is reduced. The end-point is the first drop that does not lose its blue colour, because at that point the ascorbic acid has run out.
• The mass of apple used and the volume of extract pipetted must both be fixed; if either changes between days, the DCPIP volume is not directly comparable.
• The method should be in a logical order (sampling → extract → titration → safety) so that another person could follow it; the mark scheme explicitly looks for logical set-out.