Biology 9700/33 — May/June 2013

Background Concept

Three standard biochemical tests identify which carbohydrate is in an unknown solution:

• Benedict's test detects reducing sugars (monosaccharides and some disaccharides such as maltose). On heating, reducing sugars reduce the blue Cu²⁺ in Benedict's reagent to brick-red Cu₂O. A negative result stays blue.
• Iodine test detects starch. The triiodide ion slips inside the helical amylose coil and forms a blue-black charge-transfer complex; without starch the iodine stays its original yellow/brown colour.
• Non-reducing sugars such as sucrose have no free aldehyde or ketone group, so they give a negative Benedict's result directly. They must first be hydrolysed by boiling with dilute hydrochloric acid (which splits the glycosidic bond) and then the acid must be neutralised with NaHCO₃ before Benedict's reagent will work — otherwise the acidic conditions prevent the test.

Understanding the Question

You have three solutions S1, S2 and S3, each containing one of glucose, starch or sucrose (the same molecule may be present in more than one solution). You must (i) decide which biological molecule to test for in each round, (ii) describe the method you used, (iii) record your observations and (iv) write a one-line conclusion for each test. Enzyme E is the substrate for part (b), not part (a).

The command word is essentially "identify" — your decision about which test to do first and which solutions to include is part of what earns marks.

Approach

Test for the simplest molecule first, then use the result to narrow down what to test next:

1. Test for glucose (Benedict's) on all three solutions in one go. Any solution that goes orange/red contains a reducing sugar; any that stay blue do not.
2. The solution(s) that stayed blue cannot contain glucose, so test only those for sucrose (acid-hydrolyse, neutralise, then Benedict's). If sucrose is present, hydrolysis releases glucose + fructose and Benedict's turns orange/red.
3. Any solution still unidentified is tested with iodine for starch.

This strategy minimises the number of tests and uses only the solutions that still need identifying.

Step-by-Step Reasoning

Test 1 — Glucose (reducing sugar) on S1, S2, S3

• Mix equal volumes ($2\ \text{cm}^3$) of Benedict's reagent and each solution; heat to $80\text{–}100\ ^{o}\text{C}$ for 2–3 minutes.
• Positive = colour change from blue to green, yellow, orange, red or brown.
• Negative = stays blue.
• In a typical paper, S3 turns orange/red while S1 and S2 stay blue → S3 contains glucose.

Test 2 — Sucrose (non-reducing sugar) on S1 and S2

• Test only S1 and S2; S3 has already been identified.
• Add $1\ \text{cm}^3$ HCl to $2\ \text{cm}^3$ of each, boil for ~1 min to hydrolyse sucrose → glucose + fructose.
• Neutralise with NaHCO₃ (or any named alkali) until bubbling stops.
• Add $2\ \text{cm}^3$ Benedict's reagent and re-heat.
• In a typical paper, S1 turns orange/red while S2 stays blue → S1 contains sucrose.

Test 3 — Starch on S2

• Test only S2 (the only solution still unidentified).
• Add a few drops of iodine solution. Positive = blue-black; negative = stays yellow/brown.
• In a typical paper, S2 turns blue-black → S2 contains starch.

All three solutions are now identified.

Key Takeaways

• Benedict's identifies reducing sugars directly; non-reducing sugars need acid hydrolysis + neutralisation first.
• Iodine is specific to starch.
• Strategic test ordering: glucose first (single direct test), sucrose second (acid hydrolysis), starch third (iodine) — testing only the solutions still unidentified at each stage.
• Always specify volumes, temperature ($80\text{–}100\ ^{o}\text{C}$ for Benedict's) and the named reagent (HCl, NaHCO₃, Benedict's, iodine).

Common Mistakes

• Saying "heat" without giving a temperature for Benedict's — the mark scheme credits heat only when a temperature ($80\text{–}100\ ^{o}\text{C}$, or "boil") is stated.
• Forgetting to neutralise the HCl before adding Benedict's — the acid stops the test working.
• Testing all three solutions for sucrose after the glucose test, including the one already shown to be glucose — wastes reagent and risks a false positive.
• Writing "brick red" as the positive colour for sucrose — the mark scheme ignores "brick" for the sucrose test; use orange/red/green/yellow/brown.

Things to Be Careful About

• The first Benedict's test must record results for all three solutions (S1, S2, S3).
• Volumes of Benedict's and the test solution should be equal; $2\ \text{cm}^3$ of each is the standard.
• The iodine test must NOT be heated — iodine sublimes on boiling.
• E is an irritant — avoid skin/eye contact and wash hands after use.

Background Concept

To investigate the effect of temperature on enzyme E, temperature must be the independent variable (the only thing deliberately changed) and the degree of hydrolysis must be the dependent variable (what you measure). Everything else (volumes of enzyme and substrate, the substrate used, the incubation time, the detection test) must be kept constant so that any change in the result can be attributed solely to the change in temperature.

Enzyme activity typically rises with temperature up to an optimum, then falls as the enzyme denatures; you therefore need a spread of temperatures across the range $5\text{–}100\ ^{o}\text{C}$ to capture both the rising and the falling portions of the activity curve.

Understanding the Question

The question asks you to modify the procedure used in (b)(ii) so that it becomes a fair test of temperature. The substrate (S2 / starch) and the enzyme (E) must stay the same; only the temperature changes. The mark scheme explicitly rejects any modification that also changes the substrate or the volume of Benedict's test temperature.

Approach

Lay out the modified procedure in terms of:

1. Independent variable — temperature, at least five values between $5$ and $100\ ^{o}\text{C}$.
2. Temperature control — thermostatic water bath (or hot and cold water baths) plus a thermometer; air-conditioned rooms and uncontrolled rooms are not credited.
3. Standardised variables — same volumes of E and of substrate (S2 / starch); or equilibrate E and substrate separately to the test temperature before mixing.
4. Dependent variable — test for hydrolysis after 5 minutes using Benedict's (for reducing sugars) or iodine (for remaining starch).

Step-by-Step Reasoning

1. Choose a minimum of five temperatures spread across $5\text{–}100\ ^{o}\text{C}$ (e.g. 20, 30, 40, 50, $60\ ^{o}\text{C}$).
2. For each temperature, place a beaker of E + S2 (starch) into a water bath pre-set to that temperature. The water bath is thermostatically controlled (or you use separate hot and cold baths to span the range); a thermometer verifies the actual temperature.
3. Keep the volume of E and the volume of substrate the same at every temperature (e.g. $2\ \text{cm}^3$ of each). This ensures any difference in hydrolysis is due to temperature, not to concentration.
4. To avoid the reaction starting before the substrate reaches the test temperature, equilibrate E and S2 separately in the bath for a minute or two, then combine them and start the 5-minute timer.
5. After 5 minutes, sample each mixture and run a Benedict's test (or add iodine). Record whether hydrolysis has occurred; repeat at each temperature to obtain a temperature–activity profile.

Key Takeaways

• Only temperature is changed; substrate and enzyme are held constant.
• A thermostatic water bath + thermometer is the accepted way to control and verify the temperature.
• Equilibration of E and substrate separately, then mixing, is good practice — it prevents the cold/hot reactants from altering the bath temperature and ensures the reaction begins at the intended temperature.
• Use ≥5 temperatures to span the full activity range and reveal any optimum.

Common Mistakes

• Changing another variable as well as temperature (e.g. swapping substrates between trials) — the mark scheme rejects this because it invalidates the comparison.
• Mentioning an air-conditioned room or a general "temperature-controlled room" instead of a water bath — these do not give precise enough control and are not credited.
• Boiling the Benedict's reagent at different temperatures along with the experimental temperature — the mark scheme rejects changing temperature specifically in the context of the Benedict's test.
• Omitting the thermometer — a water bath on its own without a way to verify the temperature is not credited.
• Using glucose as the substrate — glucose cannot be hydrolysed so no temperature effect would be measurable.

Things to Be Careful About

• Keep the substrate the same throughout (S2 / starch, or sucrose if you choose S1).
• "Same volume" can be any value in the range $2\ \text{cm}^3$ to $25\ \text{cm}^3$, but it must be the same at every temperature.
• The Benedict's test after incubation should be done in the standard way (heat to $80\text{–}100\ ^{o}\text{C}$), regardless of the experimental temperature — the experimental temperature affects only the enzyme reaction, not the detection test.