Biology 9700/31 — May/June 2013

Background Concept

Four standard biochemical tests identify the main organic molecules in plant extracts:

1. Starch test (iodine). Iodine (yellow-brown solution) forms a blue-black inclusion complex with the helical amylose component of starch. No heat is required. A negative result is the original iodine colour remaining.

2. Reducing-sugar test (Benedict's or Fehling's). Reducing sugars (monosaccharides and some disaccharides such as maltose) reduce blue Cu(II) ions in Benedict's solution to brick-red Cu(I) oxide when heated. The colour progresses blue → green → yellow → orange → red/brown depending on the concentration of reducing sugar.

3. Non-reducing-sugar test (sucrose). Sucrose is non-reducing, so it gives no reaction with Benedict's directly. It must first be hydrolysed with dilute hydrochloric acid to glucose + fructose (both reducing). The acid must then be neutralised (e.g. with sodium hydrogencarbonate or NaOH) before Benedict's is added — otherwise the alkaline conditions needed for Benedict's cannot be established.

4. Protein test (Biuret). Peptide bonds complex with Cu(II) in strongly alkaline solution (KOH/NaOH + $\text{CuSO}_4$, or premixed Biuret A + B) to give a violet/lilac/purple colour. A negative result is the blue colour unchanged.

Because the same green/yellow/orange/red precipitate forms in both the reducing-sugar test and the (post-hydrolysis) non-reducing-sugar test, the glucose test must be carried out first; otherwise a positive Benedict's result after hydrolysis could not be distinguished from glucose that was already present.

The xylem of a plant is dead at functional maturity and only transports water and dissolved mineral ions — it contains none of the four organic molecules listed. The phloem, by contrast, transports sucrose (the main transport sugar in plants) from photosynthetic sources to non-photosynthetic sinks.

Understanding the Question

You have five unknown plant extracts S1–S5. Four contain one of {glucose, protein, starch, sucrose} (with repeats allowed); the fifth — the xylem extract — contains none of these molecules. You must run four tests, in sequence, identifying only the as-yet-unidentified solutions each time, then state which molecule each solution contains and which is the xylem extract.

The expected outcome is:

• S1 = none (xylem)
• S2 = sucrose
• S3 = protein
• S4 = starch
• S5 = glucose

Approach

Sequence the tests so that no result is unambiguous:

• Test 1: starch (iodine) on all five solutions — a blue-black result is unambiguous because no other listed molecule gives this colour with iodine.
• Test 2: glucose (Benedict's, hot) on the four remaining solutions — once S4 is removed, any Benedict's-positive solution is glucose (sucrose does not react without prior hydrolysis).
• Test 3: sucrose (HCl hydrolysis → neutralise → Benedict's) on the three remaining solutions — only the solution containing sucrose (now hydrolysed to reducing sugars) will turn Benedict's positive.
• Test 4: protein (Biuret) on the two remaining solutions — S3 will turn lilac; S1 will stay blue, confirming it has no organic molecule (the xylem extract).

Use matched reagent volumes (Benedict's volume ≥ sample volume) and a boiling water bath (80–100 $\text{°C}$) for the Benedict's stages so that colour intensities are comparable.

Step-by-Step Reasoning

Test 1 — starch (iodine)

• Method: Add 2 drops of iodine solution to 2 $\text{cm}^3$ of each of S1–S5 in labelled tubes.
• Observation: S4 turns blue-black; the others remain yellow-brown (iodine colour).
• Conclusion: S4 contains starch. The amylose helix of starch forms an iodine inclusion complex that absorbs visible light to give the blue-black colour.

Test 2 — glucose (Benedict's)

• Method: To 2 $\text{cm}^3$ of each of the remaining S1, S2, S3 and S5, add 2 $\text{cm}^3$ of Benedict's solution. Heat in a boiling water bath (80–100 $\text{°C}$) for 2–3 min.
• Observation: S5 turns from blue to green/yellow/orange/red; the others stay blue.
• Conclusion: S5 contains glucose. The Cu(II) in Benedict's is reduced to Cu(I) oxide by the reducing sugar.

Test 3 — sucrose (HCl hydrolysis then Benedict's)

• Method: To 2 $\text{cm}^3$ of each of the remaining S1, S2, S3 add 2 $\text{cm}^3$ of dilute HCl and heat in the water bath for several minutes. Neutralise by adding sodium hydrogencarbonate until effervescence stops. Add 2 $\text{cm}^3$ of Benedict's and reheat.
• Observation: S2 turns green/yellow/orange/red; S1 and S3 stay blue.
• Conclusion: S2 contains sucrose. Acid hydrolysis cleaves the glycosidic bond, releasing glucose + fructose; both are reducing and react with Benedict's. The prior glucose test was negative for S2, so the Benedict's-positive result after hydrolysis is unambiguously due to sucrose.

Test 4 — protein (Biuret)

• Method: To 2 $\text{cm}^3$ of each of the remaining S1 and S3 add 2 $\text{cm}^3$ of Biuret reagent (or 2 $\text{cm}^3$ KOH + a few drops of $\text{CuSO}_4$).
• Observation: S3 turns lilac/purple/mauve/violet; S1 stays blue.
• Conclusion: S3 contains protein. Cu(II) in alkali complexes with peptide bonds to give the violet Biuret colour.

Final deduction
S1 is the only solution left and has been negative in every test, so it contains no biological molecule — consistent with it being the xylem extract.

Key Takeaways

• Order the four tests to remove ambiguity: starch → reducing sugar → non-reducing sugar → protein.
• Benedict's test cannot, by itself, distinguish reducing from non-reducing sugars; acid hydrolysis plus neutralisation is required.
• Always neutralise the HCl before adding Benedict's in the sucrose test.
• Use matched volumes and a boiling water bath so that Benedict's colour intensities are comparable.
• Iodine needs no heat; Biuret needs no heat; Benedict's does.
• The xylem extract contains no organic biological molecule because mature xylem vessels are dead and conduct only water and dissolved mineral ions.

Common Mistakes

• Doing the sucrose (hydrolysis) test before the glucose test. After hydrolysis, any Benedict's-positive result is ambiguous (could be glucose already present, or sucrose now hydrolysed). The mark scheme rejects sucrose conclusions unless the glucose test was done first.
• Re-testing solutions already identified — wastes reagent and confuses later readings (especially in the sucrose test, where pre-existing reducing sugar would give a false sucrose-positive).
• Forgetting to neutralise the acid in the sucrose test. In acid, Benedict's Cu(II)-citrate complex is broken down and the test fails.
• Imprecise colour descriptions: writing "blue" or "pink" instead of the accepted terms (blue-black; green/yellow/orange/red; lilac/purple/mauve/violet).
• Heating the iodine test — iodine sublimes and the test fails. The mark scheme explicitly rejects "heated" in the starch-test method.
• Omitting row lines in the test 1 results table — this is a separate marking point.

Things to Be Careful About

• Quote volumes in $\text{cm}^3$ (or mL) at least once — the mark scheme explicitly looks for this.
• Benedict's (or sum of Fehling's A + B) volume must be ≥ sample volume.
• Benedict's temperature: 80–100 $\text{°C}$ (boiling water bath). State the temperature explicitly.
• Sucrose test needs all three steps: acid + heat → neutralise → Benedict's + heat.
• Biuret positive colour is "lilac, purple, mauve, or violet" — not "pink" or "magenta".
• Starch positive colour is "blue-black, black, navy blue, or dark blue" — not just "blue".
• The final sentence ("the remaining solution") must identify S1 and say it "contains no biological molecules" or "none" — not "water" or "xylem".
• Draw horizontal lines between every row of the test 1 results table (separate marking point).

Background Concept

The phloem is the living vascular tissue that translocates organic solutes — predominantly sucrose — from "sources" (photosynthetic leaves, storage tissues) to "sinks" (growing tips, roots, fruits, seeds). Sucrose is the transport sugar of choice because it is highly soluble, chemically stable in transit, and non-reducing (so it does not react with proteins during translocation).

The xylem, by contrast, is dead at functional maturity and transports only water and dissolved mineral ions — no significant organic molecules.

Understanding the Question

From part (a)(i) you have identified the contents of each solution. You are now asked to identify the plant tissue of origin (phloem) using only the biochemical result.

Approach

Match the biochemical identity of each extract to the tissue that would carry it in the highest concentration. Sucrose is the principal transport sugar of plants, so the sucrose-positive extract must be the phloem sap.

Step-by-Step Reasoning

The mark scheme shows that the only extract giving a positive Benedict's test after acid hydrolysis (i.e. sucrose-positive) is S2. Sucrose is the molecule translocated in the phloem, so S2 must be the phloem extract.

(For completeness, S1 is negative for every test and is therefore the xylem extract; xylem is dead tissue carrying only water and minerals.)

Key Takeaways

• Phloem transports sucrose; xylem transports water and dissolved mineral ions.
• The biochemistry of an extract (which biological molecule is present) can be used to identify the source tissue.

Common Mistakes

• Choosing S5 (glucose). Glucose is rarely the main transport sugar in plants — sucrose is. Glucose-positive extracts typically come from storage tissues (e.g. ripe fruit) where free monosaccharides accumulate.
• Choosing S4 (starch). Starch is an insoluble storage polysaccharide in amyloplasts of storage organs (e.g. potato tubers, seeds), not in transport tissues.
• Choosing S3 (protein). Proteins are not the main transport solute of phloem (only small amounts of amino acids are).

Things to Be Careful About

• The transport sugar is specifically sucrose because it is non-reducing — this is what the question tests.