describe and carry out a semi-quantitative Benedict’s test on a reducing sugar solution by standardising the test and using the results (time to first colour change or comparison to colour standards) to estimate the concentration

Testing for Biological Molecules

Objective

To describe and carry out a semi‑quantitative Benedict’s test on a reducing sugar solution by standardising the test and using the results (time to first colour change or comparison to colour standards) to estimate the concentration of the sugar.

1. Principle of the Benedict’s Test

Benedict’s reagent contains copper(II) sulphate, sodium carbonate and sodium citrate. In an alkaline medium, reducing sugars (e.g., glucose, fructose) reduce Cu²⁺ ions to Cu⁺, which precipitate as a coloured cuprous oxide (Cu₂O). The colour of the precipitate varies with the amount of reducing sugar present:

• Blue – no reducing sugar
• Green – low concentration
• Yellow – moderate concentration
• Orange – high concentration
• Brick‑red – very high concentration

2. Materials and Reagents

• Benedict’s solution (freshly prepared)
• Distilled water
• Glucose (or another reducing sugar) – analytical grade
• Test tubes (clean, dry)
• Water bath (maintained at 95 °C)
• Timer or stopwatch
• Colour comparison chart (see Table 1)
• Pipettes (1 mL and 5 mL)
• Protective lab wear (gloves, goggles, lab coat)

3. Preparation of Standard Reducing‑Sugar Solutions

Prepare a series of glucose standards covering the expected concentration range of the unknown sample.

1. Weigh 0.180 g, 0.360 g, 0.540 g, 0.720 g and 0.900 g of glucose (each to 0.001 g).
2. Dissolve each mass in a 100 mL volumetric flask and make up to the mark with distilled water. The resulting concentrations are 0.10 % w/v, 0.20 % w/v, 0.30 % w/v, 0.40 % w/v and 0.50 % w/v respectively.
3. Label the flasks as Standard A (0.10 %), B (0.20 %), C (0.30 %), D (0.40 %) and E (0.50 %).

4. Standardisation Procedure

1. Place 2 mL of Benedict’s solution into each of five clean test tubes.
2. Add 1 mL of each standard solution to a separate tube (A–E). Mix gently.
3. Place the tubes in the water bath at 95 °C.
4. Start the timer as soon as the tubes are immersed.
5. Observe the time taken for the first visible colour change (from blue to green) and record the final colour after 5 minutes.
6. Compare the final colour with the colour standards in Table 1 and note the corresponding concentration.

5. Testing an Unknown Reducing‑Sugar Solution

1. Place 2 mL of Benedict’s solution into a clean test tube.
2. Add 1 mL of the unknown sample. Mix gently.
3. Immerse the tube in the 95 °C water bath and start the timer.
4. Record the time taken for the first colour change.
5. After 5 minutes, compare the final colour with the colour standards (Table 1) to obtain an approximate concentration.
6. If the colour falls between two standards, estimate the concentration by linear interpolation.

6. Recording Results

7. Colour Standards (Table 1)

8. Estimating Concentration from Time to Colour Change

For a more quantitative approach, plot the recorded times (seconds) for the standards against their known concentrations and fit a straight line (linear regression). The concentration of the unknown (\$C{\text{U}}\$) can then be estimated from its recorded time (\$t{\text{U}}\$) using the equation of the line:

\$C = m\,t + b\$

where \$m\$ is the slope and \$b\$ the intercept obtained from the standard data.

9. Safety and Waste Disposal

• Wear gloves, goggles and a lab coat at all times.
• Benedict’s solution is alkaline and may cause skin irritation – avoid direct contact.
• Dispose of copper‑containing waste in the designated heavy‑metal waste container.
• Never heat sealed tubes; use open tubes in a water bath.

10. Extension Questions

1. Why is sodium citrate added to Benedict’s reagent?
2. Explain why non‑reducing sugars (e.g., sucrose) give a negative result unless hydrolysed first.
3. How would the presence of interfering substances (e.g., ascorbic acid) affect the test?
4. Design an experiment to determine the detection limit of the Benedict’s test under the conditions described.