Colorimetry Virtual Lab: Glucose Calibration Curve

This colorimetry virtual lab follows the AQA A-level Biology required practical on glucose: a dilution series, a colorimetric calibration curve and an unknown "urine" sample. Students heat six glucose standards with Benedict's reagent, remove the red precipitate, choose a filter, zero with a blank and read every tube three times. The mean absorbances give a calibration curve, and the curve gives the unknown's concentration. Every value below was read from the simulation.

Curriculum links

• AQA A-level Biology (7402), required practical 11: a glucose dilution series and a colorimetric calibration curve to find the glucose concentration of an unknown "urine" sample (specification 3.6.4.2, control of blood glucose concentration).
• AP Chemistry, Topic 3.13 Beer–Lambert law (SAP-8.C): the same method as the guided-inquiry lab on the mass percent of copper in brass, which has its own preset (see Extension).

Simulic is not affiliated with or endorsed by AQA or the College Board.

Before the lab (5 min)

Ask students to commit to a prediction, on paper or as question 1 of the class link:

"Standards with more glucose are heated with Benedict's reagent, the red precipitate is removed, and the solution left is read through a red filter. What happens to the absorbance as the glucose concentration rises?"

Many students expect "more glucose, more color, higher absorbance".

Method in the simulation

1. Click the Calibration tab. Keep Assay: Glucose – Benedict's reagent (urine sample) and Standards: Proportional (to 10 mL), and leave Noise and Remove precipitate (centrifuge) ticked.
2. Read the dilution table: S1 to S6 hold 0 to 10 mL of 10 mmol/L stock made up to 10 mL, so 0 to 10 mmol/L.
3. Set Filter to Red 660 nm and press Zero (blank).
4. Choose Standard S1 under In colorimeter (or tap the tube) and press Read three times. Do the same for S2 to S6.
5. Choose Unknown and press Read three times. Keep Fit: Curve through points and read the concentration at the end of the readout.
6. Filter test: set Filter to Green 525 nm, press Zero (blank) again and read every tube three times.
7. Only now press Show true value and note your percentage error.

Tube	c (mmol/L)	Mean A, Red 660 nm	Mean A, Green 525 nm
S1	0
S2	2.00
S3	4.00
S4	6.00
S5	8.00
S6	10.0
Unknown	?

Expected results

The noise is seeded by the sample number: everyone on sample 1 gets these readings.

c (mmol/L)	0	2.00	4.00	6.00	8.00	10.0	Unknown
Mean A, Red 660 nm	0.898	0.780	0.676	0.583	0.498	0.432	0.521
Mean A, Green 525 nm	0.216	0.186	0.161	0.143	0.127	0.109	0.127

• The curve falls. More glucose leaves less blue copper(II) to absorb red light. It bends slightly; a straight line gave R² = 0.9911.
• Unknown: A = 0.521 lies between S4 and S5, giving 7.45 mmol/L. The true value is 7.82 mmol/L (error −4.7%).
• Filter: A fell by 0.466 across the standards with red but only 0.107 with green: the red curve is over four times steeper.
• Across all 20 sample numbers, red-filter results were never more than 0.37 mmol/L from the true value (mean 0.12); green-filter results were up to 1.05 mmol/L off (mean 0.30). S4 always read 0.576–0.602 at red.

Questions for students

1. (Prediction, asked again after the lab) What happens to the absorbance through a red filter as glucose concentration rises?
2. Which is a control variable?
3. What is the mean absorbance of S4 (6.00 mmol/L) with the red filter?
4. What is the glucose concentration of the unknown (sample 1)?
5. Why does the red filter give a more reliable result than the green filter?

Answers for teachers: (1) It falls, from 0.898 to 0.432. (2) The filter and the volume of Benedict's reagent in every tube. (3) Accept 0.56–0.62 (0.583 on sample 1). (4) Accept 7.0–8.3 mmol/L (7.45 from the curve; true value 7.82). (5) The blue solution absorbs red light, its complementary color, so the curve is steepest at 660 nm and the same reading noise gives a smaller error in concentration.

Common misconceptions

• "More glucose gives a higher absorbance." Here the blue copper(II) left over is measured, and it falls as glucose rises.
• "The filter should match the solution's color." A blue solution transmits blue light; a red filter measures what it absorbs.
• "The blank is the 0 mmol/L standard." The blank is distilled water; S1 holds Benedict's reagent and reads 0.898.

Extension

• Copper in brass (AP Chemistry): choose Assay: Copper in brass (Cu²⁺), set Red 660 nm, press Zero (blank) and keep the straight-line fit. On sample 1 the unknown gave 0.0922 mol/L, so 0.982 g of brass held 0.586 g of copper: 59.6% (true value 60.4%).

FAQ

Can the class link open on the Calibration tab?

Yes. In the link's starting values, set Screen to Calibration. Keep Assay (Calibration screen) on Glucose – Benedict's reagent (urine sample), Dilution of the standards (Calibration screen) on Proportional (to 10 mL), Unknown sample number (changes the unknown concentration) at 1 and Measurement noise (pipetting, reading) ticked, so the answers match.

What if I give groups different sample numbers?

Each sample number gives a different unknown, between 2.5 and 8.5 mmol/L. Questions 1, 2, 3 and 5 still work, but the range for question 4 only fits sample 1.

Can students just press Show true value?

Yes. Ask them to record their result first, then discuss the percentage error it shows.

Related simulations and guides

Preparing solutions – percentage concentration, molar concentration and dilution Food tests – sugars, starch, protein and fats in unknown foods

For absorbance, path length and wavelength on the other tabs, see the Beer's law virtual lab. For the qualitative Benedict's test, see the food tests virtual lab.