Required Practical 11: Dilution Series of Glucose and Calibration Curve
Laura Armstrong
Teacher
Contents
Recall Questions
This topic requires prior knowledge of biochemical tests. You can test your knowledge below.
What colour change occurs when a reducing sugar is heated with Benedict’s solution?
Blue to brick red precipitate, colour change depends on concentration.
Why is it important to control variables in an experiment?
To ensure that only the independent variable affects the outcome, improving validity.
What is the effect of increasing substrate concentration on enzyme activity?
Increases the rate of reaction up to a point, after which it plateaus due to saturation of enzyme active sites.
Topic Explainer Video
Check out this @JoeDoesBiology video that explains required practical 11 or read the full notes below. Once you've gone through the whole note, try out the practice questions!
Required Practical 11
- Create a range of known concentrations of solution.
- Use a colorimeter to measure light absorbance or transmission.
- Plot a calibration curve of absorbance/ transmission against known concentration.
- Use the curve to find the concentration of an unknown sample.
Step-by-Step: How to Prepare a Dilution Series
Goal: Create a set of known concentrations from a single stock solution (e.g. 10 mmol dm⁻³ glucose).
Each dilution is prepared by taking part of the stock solution and adding distilled water up to the same volume.
Example: All of these solutions will have a total volume of 10 cm³
| Tube | Stock (cm³) | Distilled Water (cm³) | Final Concentration |
| 1 | 10 | 0 | 10.0 mmol dm⁻³ |
| 2 | 8 | 2 | 8.0 mmol dm⁻³ |
| 3 | 6 | 4 | 6.0 mmol dm⁻³ |
| 4 | 4 | 6 | 4.0 mmol dm⁻³ |
| 5 | 2 | 8 | 2.0 mmol dm⁻³ |
Using a Colorimeter
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Perform a test that causes a colour change proportional to concentration.
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e.g. Benedict’s test for reducing sugars.
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Filter the solution to remove precipitate (optional).
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Use a cuvette and set the colorimeter to the correct wavelength (red filter for Benedict’s test).
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Calibrate using a blank (e.g. distilled water) this prevents a zero error.
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Record absorbance or transmission values for each known sample.
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Make sure to control variables, such as volume of Benedict's reagent and volume of solution being tested.
Plotting the Calibration Curve
- X-axis: Concentration of known samples.
- Y-axis: Absorbance or transmission.
- Plot your points and draw a smooth curve or line of best fit.
This curve allows you to interpolate the concentration of an unknown sample by matching its absorbance. For example if an unknown sample has a transmission of 1.2 the concentration of glucose will be 7 mmol dm-3 according to the calibration curve below.
Key Terms
- Calibration Curve: Graph showing known concentrations against absorbance/transmission.
- Serial Dilution: Series of stepwise dilutions to produce known concentrations.
- Colorimeter: Device that measures how much light is absorbed or transmitted.
- Absorbance: How much light is absorbed by the sample (increases with concentration).
- Transmission: How much light passes through the sample (decreases with concentration).
No answer provided.
Exam Tip
A colorimeter allows quantitative comparison — more accurate than observing colour changes by eye.
No answer provided.
Explain how you would create a calibration curve to determine the glucose concentration in an unknown solution using Benedict’s test and a colorimeter. (5 marks)
- Prepare a set of known glucose concentrations.
- Add Benedict’s reagent to each and heat in a water bath.
- Use same volume of Benedicts reagent and same volume of solution in each test.
- Use a colorimeter to measure the absorbance of each solution.
- Plot absorbance on y-axis against concentration on x-axis and draw the line of best fit to form a calibration curve.
- Use the curve to determine the concentration of the unknown solution from its absorbance.
Practice Question
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!