Measuring Concentration of Glucose Using a Calibration Curve (AQA A-Level Biology): Revision Notes
Measuring Concentration of Glucose Using a Calibration Curve
Purpose and principle
This practical uses a quantitative Benedict's test to determine unknown glucose concentrations in simulated urine samples. The technique relies on creating a calibration curve using solutions of known glucose concentration.
Benedict's reagent contains Cu²⁺ ions which are reduced by glucose (a reducing sugar) to form insoluble copper(I) oxide. This reaction produces a characteristic colour change from blue to brick red. The intensity of this colour change is directly proportional to the glucose concentration present.
The colour intensity can be measured using a colorimeter, which measures absorbance. By plotting absorbance values against known glucose concentrations, you create a calibration curve that allows determination of unknown concentrations through interpolation.
Apparatus and materials
- 10 mmol dm⁻³ glucose solution
- Distilled water
- Unknown 'urine' samples
- Benedict's solution
- Boiling tubes and rack
- Water bath
- Colorimeter
- Cuvettes
- Measuring pipettes
- Tongs for handling hot tubes
Method
- Prepare a dilution series using the stock glucose solution and distilled water. Create six different concentrations ranging from 0 to 10 mmol dm⁻³ in separate boiling tubes.
- Add unknown samples by placing 2 cm³ of each unknown sample into separate boiling tubes.
- Add Benedict's reagent - place 2 cm³ of Benedict's solution into each boiling tube containing either known or unknown glucose solutions.
- Heat the samples by placing all tubes in a water bath maintained at 90°C for exactly four minutes.
- Cool the samples by removing tubes with tongs and allowing them to reach room temperature.
- Calibrate the colorimeter by setting it to zero absorbance using a cuvette containing distilled water. Ensure the colorimeter uses a red philtre.
- Measure known standards by transferring each glucose standard to a cuvette and recording its absorbance value.
- Create the calibration curve by plotting absorbance (y-axis) against known glucose concentration (x-axis). Draw the line of best fit through the data points.
- Analyse unknown samples by measuring their absorbance and using the calibration curve to determine their glucose concentrations.
Ensure all samples are heated for exactly the same duration (4 minutes) to maintain consistency in the colour development reaction.
Safety considerations
This practical involves low-risk hazards that require standard laboratory precautions:
- Benedict's solution can irritate skin and eyes. Wear eye protection and wash immediately with cold water if contact occurs.
- Hot liquids present a scalding risk. Use tongs when handling tubes from the water bath, keep materials away from desk edges, and run burns under cold water while seeking assistance.
- Broken glassware creates cuts from sharp objects. Handle carefully and do not attempt to remove glass fragments from wounds - seek medical assistance.
Data collection and processing
Record absorbance readings for each known glucose concentration in a suitable table, including appropriate units (mmol dm⁻³ for concentration).
Plot the calibration curve with absorbance on the y-axis and glucose concentration on the x-axis. Draw a line of best fit through the points to enable accurate interpolation of unknown values.
Data Recording Tip: Include at least three significant figures in your absorbance measurements and ensure your table has clear headings with appropriate units.
Analysis and interpretation
The practical typically requires identification of which unknown sample indicates high diabetes risk. Normal blood glucose concentration ranges from 0-0.8 mmol dm⁻³.
Worked Example: Diabetes Detection
If an unknown urine sample shows an absorbance of 0.45, and your calibration curve indicates this corresponds to 5.2 mmol dm⁻³ glucose:
Step 1: Compare to normal range (0-0.8 mmol dm⁻³)
Step 2: Since 5.2 > 0.8, this indicates abnormally high glucose
Step 3: Conclusion - This sample suggests potential diabetes risk
High glucose concentration in urine suggests diabetes because insufficient insulin production by β islets of Langerhans and reduced insulin sensitivity in liver cells leads to elevated blood glucose. When blood glucose exceeds the kidney's reabsorption capacity, glucose appears in urine after filtration through the glomerulus because it cannot be completely reabsorbed in the proximal convoluted tubule.
Key Points to Remember:
- Benedict's test produces a blue to brick red colour change with reducing sugars
- Absorbance is directly proportional to glucose concentration
- Normal urine glucose should be 0-0.8 mmol dm⁻³
- High glucose in urine indicates potential diabetes through kidney filtration overflow
- The red philtre in the colorimeter optimises detection of the brick red copper(I) oxide product