Measuring Concentration of Glucose using a Calibration Curve

Introduction

This experiment uses a quantitative Benedict's test to determine the concentration of glucose in an unknown sample. By creating a calibration curve using known glucose concentrations, the absorbance of the unknown samples can be compared to estimate their glucose concentration.

Glucose is a reducing sugar that reduces Cu²⁺ ions in Benedict's solution to form insoluble copper(I) oxide, resulting in a colour change from blue to brick red. The intensity of this colour is directly proportional to the glucose concentration and can be measured using a colorimeter.

Equipment List

• 10 mmol $dm⁻³$ glucose solution
• Distilled water
• "Urine" samples with unknown glucose concentrations
• Benedict's solution
• Boiling tubes
• Boiling tube rack
• Water bath
• Colorimeter
• Cuvettes

Method

1. Prepare a dilution series of glucose using distilled water. Create six concentrations ranging from 0 to 10 mmol $dm⁻³$.
2. Place 2 cm³ of each dilution into separate boiling tubes.
3. Add 2 cm³ of Benedict's solution to each boiling tube.
4. Heat the boiling tubes in a water bath at 90°C for 4 minutes.
5. Remove the boiling tubes from the water bath using tongs and allow them to cool.
6. Zero the colorimeter using a cuvette filled with distilled water. Set the colorimeter to the red filter.
7. Transfer each sample into a cuvette and measure its absorbance using the colorimeter.
8. Plot the calibration curve: Absorbance (y-axis) against glucose concentration (x-axis).
9. Measure the absorbance of the unknown samples and use the calibration curve to determine their glucose concentrations.

Risk Assessment

Hazard	Risk	Safety Precaution	In Emergency	Risk Level
Benedict's solution	Irritant to skin and eyes	Wear eye protection	Wash eyes/skin with cold water	Low
Broken glass	Cuts from sharp objects	Take care when handling glassware; keep away from edges of desk	Elevate cuts; apply pressure; seek medical assistance	Low
Hot liquids	Risk of scalding	Use tongs to handle boiling tubes; wear heat-proof gloves	Run burn under cold water; seek medical assistance	Low

Graph

• Plot absorbance against known glucose concentrations to form the calibration curve.
• Add a line of best fit to identify the relationship.

Explanation

1. High glucose concentration in urine occurs because the β-cells of the islets of Langerhans in the pancreas produce insufficient insulin. This causes reduced uptake of glucose by liver cells, resulting in hyperglycaemia.
2. Glucose is filtered out of the blood in the glomerulus of the kidney, but its high concentration prevents full reabsorption by the proximal convoluted tubule, so it appears in the urine. This experiment effectively links practical skills with understanding of glucose metabolism and the biological basis of diabetes.