Colorimetric Determination of Glucose

Measuring blood glucose analog using the DNS method

Colorimetry Intermediate (High School) 60 min ~$10.00

Objective

Determine glucose concentration using the dinitrosalicylic acid (DNS) colorimetric method and understand reducing sugar chemistry.

Background

The DNS method is a classic colorimetric assay for reducing sugars. 3,5-dinitrosalicylic acid (yellow) is reduced by the free aldehyde group of glucose to 3-amino-5-nitrosalicylic acid (orange-red). The color change occurs upon heating, and the intensity at 540 nm is proportional to the reducing sugar concentration. This method is widely used in food science and fermentation monitoring.

Safety Warnings

  • DNS reagent contains NaOH — corrosive to skin
  • Boiling water bath — handle tubes with tongs
  • Cool tubes before opening to prevent splattering
  • DNS reagent stains skin yellow

Required PPE

goggles gloves lab_coat

Materials

  • DNS reagent (100 mL)
    1% DNS in NaOH with sodium potassium tartrate
  • Glucose stock solution (100 mL)
    10 mg/mL in water
  • Unknown sugar solution (20 mL)
  • Distilled water (200 mL)

Equipment

Spectrophotometer or colorimeter Cuvettes Boiling water bath Test tubes (10) with caps Pipettes (1 mL, 5 mL) Ice bath Timer

Procedure

1

Prepare glucose standards: 0, 1, 2, 4, 6, 8, 10 mg/mL by diluting the stock solution.

10 min
2

Add 1 mL of each standard and the unknown to labeled test tubes. Add 1 mL of DNS reagent to each tube.

5 min DNS contains NaOH
3

Place all tubes in a boiling water bath for exactly 5 minutes. The color changes from yellow to orange-red.

5 min Use tongs for hot tubes
4

Transfer tubes immediately to an ice bath to stop the reaction. Cool for 2 minutes.

2 min
5

Add 8 mL of distilled water to each tube and mix well.

5 min
6

Measure absorbance at 540 nm. Zero with the blank (0 mg/mL glucose).

15 min
7

Plot the calibration curve and determine the glucose concentration of the unknown.

18 min

Expected Results

A linear calibration curve from 1–10 mg/mL glucose. The color intensifies from pale yellow (blank) to deep orange-red (10 mg/mL). Non-reducing sugars like sucrose give no color change.

Cleanup

Neutralize DNS waste with dilute HCl before disposal. Clean test tubes immediately as dried DNS residue is difficult to remove.

Details

Category
Colorimetry
Difficulty
Intermediate (High School)
Duration
60 min
Est. Cost
$10.00
Steps
7
Materials
4

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Frequently Asked Questions

What is the objective of Colorimetric Determination of Glucose?
Determine glucose concentration using the dinitrosalicylic acid (DNS) colorimetric method and understand reducing sugar chemistry.
How difficult is Colorimetric Determination of Glucose?
This experiment is rated as Intermediate (High School). It takes approximately 60 minutes to complete.
What safety precautions are needed for Colorimetric Determination of Glucose?
Key safety precautions include: DNS reagent contains NaOH — corrosive to skin; Boiling water bath — handle tubes with tongs; Cool tubes before opening to prevent splattering.
What materials are needed for Colorimetric Determination of Glucose?
The main materials required are: DNS reagent, Glucose stock solution, Unknown sugar solution, Distilled water.
What results should I expect from Colorimetric Determination of Glucose?
A linear calibration curve from 1–10 mg/mL glucose. The color intensifies from pale yellow (blank) to deep orange-red (10 mg/mL). Non-reducing sugars like sucrose give no color change.