Determination of total reducing sugars - DNS method
Sucrose measurement
I. Introduction
The 3,5-dinitrosalicylic acid (DNS) method is the most widely used procedure for the
estimation of reducing sugars. This colorimetric assay involves the oxidation of the
aldehyde functional group present to the corresponding acid while DNS is simultaneously
reduced to 3-amino-5-nitrosalicylic acid under alkaline conditions (Figure 1). After
completion of reaction, it gives an intense red-brown color and intensity of color can be
read at 540 nm. Total reducing sugar content can be estimated from standard curve.
Figure 1. Colorimetric reaction between DNS and glucose.
Sucrose, commonly found in foods and vegetables, is a non-reducing disaccharide.
Consequently, DNS does not react with sucrose. However, indirect quantification of this
non-reducing sugar using DNS can be performed by first hydrolyzing it with
hydrochloric acid to yield glucose and fructose which are reducing sugars.
In this practical experiment, you will be given beverage samples to determine their
sucrose content.
II. Materials
- Glucose solution: 2 mg/mL
- Distilled water
- 5% HCl
- 5% NaOH
- Erlenmeyer (conical) flask, volumetric flask, micropipette
- DNS reagent
III. Procedure
3.1. Preparation of Standard Curve
- Prepare six test tubes containing varying concentrations of glucose according to
Table 1. For greater accuracy, run this step in duplicate.
Table 1. Preparation of Glucose Standard Solutions
Tube 1 2 3 4 5 6
2 mg/mL glucose solution (mL) 0 0.1 0.2 0.3 0.4 0.5
Distilled water (mL) 0.5 0.4 0.3 0.2 0.1 0
Vortex
Glucose concentration
(mg/mL)
- Add 1.5 mL of DNS solution.
- Vortex vigorously.
- Place the tubes in a boiling water bath for 5 minutes.
- Cool quickly the tubes with cold water.
- Measure the solution absorbance at 540 nm. Note: Read carefully the instruction
for use of the spectrophotometer before use and set the spectrophotometer to
zero by using the blank solution (0 mg/mL glucose)
- Complete the following Table 2.
Table 2. Absorbance versus glucose concentration
Glucose
concentration
Ci (mg/mL)
A540nm
- Prepare the standard curve by plotting the absorbance value for each glucose
standard vs. its concentration in mg/mL.
3.2. Hydrolysis of sucrose
- Add 0,4 mL of test sample (using a micropipette) to a Florence flask.
- Add 10 mL of 5% HCl (using a measuring cylinder).
- Add 19 mL of distilled water (using a measuring cylinder).
- Apply an air condenser to the flask.
- Place the flask into a boiling water bath for 30 minutes.
- Cool the flask with tap water.
- Neutralize the solution with 5% NaOH solution. Check the pH using pH paper.
- Transfer the entire mixture to a 100 mL volumetric flask.
- Carefully add distilled water to the mark, and mix well (solution A).
3.3. Dilution of the original sample
- Add to a 100 ml volumetric flask 0.4 ml of test sample.
- Carefully add distilled water to the mark, and mix well (solution B).
3.4. Determination of reducing sugars in solution A and solution B by DNS
method
Solution A:
- Add 0.5 mL solution A to a test tube.
- Add 1.5 ml of DNS reagent.
- Votex well.
- Place the tube into boiling water for 5 minutes.
- Cool quickly the tube with cold water.
- Measure the solution absorbance at 540 nm. Note: Read carefully the
instruction for use of the spectrophotometer before use and set the
spectrophotometer to zero by using the blank solution (0 mg/mL glucose)
Solution B:
- Add 0.5 mL solution B to a test tube.
- Add 1.5 ml of DNS reagent.
- Votex well.
- Place the tube into boiling water for 5 minutes.
- Cool quickly the tube with cold water.
- Measure the solution absorbance at 540 nm. Note: Read carefully the
instruction for use of the spectrophotometer before use and set the
spectrophotometer to zero by using the blank solution (0 mg/mL glucose)
Blank solution:
- Add to a suitable tube 0.5 ml of distilled water.
- Add 1.5 mL of DNS solution.
- Votex well.
- Place the tube into boiling water for 5 minutes.
- Cool quickly the tube with cold water.
- Use this solution to set the spectrophotometer to zero.
3.5. Calculation
Calculate the sucrose concentration (w/v) in the beverage samples.
