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Estimation of Reducing Sugar by DNSA Method

Estimation of reducing sugar by DNSA method post we briefly summarises about: principle, reagents requirements, procedure, result, application and limitations of DNSA method.

Estimation of Reducing Sugar by DNSA Method

DNSA method provides a quick and simple estimate of the extent of saccharifications. By measuring the total amount of reducing sugars in the hydrolysate. Sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as reducing sugars. All monosaccharides are reducing sugars, along with some disaccharides, oligosaccharides, and polysaccharides.

The monosaccharides can be divided into two groups: the aldoses, which have an aldehyde group, and the ketoses, which have a ketone group. Ketoses must first tautomerize to aldoses before they can act as reducing sugars.

The common dietary monosaccharides galactose, glucose and fructose are all reducing sugars. Accurate determination of reducing sugars is necessary for various applications in food, agriculture and health industry. DNSA method is one of the classical method for estimation.


DNSA method for determining reducing sugar concentration was developed by Sumner and later modified by Miller.

When alkaline solution of 3, 5- dinitrosalicylic acid reacts aldehyde groups on the with reducing sugars (eg. Glucose, lactose) it is converted into 3-amino-5-nitrosalicylicacid which gives orange color which can be read at wavelength of light 540nm using spectrophotometer.

Water is used up as a reactant and oxygen gas is released during the reaction. Intensity of the colour is an index of reducing sugar.



  1. 1000 ml beaker
  2. Cuvettes
  3. Spectrophotometer
  4. Test tube stand
  5. Test tubes
  6. Water bath


  1. 0.05 M Acetate Buffer
  2. 3,4-Dinitrosalicyclic Acid
  3. Glucose
  4. Potassium Sodium Tartrate Tetrahydrate
  5. Sodium hydroxide


In 1000 ml beaker dissolve 10gm of 3,4-dinitrosalicyclic acid in 200 ml H2O. Followed by continuous stirring slowly adds a solution of NaOH dissolved in 150 ml distilled water. Incubate mixture in 50°C with stirring to obtain a clear solution. In small portions add 403 g of potassium sodium tartrate tetrahydrate. Filter mixture using paper filter and make up the volume to 1000 ml with water. Store in dark glass bottle at temperature below 20°C.


  1.  Prepare glucose standard solutions in 0.05 M acetate buffer (pH 4.8) ranging from 0.6-4.00 µmol/ml. Add 1 ml of each standard to separate tubes. To the tubes used as the blanks, add 1 ml of 0.05 M acetate buffer (pH 4.8).
  2. Prepare the unknown samples in an appropriate dilution. To each tube, add 1 ml of 0.05 M acetate buffer (pH 4.8) and mix. Add 3 ml DNS reagent to all the test tubes and mix well. Place the tubes in boiling water for 5 minutes. Cool the tubes to room temperature and measure the absorbance at 540 nm.


Estimation of Reducing Sugar by DNSA Method

The amount of carbohydrate in 1 g sample = —– mg/dl x dilution factor x 100

Dilution factor = final volume/initial (aliquot) volume.

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