Estimation of Reducing Sugars by DNSA method

/Estimation of Reducing Sugars by DNSA method
Estimation of Reducing Sugars by DNSA method2018-06-05T07:49:05+00:00

Estimation of Reducing Sugars by DNSA method

Estimation of Reducing Sugars by DNSA method

Introduction

3,5-Dinitrosalicylic acid (DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm (In case of glucose). This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions

Requirements

  1. DNSA
  2. Sodium potassium tartarate
  3. 2 N sodium hydroxide (2N NaOH)
  4. Dinitro salicylic acid (DNSA)
  5. Distilled water
  6. Sample

Procedure

  • Prepare 20 mL of 2N NaOH.
  • Weigh 1 g DNSA and dissolve in 20 mL NaOH with the help of a magnetic stirrer
  • Weigh 30 g of sodium potassium tartarate and dissolve in 50 mL dH2O.
  • Slowly pour sodium potassium tartarate solution in the DNSA and NaOH solution and made the volume up to 100 mL (Note: Wait for the two to mix properly).
  • Decant the contents in a brown bottle. Filter if necessary.

Protocol

  1. Take eight tubes and label them as Blank and 1 to 7.
  2. Make dilutions of glucose standards
  3. Add 3 ml of DNSA reagent to all the eight test tubes. Mix well.
  4. Keep in boiling water bath for 15 minutes.
  5. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer at 540nm.
  6. First, take the absorbance (OD) of Blank and make it zero.
  7. Take the OD of all the tubes (No. 1-7). Wash the cuvettes each time after taking OD.
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