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Estimation of Reducing Sugar by DNS Method

In this estimation of reducing sugar by DNS method post we briefly summarise about DNS method principle, reagents requirements, procedure, result, application and limitations of estimation of reducing sugar.

Estimation of Reducing Sugar by DNS Method

Carbohydrates are the most abundant class of organic compounds found in living organisms. Carbohydrates are a major source of metabolic energy, both for plants and for animals and they also serve as a structural material (cellulose), a component of the energy transport compound ATP, recognition sites on cell surfaces, and one of three essential components of DNA and RNA. In order to measure the concentration of carbohydrates present in a solution, either Dinitro Salicylic Acid (DNS) method can be followed.

Principle

3, 5-Dinitrosalicylic acid (DNS) is used extensively in biochemistry for the estimation of reducing sugars. It detects the presence of free carbonyl group (C=O) of reducing sugars. This involves the oxidation of the aldehyde functional group (in glucose) and the ketone functional group (in fructose). 

During this reaction dinitrosalicylic acid method is reduced to 3- amino5-nitrosalicylic acid (ANSA) which under alkaline conditions is converted to a reddish brown coloured complex which has an absorbance maximum of 540 nm.

Estimation of Reducing Sugar by DNS Method

Estimation of Reducing Sugar by DNS Method

Requirements

Equipment’s

  1. Spectrophotometer
  2. Test tube stand
  3. Test tubes
  4. Water bath

Chemicals

  1. Sodium hydroxide
  2. Distilled water
  3. Sample
  4. Sodium potassium tartarate

Reagents

1. DNS reagent

In 1000 ml beaker dissolve 10 g of 3,4-dinitrosalicyclic acid in 200 ml H2O. Followed by continuous stirring slowly add 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

2. Sugar solution

Stock standard sugar sodium: 250 mg of glucose in water and make up the volume to 100 mL.

Working standard sodium: Take 10 mL from this stock solution and make up the volume to 100 mL.

Procedure

  1. 7 clean, dry test tubes are required for DNS method. Pipette out standard sugar solution in different test tubes in the range of 0 to 3 mL and fill all test tubes to 3 mL with distilled water concentrations ranging from 0 to 750 mg.
  2. Add 1 mL Dinitro salicylic Acid DNS reagent to each test tube and mix. Plug the test tube with cotton or marble and place it in a boiling water bath for 5 minutes.
  3. Take the tubes and allow them to cool to room temperature. Extinction was measured at 540 mm against a blank.
  4. Please keep in mind that because absorbance is temperature sensitive, all tubes must be cooled to room temperature before reading.
  5. Create standard curves for the sugars supplied and use them to estimate the concentration of the unknowns supplied.

Calculation

Dinitrosalicylic Acid Method

Carbohydrate Estimation by DNS method showing increasing amount of sugar concentration

The 100 mL of unknown solution contains……….. mg of glucose.

Dinitrosalicylic Acid Method

Application

DNS method is a more sensitive and user friendly reagent than Benedict’s reagent. It is ideal for measuring the action of enzymes that produce reducing sugars, such as invertase, cellulase, and amylase, when used in conjunction with a colorimeter.

Advantages

DNS method is a simple and inexpensive method. If glucose is the only product, the DNSA assay can be used as an accurate analytical method for evaluating reducing sugars in both pure solutions and supernatants from enzymatic saccharifications of purified cellulosics.

Limitations

Because the DNS method has a low specificity, it is critical to run blanks diligently if the calorimetric results are to be interpreted correctly and accurately. Because DNS method reagent is destructive to polysaccharides, it cannot be used to measure pectinase activity against pectins. Overestimation is also associated with the DNS method.

Further Readings

Reference

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