Dear Santosh,

Please read the following text:

GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC METHOD

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:

oxidation

aldehyde group ----------> carboxyl group

reduction

3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen.The above reaction scheme shows that one mole of sugar will react with one mole of 3,5-dinitrosalicylic acid. However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is more complicated than that previously described. The type of side reaction depends on the exact nature of the reducing sugars. Different reducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of sugar also competes for the availability of 3,5-dinitrosalicylic acid. As a consequence, carboxymethyl cellulose can affect the calibration curve by enhancing the intensity of the developed color.

Although this is a convenient and relatively inexpensive method, due to the relatively low specificity, one must run blanks diligently if the colorimetric results are to be interpreted correctly and accurately. One can determine the background absorption on the original cellulose substrate solution by adding cellulase, immediately stopping the reaction, and measuring the absorbance, i.e. following exactly the same procedures for the actual samples. When the effects of extraneous compounds are not known, one can effectively include a so-called internal standard by first fully developing the color for the unknown sample; then, a known amount of sugar is added to this sample. The increase in the absorbance upon the second color development is equivalent to the incremental amount of sugar added.

References

Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426, 1959.

Hoping this will be helpful,

Rafik

Dear Santosh Kumar,

There are many methods by which we can determine the reducing sugar. Several colorimetric methods are phenol-sulfuric acid, anthrone-sulfuric acid, 3,5-dinitrosalicylic acid (DNS), potassium ferric hexacyanide reagent (Prussian blue), and the Nelson-Somogyi (molybdenum blue) methods. According to me the DNSA method is more suitable and easy for reducing sugar determination and widely accepted too. You can follow the above link given by Dr. Rafik. All The Best.

-AKM

Hi Dr Rafik, glucose has the carbonyl group when it's straight-chained. And i read on Quora that straight-chain glucose predominates in the solid form but in aqueous solutions, it converts to the ring form. How then does this DNS method work on glucose in aqueous solutions? Thanks a lot!

Dear Santosh

I have attached the article which explain the reaction between DNS and reducing sugar

which test sample should be take for estimate reducing sugar by DNSA method protocol

Germinating seed. ( any - Oats, Phaseolus Mungo etc. work great )@ Deepak Pawar