oxidation aldehyde group ----------> carboxyl group reduction 3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acidBecause 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.
3ml 1ml O.D. reagent --->----+ Rochelle soln --->----++-------> at 575nm | | | | || | +-+--+ +-++-+ | | heat | | | | --------> | | | | | | | | | | |____| |____| 3ml sample soln