2-Aminoethanaminium 2-(ethoxycarbonyl)-4,6-dinitrophenolate as a greener route in reducing sugar quantification

Graphical abstract


Specification
Procedures: 3 ml of glucose solution 0.1-0.5 mg/ml is mixed with 3 ml of AED reagent (1 gm AED dissolved in 100 ml 1% sodium hydroxide contains 0.05 gm sodium bisulfite), the reaction mixure was heated at 90 C for 15 min then cooled and the absorbance is measured at 545 nm.
In this laboratory [2] the reducing sugar is extensively and routinely analyzed in sugarcane molasses, biomass pretreatment hydrolysate, and woody residual enzymatic hydrolysate and due to the lower Pk a of DNS that might be in relation to skin irritation in case of contact and its relatively high price we investigated this reagent AED under study in comparison with DNS in reducing sugar quantification prepared simply starting with a cheap available chemicals, ethyl salicylate, sulfuric acid, nitric acid and ethylenediamine and its intense non reduced and reduced color was successfully utilized to substitute DNS as well as the elimination of phenol a skin burning compound and rochelle salt from the redox reaction mixture as shown in the following table.
Comparison of DNS and its ethyl ester ethylendiamine proton transfer salt, AED, in assay parameters.

Additional information:
The presence of four nitro groups in the molecular structure of 2-aminoethanaminium 2-(ethoxycarbonyl)-4,6-dinitrophenolate [3] is responsible for its reduction under alkaline conditions at 90 C by the reducing action of glucose for 15 min in a basic medium giving a red optical density directly proportional to glucose concentration at 545 nm. Making a comparison between this salt and DNS, Miller procedures [4], in reducing sugar quantification concluded the higher sensitivity indicated by the absence of phenol in the redox reaction to obtain a red reduced color as well as the stability of this produced red optical density upto 6 h with a slight decrease in absorbance value noticed after 2 h then relatively became constant without any addition of both of phenol responsible for raising absorbance value fifth times in glucose concentration range 0.1-0.5 mg/ml than its absence and Rochelle salt responsible for reduced form color stabilization as it was reported in DNS Miller methodology reveals a superior greener route in reducing sugar analysis using this organic proton transfer salt.
The produced red optical density measured at 545 nm was found to obey accurately Beer-Lambert law and the linearity extends to a greater absorbance value upto 3, Beer-Lambert plot for both of 3,5-dinitrosalicylic acid using Miller procedures and AED aminium salt has been shown in Fig. 1.
The sensitivity of this salt in reducing sugar determination is related to its intense color a unique property of all proton transfer complexes and/or salts [5,6] that is in turn definitely gives a reduced form intense color as well as its molecular structure has a four nitro groups. On the other hand, the reduced form color stability may be attributed to its good solubility without adding a color stabilizer.
It is noteworthy 3,5-dinitrosalicylic acid and ethylenediamine reaction afforded di proton transfer from both of carboxylic and hydroxyl groups into one molecule of ethylenediamine and it was found in a monomer form containing also two nitro groups [7] in contrary to this case of ethyl 3,5-dinitrosalicylate ester (1) and ethylenediamine reaction afforded dimeric form (2).