For the determination of reducing sugars the method with 1% DNSA seems fine, but two wavelengths at 540 nm (maximum absorbance?) And 575 nm are recommended as described by Miller 1959 in the original paper. Please tell me: 540 or 575 nm, which might be the right one?
Thanks for the reply. Indeed 540 is more often used, but 575 nm is also used. If possible, please provide some scientific explanations. I found that: "DNS reacts with free carbonyl group of the RS under alkaline condition, forming
3-amino-5-nitrosalicylic acid, an aromatic compound with maximum absorption at 540 nm, allowing a quantitative spectrophotometer measurement of the amount of RS present". Initially Miller (1959), who is cited by most researchers, made the determination at 575 nm. From our experiences the values are much higher (even 3 times) at 540 nm (maximum absorbance), compared to 575 nm. Several methods of profile universities recommend 575 nm for RS determination:
https://user.eng.umd.edu/~nsw/ench485/lab4a.htm
http://2018.igem.org/Team:UBonn_HBRS/Description/Enzyme_Assays
https://pdfs.semanticscholar.org/e697/fb2ceefbff1c296b2d62ceb4c3fa42fd3590.pdf
So, the question remains 540 (RS maximum absorption) or 575 nm (why?) ?
Also, more topics are open:
https://www.researchgate.net/post/Why-cant-I-get-the-trendline-in-the-standard-curve-for-estimation-of-reducing-sugars-by-DNS-methodMiller-1958
https://www.researchgate.net/post/How-to-prepare-stock-standard-sugar-for-DNS-method
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