The analytical procedures by NREL including "Determination of structural carbohydrates and lignin in biomass" are very well established and widely used. Please see link below for further info and details of procedures:

https://www.nrel.gov/bioenergy/biomass-compositional-analysis.html

Hope this is of help.

Cheers.

Methods are available for the efficient fractionation of ligninocellulosic biomasses into cellulosic, hemicellulosic and lignin fraction, wherein concentrated organic vapours are applied to the biomass at elevated temperatures to at least partly depolymerize or substantially solubilize the hemicellulose and lignins in the biomass. The organic acid - treated biomass may be processed into desired chemicals, fuels and/or fuel additives at a second facility. For more details consult Google Patents US8637282B2 US grant

The method that uses 72% H2SO4 will hydrolyze and dissolve carbohydrates, while acid-insoluble lignin will be obtained in the solids form. For fractionation of lignocellulosic materials, especially for bioethanol production, you can use the kraft process using NaOH and Na2S solutions. The maximum temperature of cooking is around 170 oC with 4 hours of cooking time. After that, pulp will be obtained from delignification of lignocellulosic materials. Carbohydrates will be obtained in the solids form, while lignin will dissolve. The process of saccharification and fermentation using cellulase enzymes was about 8% of the dry weight of the sample while fermentation using S.cereviciae fungus extract (1.5 x109 CFU / cc) using simultaneous saccharification and fermentation method (SSF).

Please see link below:

Article Bioethanol Production from Several Tropical Wood Species usi...

Thanks for your suggestions. Will follow up for next experiments.

My problem was solved by constant stirring in shaker, for 3 hours at 50 C, using 72% H2SO4. I got the same colors of the replicates after the digestion. But for proper quantification of the sugar content, I am follwoing this method. Please guide me to perform the microplate assay.

For fractionation of the lignocellulosic biomass, I need to perform quantification of the cellulose and hemicellulose content. For this purpose, I have performed acid digestion of the sample (72% H2SO4), and filtered the samples. Then prepared the DNS follwing this method: (fOR 100ml)

3, 5 DNS 0.63g, Phenol 0.5g, NaOH 0.5g, Na-tartrate 18g. Dissolved in 100ml water and heated for 5 mins at 70 C.

Now, for 96-well plate assay, I need help that how much DNS should be put in the microplate with the filtered digested sample?

The below mentioned researcher has many articles on extraction of cellulose, hemicellulose, and lignin from many types of biomass. Happy new year.

https://www.researchgate.net/profile/Yehia_Fahmy

Thanks for your answer Tamer

the best method i have ever tried and got great result was washing with hot water follow by organic sulfonic acid treatment in low temperature (