I was trying to pretreat rice straw with 5-10 % NaOH at 90 degrees. After that, I used the NREL method (simplified) for lignin estimation as follows:

The pH of the pretreated biomass slurry was first neutralized using 1 N HCL before lignin estimation.

The extractive free biomass (acetone wash and boiling in water) was treated with 72% sulfuric acid for 4 hours, followed by boiling the slurry with 150 ml of 1 N sulfuric acid for an hour. Then to the mixture, 400 ml of water is added for neutralization, filtered, and dried. Normal cellulose-acetate filter paper was used for filtration here.

The raw rice straw shows Lignin conteny in the range of 20-22 %, which is near to that of reported values in the literature. However, the pretreated biomass was always showing values more than the raw rice straw. I made several trial to check for any improvement. But most of the time, the lignin cotent of pretreated biomass was higher.

Why does the lignin in pretreated samples increase above or comes near to the value of raw samples? Am doing something wrong or missing something in the process?

Is there any alternative to the NREL method?

If somebody could help, it will be very helpful.