In Colorado, the National Renewable Energy Laboratory has developed a widely used series of laboratory analytical procedures (LAPs) for lignocellulosic analysis. The LAP used to determine constituent sugars and lignin takes approximately three days for the two-stage acid hydrolysis procedure. Before hydrolysis, however, it is usually necessary to remove extractives from the sample. In trials in our laboratory, we have found that not removing the extractives prior to hydrolysis can lead to significant overestimations of the Klason lignin. While various solvents can be used to remove extractives, the NREL LAP recommends water and ethanol. Removing extractives using soxhlet requires up to 24 hours per sample. The remaining solid needs to air-dry prior to hydrolysis and the extractives content can be determined indirectly from the solid mass loss or directly by evaporating the solvent and weighing.
Once the extractives are removed, the two-stage acid hydrolysis procedure specified in the NREL LAP involves mixing the sample with 72 percent sulphuric acid and incubating it in a water batch at 30 degrees Celsius for one hour, during which the sample needs to be periodically mixed. The analyst’s mixing technique is key in ensuring that the sample is fully hydrolyzed. After one hour, the hydrolysate is diluted to 4 percent acid, the pressure tube sealed and placed in an autoclave at 121 C for another hour. The hydrolysate is then filtered and analyzed using chromatography equipment to identify the sugars. The residue is dried and ashed to determine the Klason lignin content, while acid-soluble lignin content is determined using ultraviolet spectroscopy.