I am looking for answer why specifically 72% w/w H2SO4 is used as a reagent to determine klason lignin, why not some other higher or lower percent value?.
Lower concentrations will not dissolve the whole sample, so 4% hydrolysis will not work. Higher concentration will start carbonizing carbohydates.
So I guess both lower and higher concentrations will lead to overestimation of Klason lignin.
This is the optimal condition to solubilize all the carbohydrate (hydrolisis of cellulose and hemicellulose)
72 % H2SO4 hydrolyzed all the cellulose and hemicellulose rather than lignin which is optimized by Klason. In this process part of lignin has been dissolved. Therefore, after 2 h when hydrolysis completed 560 ml water has been added due to dilution. This dilution has been done because of determining the dissolved lignin after 1h digestion in autoclave at 121 oC.
Digestion of plant biomass in 72% H2SO4 at 30oC for 1 hr and hydrolysis in 2 - 4% H2SO4 at 121oC for 60 min is an optimum protocol for reproducible hydrolysis of cellulose and hemicellulose in plant. I carried a comparative analysis with 18, 36 and 72% H2SO4 using standard protocol and determined that biomass hydrolysis directly correlated with acid concentration. The poor hydrolysis of the biomass with the lower acid concentration was attributed to inefficient digestion of the biomass at this concentrations. The result indicated digestion step which denature and homogenized the cell wall components was critical for efficient acid hydrolysis of biomass. However, I believe that lower acid concentration could used for Klason lignin determination, if the biomass digestion time and probably the incubation time in the autoclave are increased to ensure efficient hydrolysis of the biomass.
Because,concentration below 72% will not break cellulose totally, and above 72% may have side reactions and the yield of the consumption of cellulose will diminish.
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