## 3 Results and Discussion
### Initial Experiments to Define Lignin Isolation Variables and Further Optimization by RSM
This interactive behavior is due to the fact that the higher biomass loading needed some additional thermomechanical and/or thermochemical treatments to expose the inner biomass surfaces for the catalyst transport as the outer biomass layer may swell, due to the acidic environment, on the surface offering a hindrance for the catalyst to penetrate deep inside [44]. For the interactive effects, it was found that only the effects of time and catalyst dose have \(p\) values (0.0030) less than 0.005. This interactive effect is quite obvious as both are significant even individually to cause the enhanced lignin yield. This similar trend was also obvious in our preliminary trials. Additionally, there are some previous studies where the similar trend was exhibited [45]. Therefore, it can be suggested that the synergism of both the time and HCl dose can lead to a better lignin yield but at room temperature. Regarding the interactive effects of time and solids loading, its \(p\) value was 0.0142 showing not a significant effect and a similar trend was also observed in the case of the solids loading and the catalyst, where the \(p\) value was also not significant and showed a value of 1.0000. Under all of the aforementioned conditions, the optimal lignin isolation combination was found out to be at 48 h of time, 10% of solids loading, with 10 mL of catalyst dose with a predicted lignin yield of 8.33%. The predicted value was confirmed experimentally which yielded the lignin with 8.10%. Our results are fairly comparative with previously published studies. For example, in one similar study, almost 15% lignin yield was obtained by adapting typical Soxhelet apparatus. In that study, they used dioxane with 0.4% \(v/v\) HCl and 3% \(v/v\) water for the extraction of low molecular weight lignin [46]. In another study, a lignin yield of 5% lower than our yield was obtained when imidazolium ionic liquids were used to treat woody biomass around 100 \({}^{\circ}\)C temperature and long retention times from 8 to 24 h [41]. In another study a lignin yield of 7.2% was obtained when sugar cane bagasse was dissolved in different concentrations of dioxane in 1% NaOH to extract the lignin [47]. In one such study a lignin yield of [PERSON]'s native lignin was almost 8% obtained from the woody biomass taking into account the proto lignin structure of the extracted lignin [48]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Scheme 2** & **Sum of Squares** & **df** & **Mean Square** & **F Value** & \(p\)**Value Prob \textgreater{} F** & \\ \hline Model & 103.04 & 9 & 11.45 & 108.18 & \textless{}0.0001 & Significant \\ \(\chi_{1}\)—Time & 59.95 & 1 & 59.95 & 566.47 & \textless{}0.0001 & Significant \\ \(\chi_{2}\)—Solid loading & 1.62 & 1 & 1.62 & 15.31 & 0.0113 & \\ \(\chi_{3}\)—Catalyst & 22.78 & 1 & 22.78 & 215.26 & \textless{}0.0001 & Significant \\ \(\chi_{1}\)\(\chi_{2}\) & 1.44 & 4 & 1.44 & 13.61 & 0.0142 & \\ \(\chi_{1}\)\(\chi_{3}\) & 3.06 & 1 & 3.06 & 28.94 & 0.0030 & \\ \(\chi_{2}\)\(\chi_{3}\) & 0.000 & 1 & 0.000 & 0.000 & 1.0000 & \\ \(\chi_{1}^{2}\) & 11.69 & 1 & 11.69 & 110.44 & 0.0001 & \\ \(\chi_{2}^{2}\) & 0.60 & 1 & 0.60 & 5.70 & 0.0626 & \\ \(\chi_{3}^{2}\) & 3.19 & 1 & 3.19 & 30.12 & 0.0027 & \\ Residual & 0.53 & 5 & 0.11 & & & \\ Lack of fit & 0.40 & 3 & 0.13 & 2.12 & & Not significant \\ Pure error & 0.13 & 2 & 0.063 & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: ANOVA for response surface optimization of the variables.