## 3 Results and Discussion  
### Initial Experiments to Define Lignin Isolation Variables and Further Optimization by RSM  
Initially to find out the experimental hotspot we performed some laboratory trials and tried different reagent combinations on the basis of previous literature survey. The list of the adapted reagent combinations of dioxane-acetic acid, GVL-acetic acid and GVL-dioxane catalyzed by HCl along with their operating conditions are mentioned in Table 1. As it can be seen in the table, the values in terms of lignin yield were quite noticeable for all the combinations with the highest value of 42% for GVL: acetic acid combination and the lowest one 2.5% with dioxane acetic acid conditions. These values are quite comparable with the previously published literature. For example, in a previous study, a lignin yield of 43% was obtained when organosolv reagent (H2O: EtOH/35:65 \(v/v\)) combination was employed at 162 \({}^{\circ}\)C for 78 min and with 1.6% H\({}_{2}\)SO\({}_{4}\)[38]. In a similar other study, a lignin yield of 23.8% was obtained from corn stover when deep eutectic solvent (choline chloride and formic acid (1:2)) were employed at a higher temperature of 130 \({}^{\circ}\)C for 2 h of time [39]. In our reagent combination of dioxane and acetic acid with 10 mL of HCl dose and at 70 \({}^{\circ}\)C of temperature for 6 h of process time a lignin yield of 11% was obtained and in a previous study a slightly higher yield of 14% was obtained when flax fiber was subjected to a two-step lignin extraction process with the help of formic acid and hydrogen peroxide [40]. For our case of dioxane and acetic acid although the yield is much lower, this lower yield can be anticipated due to the rather less harsh thermal conditions and such low yields are possible as in one previous study a lignin yield of 5% 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]. Since our objective was to extract high quality lignin, all these extracted samples were further subjected to the DFRC analysis and their results were quite different within \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Reagent (50\% \(v/v\))** & **Temperature (\({}^{\circ}\)C)** & **Time (h)** & **Lignin Yield (\%)** & **G Units \({}^{*}\)** & **Mw** & **Mn** & **PDI \({}^{*}\)** \\ \hline Di:AA 1\({}^{*}\) & 70 & 6 & 11 & 200 \(\pm\) 20 & 4732 & 2290 & 2.06 \\ Di:AA 2\({}^{*}\) & Room temp. & 48 & 2.5 & 332 \(\pm\) 30 & 4718 & 2473 & 1.90 \\ GVL:AA 1\({}^{*}\) & 70 & 6 & 42 & 100 \(\pm\) 35 & 3604 & 1851 & 1.94 \\ GVL:Di 2\({}^{*}\) & 70 & 6 & 38 & 110 \(\pm\) 40 & 4562 & 2064 & 2.21 \\ \hline \hline \end{tabular}  
* dioxane acetic acid with 5 mL HCl; GVL: AA 1\({}^{*}\)
- GVL acetic acid with 10 mL HCl; GVL: Di 2\({}^{*}\) = GVL dioxane with 10 mL HCl; PDI \({}^{*}\) = Mw/Mn; G Units \({}^{*}\) = mol/g.  
\end{table} 
Table 1: Different reagent combinations for lignin yield, G units, M\({}_{\rm w}\) and polydispersity values. themselves. As can be seen in the table, the highest DFRC value was for dioxane: acetic acid combination when they were treated at room temperature for about 48 h with a value of 332 units. However, this value dropped significantly to 200 units when this combination was subjected to mild thermal severity 70 \({}^{\circ}\)C for about 6 h of time. Although in this case the yield was enhanced greatly to 11%, the DFRC value was dropped as well indicating that this combination did not preserve well the prototype structure of the isolated lignin. Moreover, the synergism of the gamma valerolactone (GVL) with both the acetic acid and dioxane also did not preserve well the higher DFRC values and these were 100 and 110, respectively, mainly due to the high thermal severity (70 \({}^{\circ}\)C) and high HCl dose (10 mL). For these two combinations, we did not observe any lignin yield with either 5 mL HCl or at room temperature.