*3.3. E*ff*ect of S*/*L Ratio*

The S/L ratio is the ratio of solid phase (biomass) and liquid phase (solvent plus water) in the organosolv pretreatment. In the first approach, different S/L ratios, namely, 1:20, 1:10, and 1:5, were applied for 60 min at 210 ◦C. The lignin purity obtained from pretreatment experiments at different S/L ratios of 1:20, 1:10, and 1:5 was 66.73 ± 2.55, 77.60 ± 2.80, and 80.26 ± 2.09%, respectively. Changing the S/L ratio from 1:20 to 1:10 increased the purity of lignin, however, no further improvement on lignin purity was achieved at S/L ratio of 1:5 (Figure 3a).

**Figure 3.** Effect of solid to liquid ratio and retention time of (**a**) 60 min and (**b**) 120 min (pretreatment at 210 ◦C) on lignin purity (*p*-value = 0.009 and *p*-value = 0, respectively) and recovery (*p*-value = 0 and *p*-value = 0.002, respectively). Error bars represent ± 2× the standard deviation.

However, such a trend was not observed for lignin recovery (Figure 3a), where a significant decrease was observed when the S/L ratio was changed from 1.10 to 1:5 (55.86 ± 0.06% vs. 35.99 ± 0.63%, respectively). Accordingly, the retention time was extended to 120 min (Figure 3b); similar trends were found for lignin purity and recovery although an overall increase in the later was observed. These results together with those obtained throughout the optimization approach, culminating in higher purity and recovery, indicate the interaction between S/L ratio and retention time which should be the focus of future research.

In a further optimization experiment, a retention time of 90 min was also investigated for pretreatment at an S/L ratio of 1:10. This retention time resulted in a significantly higher lignin recovery of 64.94 ± 1.09% than that obtained from the pretreatment at 60 min which was 55.85 ± 0.06%. A similar lignin recovery value of 63.94 ± 1.25% was obtained at 120 min (Figure 4); therefore, shorter retention times can be used while achieving similar recovery and purity of lignin. In summary, the best conditions found among those tested in this work, for the recovery and purity of lignin, include sulfuric acid as catalyst, temperature of 210 ◦C, retention time of 90 min, and S/L ratio of 1:10. In these conditions, lignin purity of 70.56 ± 4.48% and lignin recovery of 64.94 ± 1.09% were obtained.

**Figure 4.** Effect of retention time at S/L ratio of 1:10 on lignin purity (*p*-value = 0.118) and recovery (*p*-value = 0.002). Error bars represent ± 2× the standard deviation.

Analysis of the composition of the lignin-rich fraction and the glucan-rich fraction, obtained under the best conditions mentioned, was carried out and the result is presented in Table 2. Based on the recovery of lignin in the glucan-rich fraction (around 10%), delignification of about 90% was obtained which shows some material loss during recovery through centrifugation.


**Table 2.** Composition of glucan-rich fraction and lignin-rich fraction obtained after acid-catalyzed organosolv pretreatment at 210 ◦C, for 90 min, and using an S/L ratio of 1:10.
