*3.2. FTIR Analysis of Pretreated Feedstock*

FTIR is generally used to assess chemical functional group changes in biomass and this information is used to compare the structural changes of the biomass before and after chemical pretreatments. The alkaline pretreated CC and SSB biomasses were analysed with FTIR to validate the compositional analysis and SEM results. Figure 2 demonstrates that the alkaline pretreated CC and SSB biomasses possessed more cellulose II than crystalline cellulose I, which is an indication of reduced biomass crystallinity. The positive control for cellulose Iβ (Avicel PH101 microcrystalline cellulose) showed high intensity peaks at 1428.66, 1160.54 and 1104.67 cm−<sup>1</sup> . These peaks represent the presence of predominately crystalline cellulose Iβ and small quantities of cellulose II in the biomass. Similar peaks (1428.06, 1157.46 and 1111.00 cm−<sup>1</sup> ) were present in the untreated SSB biomass, which suggest that there was a presence of crystalline cellulose I and cellulose II in this sample. However, only one peak was present in the Ca(OH)<sup>2</sup> (1158.11 cm−<sup>1</sup> ) and NaOH (1157.23 cm−<sup>1</sup> ) pretreated SSB biomass (Figure 2). These results demonstrated that after pretreatment, the SSB biomass had a reduced content of crystalline cellulose Iβ by producing cellulose II. In addition, the Ca(OH)<sup>2</sup> pretreatment shifted the 1428.66 cm−<sup>1</sup> peak to 1410.45 cm−<sup>1</sup> , while NaOH pretreatment shifted the 1428.66 cm−<sup>1</sup> peak to 1421.90 cm−<sup>1</sup> . This shift is characteristic to the formation of cellulose II and amorphous cellulose, and the disappearance of the crystalline cellulose I in the biomass. Figure 2B showed that the similar spectra were identified in the untreated and alkaline pretreated CC biomass, suggesting that alkaline pretreatment removed lignin and changed the CC biomass crystallinity in a similar fashion to the SSB biomass.

**Figure 1.** Topological analysis of pretreated sweet sorghum (SSB) and corncob (CC) feedstocks using scanning electron microscopy (SEM). The captions above the images indicate the control (untreated), type of treatment and the biomass in each SEM image. The scale bar was 20 µm and the magnification was 2.22 kx.

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− **Figure 2.** Comparative FTIR in the spectra range 4000–650 cm−<sup>1</sup> corresponding to Avicel PH101 (i), untreated (ii), Ca(OH)<sup>2</sup> pretreated (iii) or sodium hydroxide pretreated biomass. (**A**) represents the SSB biomass and (**B**) represents the corncob biomass. Keys: (**A**) in the *y*-axis represents arbitrary units.

The peaks at 1515.45 cm−<sup>1</sup> and 1519.97 cm−<sup>1</sup> in the untreated SSB and CC biomasses, respectively, represent the C≡C vibration of the aromatic rings of the lignin. In contrast, these bands were absent in the IR spectrum of the positive control (Avicel) and NaOH pretreated SSB and CC biomasses (Figure 2). These results supported the biomass composition analysis and SEM findings which revealed that NaOH pretreatment removed the lignin from the biomass. However, the spectrum of the Ca(OH)<sup>2</sup> pretreated SSB and CC biomass samples had a broad and higher intensity peak starting from 1640 cm−<sup>1</sup> , reaching a maximum at 1410 cm−<sup>1</sup> and ending at 1370 cm−<sup>1</sup> (Figure 2). This observation suggested that lignin was not completely removed by the Ca(OH)<sup>2</sup> pretreatment but was re-distributed as revealed by SEM (Figure 1). In addition, the untreated and the alkaline pretreated SSB and CC biomasses displayed one peak between 1033 and 1027, which represented the hemicellulose. These results revealed that the hemicellulose was still present in the SSB and CC after alkaline pretreatment.

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