*3.4. Structural Characterization of OS*

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Figure 3 shows the HPAEC–PAD elution profiles corresponding to AL210 and retentate. Data concerning commercial XOS in the range 2–6 are also included for comparison. It must be noted that the alkaline mobile phase used for HPAEC–PAD analysis caused the saponification of AG. Because of this, this technique provided useful information about the DP distribution of the oligomers, but not about the substitution pattern [33,43]. The elution profiles of AL210 and retentate showed similar patterns for compounds with DP > 3, peaks of oligomers with the same size. Oppositely, the peaks observed for DP2 and DP3 compounds were smaller in the case of retentate. This finding is in agreement the high recovery of XOS in the retentate, as discussed above. – – –

**Figure 3.** HPAEC–PAD elution profiles for the autohydrolysis liquors at 210 ◦C and retentate.

– Additional information on the structures of oligomeric saccharides contained in retentate was obtained using MALDI–TOF MS (see Table 4). This technique is a powerful tool widely used for carbohydrate characterization, as oligomers can be investigated with minimal fragmentation [45,46]. The experimental data allowed the assessment of the pentose chain lengths, as well as their substitution pattern. The data reported correspond to sodium and potassium adducts. As 2,5 dihydroxybenzoic acid (DHB) was used in the analyses as a matrix, the components with *m*/*z* < 500 could not be identified with this method [23,24,34].

Based on the compositional information shown in Figure 2, the OS in the retentate were constituted mainly by pentose units. Based on the HPLC data obtained for arabinose and xylose, it can be concluded that the pentoses in OS backbones corresponded to xylose. The pentose chains were highly substituted by acetyl groups (AG) and/or *O*-methylglucuronic groups (U), following the patterns [mP nAG] or [mP nAG oU]. The spectra confirmed the presence of OS composed by pentose chains with DP in the range 3–16, containing up to 6 AG groups, and 1 or 2 U groups. These results are in agreement with the compositional data and with the molar ratios XOS:AG:U discussed above.


**Table 4.** OS structures in the retentate stream identified by MALDI–TOF MS.

P: pentoses; AG: acetyl groups; U: uronic groups. Na+: sodium; K+: potassium.
