# Determination of Cellulose Degree of Polymerization in Historical Papers with High Lignin Content
## 3 Results and Discussion
### Stability of Cellulose during Delignification
The distribution in the hemicellulose range narrowed after each delignification cycle, indicating a significant decrease in hemicellulose content in the pulp [19]. In this study, the chromatogram of the untreated sample is not shown. The differences in the curves between untreated and treated paper samples can be explained by the fact that none of the methods, neither derivatization in the form of cellulose tricarbanilates nor SEC of cellulose samples in LiCl-DMAc, seem to be directly suitable for the case of impure cellulosic samples, such as mechanical puls or paper with a high content of hemicelluloses and lignin [9]. Therefore, also before SEC analysis, delignification of cellulosic samples was attempted using chlorite-based pretreatments either at 70 \({}^{\circ}\)C and pH 4.8 (holocellulose treatment) or using cold holocellulose treatment [9]. It was shown that the holocellulose treatment resulted in a significant decrease in cellulose average DP, but the presence of lignin counteracted this effect [9]. A high lignin content could interfere with the SEC measurements made with a UV-VIS detector, since lignin absorbs in the UV-VIS region due to its aromatic nature [1,4]. However, in Figure 1, which shows the SEC chromatograms of the paper sample No. 1 with the highest lignin content of the samples studied (Table 1) at different stages of delignification, almost no difference can be observed between the relative signals in the molecular mass range of cellulose. Therefore, it can be concluded that lignin content does not interfere with the determination of cellulose M\({}_{\mathrm{w}}\) after CTC derivatization and analysis using our SEC setup. SEC with UV-VIS and MALS detectors have already been used for DP determinations of cellulose in lignin-containing paper samples [4]. Analyses of CTC derivatives have also been performed for papers with much higher lignin contents (up to 30%) using SEC with MALS and UV-VIS detector [35]. For all paper samples, the average molecular mass of CTC derivatives was determined using SEC before and after delignification to evaluate cellulose degradation due to delignification (Table 4). The differences between the determined weight average molecular masses of the papers before and after delignification were less than 10%. In addition to the above explanation of the differences in SEC chromatograms of untreated and treated cellulose samples, previous studies have shown that when derivatized tricarbanilate cellulose samples are analyzed in the same laboratory, the average RSD between measurements of the \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Monosaccharide** & **First Filtrate, 3 h** & **First Filtrate, 6 h** & **Second Filtrate, 9 h** \\ \hline glucose & 0.11 mg\(\cdot\)L\({}^{-1}\) & 0.12 mg\(\cdot\)L\({}^{-1}\) & \textless\textless\textless\textgreater{}0000 \\ mannose & nd & nd & nd \\ galactose & nd & \textless\textless\textless\textgreater{}0000 & nd \\ xylose & nd & \textless\textless\textless\textgreater{}0000 & nd \\ arabinose & \textless\textless\textless\textless\textgreater{}0000 & 1.16 mg\(\cdot\)L\({}^{-1}\) & nd \\ \hline nd = not detected. & & & \\ \end{tabular}
\end{table} 
Table 3: Monosaccharide content in the first filtrates after 3 or 6 h of delignification and second filtrates after additional 3 h of delignification (9 h altogether). same sample is 8%, but RSD between laboratories is higher with a value of 15% [11]. The differences in the obtained molecular masses can also be attributed to the uncertainty due to the preparation of the cellulose tricarbanilate derivatives, which is the common sample preparation for cellulose dissolution before SEC analysis. A precondition for derivatization prior to SEC analyses is that the chemical reaction proceeds with quantitative yields, which is suggested for the CTC derivative and pure cellulose. Derivatization is not straightforward if the lignin content is high; a low content does not interfere.