# Comparison of Analytical Methods for Determining Methylesterification and Acetylation of Pectin
## 3 Results and Discussion
### Determination of DM and DAc by HPLC
These values are consistent with results from the titration method. We also measured the DM of CP as 48.0 \(\pm\) 0.43%, which had no significant differences compared with the DM of 47.0% and 47.6%, determined by the titration and FT-IR methods, respectively (\(p\) > 0.05). Comparison of pectin with different DM determined by HPLC is shown in Figure 4b. Meanwhile, the DAc content of CP was determined to  
Figure 3: (**a**) Elution curves of methanol and acetic acid with known concentrations on HPLC. (**b**) Standard curve determination between the concentration and peak area of MeOH. (**c**) Standard curve determination between the concentration and peak area of HOAc. The equations and squared correlation coefficients (R\({}^{2}\)) of the linear fit are displayed on the graphs. Values are a mean of three independent experiments. be 1.6 \(\pm\) 0.14%, indicating that there are only small amounts of acetyl groups in CP. Thus, both DM and DAc could be determined simultaneously by using HPLC. According to the above analysis, the DM content of pectin can be determined by the titration, FT-IR, and HPLC methods, and the DAc content can be determined by using the HPLC approach. Comparisons of these three methods are summarized in Table 2. Titration is easy to perform and can be used to determine the DM content in pectin when other instruments are not available in the laboratory; however, it is not suitable with small quantities of samples. The FT-IR method is simple and accurate, and it only needs a few milligrams of a sample. In addition, this method does not destroy the sample; it can be recovered. However, neither the titration nor FT-IR methods can be used to determine DAc content. When the degree of acetylation in the sample is high, the result of DM determination by the titration or FT-IR method approach will be affected. Therefore, these two methods are not suitable for pectin with high acetylation content. Compared with the titration and FT-IR approaches, the HPLC method can be used to determine DM and DAc content in pectin simultaneously, and only a few milligrams of sample will be required. Therefore, if the pectin sample has a high degree of acetylation and HPLC is available, this method is better to use for determining DM and DAc content. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Titration** & **FT-IR** & **HPLC** \\ \hline Determination of DM & Yes & Yes & Yes \\ Determination of DAc & No & No & Yes \\ Sample amount & 500 mg & 2 mg & 5 mg \\ Instrument & Burette & Infrared spectrometer & HPLC \\ Operation & Simple & Simple & Complicated \\ Sample recyclability & No & Yes & No \\ Standard sample required & No & \
\begin{tabular}{c} Pectin with known \\ DM \\ \end{tabular} &
\begin{tabular}{c} MeOH and HOAc with known concentration \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Comparison of different methods for the determination of DM and DAc. Figure 4: (**a**) Elution profiles of methanol and acetic acid released from different pectins on HPLC. (**b**) The DM results of different pectin by HPLC detection.