# Comparison of Analytical Methods for Determining Methylesterification and Acetylation of Pectin
## 3 Results and Discussion
### Determination of DM and DAc by HPLC  
HG-type pectin is usually methyl-esterified by methanol at _O-6_ carboxyl of GalA, but it can sometimes be acetylated at _O-2_ or _O-3_ of GalA by acetic acid. The titrimetric and FT-IR methods can determine the DM content of HG pectins, whereas these methods cannot be used to determine DAc content. For the characterization of HG-type pectin, it is better to find a way to simultaneously determine levels of DM and DAc. According to the literature, HPLC analysis following alkali saponification is a method used for determining pectin DM and DAc content simultaneously. In the present study, we adopted this approach with some modifications. In this regard, methyl-ester and acetyl groups were hydrolyzed by alkali to produce methanol and acetic acid, and these compounds were then detected by HPLC with an RI detector. NaOH neutralization was performed by using 0.2 M H\({}_{2}\)SO\({}_{4}\) rather than cation exchange resin. H\({}_{2}\)SO\({}_{4}\) was also the eluent for HPLC analysis; therefore, neutralization was simplified by using our procedure. We referred to [PERSON] and [PERSON]'s method for improvement, shortening the sample pretreatment and injection time on this basis [24]. Then, de-esterified pectin was precipitated by adding CuSO\({}_{4}\) or isopropanol, as in the literature [17]. However, pectin cannot be completely precipitated by using this approach. At the same time, CuSO4 or isopropanol can also produce higher solvent peak, which may cause error to the DM measured. Column and liquid phase systems may also  
Figure 2: (**a**) FT-IR spectra of HG pectin standards with different DM values. (**b**) The two peak areas of A1740 and A1630 with the Levenberg–Marquardt algorithm and the Gaussian function by OMNIC. (**c**) Linear correlation between DM and A1740/(A1740 + A1630) of standard pectins. The equations and squared correlation coefficients (R\({}^{2}\)) of the linear fit are displayed on the graphs. (**d**) FT-IR spectrum of CP pectin. Values are the mean of three independent experiments. be damaged. Here, we did not remove pectin before HPLC analysis, because pectin will not be preserved on the C18 column or guard column. Pectin was usually eluted in under 5 min. Therefore, the modified procedure is simpler and more convenient. In order to quantify the content of released methanol and acetic acid, external standards were used, and the calibration curve was based on the peak area of standard methanol or acetic acid solutions from HPLC. The results of the determination of methanol and acetic acid at different concentrations by HPLC are shown in Figure 3a. As can be seen, the elution times for methanol and acetic acid were 6.1 min and 9.2 min on HPLC, respectively, which was less than 10 min for each analysis. The peak area was proportional to the concentration of methanol or acetic acid (Figure 3b,c). Therefore, the standard curve was drawn between methanol or acetic acid to their peak areas. The regression equation used for the calibration curve was C(MeOH) = A(MeOH)/906.47 (R\({}^{2}\) = 0.9993) and C(HOAc) = A(HOAc)/5032.3 (R\({}^{2}\) = 0.9997), where C represents the concentration of methanol or acetic acid, and A represents the peak area for methanol or acetic acid. HG pectin standards with different DMs measured by titration (DM = 0.0%, 22.0%, 44.0%, 66.0%, 88.0%) were saponified with alkali and then determined by HPLC. The elution peaks of methanol from different HGs are shown in Figure 4a. No elution peaks were detected at 9.2 min for acetic acid in HPLC, suggesting that there was no acetylation in these HG pectins. By calculating the peak areas of methanol, their concentrations could be determined according to the above calibration curve. Based on Equation (2) in Section 2.6, the DMs of HG standards were calculated as 0%, 23.7 \(\pm\) 0.24%, 44.5 \(\pm\) 0.56%, 67.3 \(\pm\) 0.38% and 84.2 \(\pm\) 0.67%, respectively. The results between HPLC and titration were not significantly different (\(p\) > 0.05).