# Red Orange and Bitter Orange IntegroPectin: Structure and Main Functional Compounds
## 3 Results and Discussion  
### DRIFT Structural Analysis  
The DRIFT spectra in Figure 1 (500-2000 cm\({}^{-1}\) region) show the typical features of pectin, with differences in the relative intensities and frequencies of some fingerprint bands. \begin{table}
\begin{tabular}{c c} \hline Sample/Matrix: & 0.362 g freeze-dried, ground red orange IntegroPectin; 0.683 g freeze-dried, ground bitter orange IntegroPectin \\ \hline SPME fiber: & 50/30 \(\upmu\)m DVB/CAR/PDMS \\ \hline Sample equilibration: & 24 h, room temperature \\ \hline Extraction: & 2 min for red orange IntegroPectin, headspace, room temperature; 20 s for bitter orange IntegroPectin, headspace, room temperature \\ \hline Column: & ZB-WAX Phenomenex, L = 30 m \(\times\) I.D. = 0.25 mm \(\times\) df = 0.25 \(\upmu\)m \\ \hline Oven: & 50 \({}^{\circ}\)C (0 min), 10 \({}^{\circ}\)C/min to 250 \({}^{\circ}\)C (5 min) \\ \hline Injection T: & 260 \({}^{\circ}\)C \\ \hline Detector: & Triple quadrupole \\ \hline Scan range: & Full scan, \(m/z\) 50–500 \\ \hline Carrier gas: & He 99.9999\%, 1 mL/min constant flow \\ \hline \end{tabular}
\end{table} 
Table 1: Conditions employed for the SPME-GC/MS analysis. The strong bands in the 1550-1800 cm\({}^{-1}\) region, with maxima at 1610, 1643 (IP-bitter) and 1730 cm\({}^{-1}\), are assigned to the stretching modes of carboxylate groups (\(\
u_{\rm as}\)COO\({}^{-}\)), carbonyl groups from non-esterified hydrogenated acidic carbonyl groups (\(\
u\)C=O\({}_{\rm acid}\)) and esterified galacturonic acid (\(\
u\)C=O\({}_{\rm ester}\)), respectively [14]. In this region, the relative intensities of the ester-related components are much higher for IP-bitter pectin, suggesting a higher degree of esterification. The 1200-1550 cm\({}^{-1}\) region is dominated by CH\({}_{\rm x}\) and C-O-H deformation modes, partially overlapped with ester-related stretching modes [15; 16]. These include: the \(\delta_{\rm ip}\)C-O-H (from alcohol hydroxyl groups in the pyranose rings of the pectin chain) at \(\sim\)1236 cm\({}^{-1}\), the \(\
u\)C-O-C\({}_{\rm ester}\) at \(\sim\)1330 cm\({}^{-1}\), the \(\
u_{\rm s}\)COO\({}^{-}\) at \(\sim\)1420 cm\({}^{-1}\), and the \(\delta_{\rm s}\)CH\({}_{3}\) and \(\delta_{\rm as}\)CH\({}_{3}\) (from ester methyl groups in the galacturonic rings and rhamnose rings of the pectin backbone) at 1336 and 1520 cm\({}^{-1}\). The 950-1200 cm\({}^{-1}\) region contains a set of very intense bands typical of pectin, partially overlapped, assigned to skeletal stretching modes (\(\
u\)C-C and \(\
u\)C-O-C) of the pyranose rings and of the glycosidic bonds, and to a combination of the \(\
u\)C-OH and \(\
u\)C-C modes from the pyranose rings [17; 18]. The 500-950 cm\({}^{-1}\) region contains the bands related to the external deformation vibrations of methyl, methylene, and methine groups (\(\rho\)CH\({}_{\rm x}\) and \(\delta\)C-H) [14]. The degree of esterification of each pectin (percent of esterified carboxyl groups) was obtained by spectral analysis of the 1550-1800 cm\({}^{-1}\) region, as the ratio of ester carboxyl to total carboxyl peak areas (Equation (1)) [15; 19]:  
\[{\rm DE}=\Sigma A_{\
u\rm C-Oester}/(\Sigma A_{\
u\rm C-Oester}+A_{\
u\rm C- Oacid}+A_{\
u\rm assCOO}{}^{-}) \tag{1}\]  
Figure 1: DRIFT spectra of the pectin samples in the 500–2000 cm\({}^{-1}\) region, normalized to the \(\
u_{\rm as}\)COO\({}^{-}\) band carboxylate groups, at \(\sim\)1610 cm\({}^{-1}\). The amount of galacturonic acid-rich (HG) regions of pectin are not calculated, but they are proportional to the ratio described by Equation (2) [15]:  
\[\text{HG }\alpha(\Sigma A_{\text{vC=O}}+A_{\text{vasCOO}}-)/(\Sigma A_{\text{vC -O-C}}+\Sigma A_{\text{vC=O}}+A_{\text{vasCOO}}-) \tag{2}\]  
The vC=O and vasCOO\({}^{-}\) band areas of the samples were estimated by decomposing the 850-1800 cm\({}^{-1}\) region into a sum of Gaussian components, using a nonlinear least-squares fitting [15].