br Monosaccharide composition br Monosaccharide composition was

2.5.2. Monosaccharide composition
Monosaccharide composition was performed using a high-performance anion-exchange chromatography coupled with a pulse amperometric detector (HPAEC-PAD) [27,28]. Samples (1 mg) were hy-drolyzed with 2 M trifluoroacetic KIN-59 at 120 °C for 90 min. Then t-butyl alcohol was added, and the mixture was evaporated under N2 flow, sol-ubilized in water, and filtered. Next, samples were analyzed in a DX 500 system (Dionex, Sunnyvale, CA, USA) equipped with a CarboPac PA10 column (250 × 4 mm). For the analysis of neutral sugars, a post column adjustment with 300 mM NaOH was performed. For the uronic acid analysis, the same system was used with 150 mM NaOH (1 mL/min; 30 min) with a 0–220 mM sodium acetate gradient as the eluent. Neu-tral sugars and uronic acids were used as standards.
2.5.3. Fourier transform infrared (FTIR) attenuated total reflectance ATR The Fourier transform infrared (FTIR) spectroscopy was applied to
infer some structure characteristics from papaya pectin and to deter-mine the degree of O-methyl esterification [29] using an Alpha FTIR spectrometer (Bruker Optic, Ettlingen, Germany) equipped with a deu-terated triglycine sulfate detector and a single-bounce attenuated total reflectance (ATR) accessory (diamond crystal). FTIR–ATR spectra of samples were obtained with a resolution of 4 cm−1 and 50 scans. GRAMS/AI 9.1 software (Thermo Scientific) was used for spectra analy-sis. Methyl esterified and free uronic acids corresponded to bands at 1749 cm−1 and 1630 cm−1, respectively. Commercially available pec-tins with known degrees of O-methyl esterification (28%, 64%, 91%) and their mixtures (14%, 46%, 78%) were used to produce a standard curve of O-methyl esterification.
Samples diluted in water (2.5 μL/mL) were sonicated, dropped onto freshly cleaved mica, and dried in a vacuum at 30 °C for 20 min. Samples were maintained in a desiccator until analysis. To-pography images were obtained in an NX-10 Atomic Force Micro-scope (Park Systems, Suwon, South Korea) in an acrylic glove box with controlled temperature (~22 °C) and humidity (~3%). AFM im-aging was acquired on tapping mode using an NCHR probe (NanoWorld) with a spring constant of 42 N/m and 320 kHz reso-nance frequency. The scan speed and scanning resolution were
0.5 Hz and 512 × 512 points, respectively. For each sample, at least ten images were collected. Image measurements and automatic pro-cessing (plane subtraction and row alignment) were performed using Gwyddion 2.47 software (http://gwyddion.net/).
1D and 2D 1H spectra of 3CSF were recorded using a 500 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) with a triple res-onance probe. Approximately 5 mg of each sample were dissolved in 0.5 mL of 99.9% deuterium oxide (Cambridge Isotope Laboratory, Cam-bridge, MA). All spectra recorded at 35 °C with deuterated water exhib-ited a peak due to exchange with residual H2O (HOD), suppressed by presaturation. For 1D 1H NMR (zgpr from Bruker Library) spectra, 64 scans were recorded using an interscan delay equaling 1 s. 2D 1H-1H COSY (cosyphpr from Bruker Library) and 1H\\1H TOCSY (mlevphpr from Bruker Library) spectra were recorded using states time propor-tion phase incrementation for quadrature detection in the indirect di-mension. TOCSY spectra were run with 4096 × 512 points with a spin lock field of 10 kHz and a mixing time of 60 ms. The 1H/13C Multiplicity-edited HSQC (HSQC) spectra were recorded with 1024
× 512 points and 256 scans using an Echo-Antiecho acquisition mode
with globally optimized alternating phase rectangular pulses for decoupling. The 1H/13C HMBC spectra were recorded with 2048 × 200 points and 512 scans, with a 50 ms delay for evolution of long-range couplings and set with no decoupling during the acquisition time, a
low-pass filter, and a QF magnitude acquisition mode. Chemical shifts were displayed relative to external trimethylsilylpropionic acid at 0 ppm for 1H. The data were processed using TopSpin3.1 (Bruker Biospin) [30].