Carbopac pa20 column

Following sample preparation, free and total concentrations of Neu5Ac and Neu5Gc acids were obtained by ion chromatography with pulsed amperometric detection (ICS-5000, Dionex, Sunnyvale, CA) with a gold cell electrochemical detector and a multi-step gradient of 7–300 mM sodium acetate in 100 mM sodium hydroxide at 30°C. Separation was conducted utilizing a CarboPac PA20 Column, 3 × 150 mm (Dionex, Sunnyvale, CA) with a flow rate of 0.5 mL/min and a 10 μL injection volume (28 (link), 29 ). Free and total sialic acid for both diet and tissue samples were determined by multiplying the sample hydrolyzed value by the appropriate dilution factor and as-is sample weight and dividing this value by the sample weight (as-is for tissue samples, dry matter-basis for diet samples). For plasma samples, the sample hydrolyzed value was multiplied by the appropriate dilution factor and divided by the sample volume. Concentrations of bound SA were determined by subtracting free SA from the total SA concentrations for each subject.

Monosaccharide composition analysis was determined based on a previous report [51 (link)]. The sample (2 mg) was first hydrolyzed with 2 M TFA, and the hydrolysate was dried with nitrogen. Then, methanol was added to facilitate the volatilization of TFA, and the dried hydrolysate was dissolved with ultra-pure water. Finally, the solution was analyzed using high-performance anion exchange chromatography (HPAEC, ICS2500, Dionex, Sunnyvale, CA, USA) with a pulsed amperometric detector (PAD) and a Carbopac PA-20 column (3 mm × 150 mm, Dionex, Sunnyvale, CA, USA).

UDP-Arap and UDP-Araf were obtained from CarboSource Services (Athens, USA) and Peptide Research Institute (Osaka, Japan), respectively. The RGP1 mutase activity assay and product detection were modified from methods previously described [17 (link)]. Briefly, 3 μg recombinant Arabidopsis thaliana RGP1 and 1 mM UDP-Araf were added to 20 mM Tris, 5 mM MnCl₂, 250 mM NaCl, pH 6.8, and incubated at 30°C for 1 h. The product was detected with high-performance anion-exchange chromatography using a Dionex Ultimate 3000 with CarboPac PA20 column (3 × 150 mm; Dionex; Sunnyvale, USA). Nucleotide sugars were separated with 0 to 2.1 min, 50 mM ammonium formate, isocratic; 2.1 to 40 min, 50 mM to 1 M ammonium formate, linear, 40 to 45 min, 1 M to 50 mM ammonium formate, linear, 45 to 55 min, 50 mM ammonium formate, isocratic, all at a flow rate of 0.5 mL/min. Nucleotide sugars were detected with UV light at 262 nm.

The monosaccharide composition was determined by HPAEC. The polysaccharides were hydrolyzed in a sealed chromatographic bottle with 2 M trifluoroacetic acid (TFA) for 2 h at 121 °C and redissolved with water and analyzed by HPAEC following the removal of TFA. The retention times and standard curves of monosaccharide standards, including Rha, Ara, Glc, Gal, Fuc, Xyl, Man, Fru, Rib, Gal-UA, Glc-UA, Man-UA, and Gul-UA, were determined. By comparing the chromatographic peak and retention time of the sample with that of the standards, the composition and content of the monosaccharide in the sample can be determined.
Chromatographic conditions were as follows: Dionex™ CarboPac™ PA-20 column (3.0 mm × 150 mm, 10 µm); Dionex™ ICS-5000 system with pulsed amperometric detector (PAD); flow rate: 0.5 mL/min; column temperature: 30 °C; injection volume: 5 µL; mobile phase: ddH₂O (A); 0.1 M NaOH (B); 0.1 M NaOH, 0.2 M NaAc (C); gradient elution (0–26 min, 95–85% A, 5–5% B, 0–10% C; 26–42 min, 85% A, 5% B, 10% C; 42–42.1 min, 85–60% A, 5–0% B, 10–40% C; 42.1–52 min, 60–60% A, 0–40% B, 40–0% C).

The monosaccharide composition of extracted pectin samples was analysed using methylation, followed by high-pH anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD) and GC-MS analysis (triplicate runs) [68 (link),69 ].
Samples (1.00 ± 0.02 mg) of dried pectin were mixed with 2 M HCl in methanol and flushed with argon. Methanolysis was then performed at 100 °C for 4 h. Samples were then neutralized with pyridine and dried under compressed air. Dried samples were subsequently hydrolyzed using 2 M trifluoracetic acid at 120 °C for 1 h, dried under compressed air and re-suspended in water. Hydrolysed monosaccharides were analyzed using HPAEC-PAD on an ICS6000 system (Dionex, Sunnyvale, CA, USA) using a Dionex CarboPac PA20 column, at 30 °C at a flow rate of 0.4 mL min⁻¹.

The non-cellulosic monosaccharide composition of leaf and stem cell walls before and after pretreatment and fermentation was analyzed by HPAEC-PAD on an ICS-5000 system (Dionex, USA) equipped with electrochemical detector and a CarboPac PA 20 column (3×150 mm, Dionex, USA), according to the description in Ellinger et al. [29] (link).