Dionex carbopac pa1 column

2 mL hydrolysis reactions containing 0.5% (w/v) ball milled corncob, 0.1 mg Ultraflo^® (Novozymes, Denmark)/g DW, without or supplemented with 175 nM CE15 enzyme (SlCE15A, SuCE15A or SuCE15C) were performed in triplicate experiments, in 50 mM sodium acetate (pH5.5) at 35 °C with 1000 rpm mixing. Reactions were stopped after 10 min by heating at 95 °C. A low concentration of Ultraflo^® was chosen with to obtain limiting enzymatic hydrolysis conditions. No BnzGlcA-cleaving activity was detected in Ultraflo^®. Released monosaccharides were monitored by high-performance anion exchange chromatography with pulsed amperometric detection on an ICS3000 system equipped with a 4 × 250 mm Dionex Carbopac™ PA1 column with a 4 × 50 mm guard column maintained at 30 °C, (Dionex, Sunnyvale, CA, USA). 25 μL samples were injected. The eluents were—A: Water; B: 300 mM sodium hydroxide, and C: 100 mM sodium hydroxide and 85 mM sodium acetate. The samples were eluted isocratically with 100% eluent A for 40 min (1 mL/min) and detected with postcolumn addition of 0.5 mL/min of solvent B. Thereafter, a cleaning step with 40% eluent A and 60% eluent B was performed at 1 mL/min for 10 min. Peak analysis was performed using the Chromeleon software. Peaks were quantified against pure monosaccharide standards, and 10 mg/L fructose was added as an internal standard.

The main monosaccharides present in the MF, PF and PMF fractions were evaluated after acid hydrolysis by TFA, following the methods reported by Fengel & Wegener [46 ]. The hydrolysate mixtures were dried under vacuum and the residue was solubilized in 10 mL of H₂O, and filtered. The carbohydrate analysis was performed with a HPLC Dionex DX500 system equipped with a GP50 gradient pump, an ED40 Electrochemical Detector and Dionex Peaknet 5.11 chromatographic Software (Dionex corporation, Sunnyvale, CA, USA). The monosaccharide separation was obtained using a Dionex CarboPac PA1 column (Dionex corporation, Sunnyvale, CA, USA) with a PA1 guard column; the mobile phases were sodium hydroxide (45 mM) and water at isocratic mode (10:90), the time course was 30 min at 35 °C and the flow rate was 1.0 mL/min. Meanwhile, the standard solutions of rhamnose, arabinose, galactose, glucose, mannose and xylose were used for the identification and quantification of the main monosaccharides.

The composite monosaccharides in fucoidans F85 and F95 were quantitatively measured using high-performance anion exchange chromatography equipped with a pulsed amperometric detector (Dionex ICS-5000; Thermo Fisher Scientific, Waltham, MA, USA). Before analysis, a 10 mg sample was separately hydrolyzed with 100 µL of 72% H₂SO₄ at 30 °C for 2 h, further treated at 121 °C for 1 h with addition of 2.8 mL DW, and filtrated using a 0.2 µm polytetrafluoroethylene syringe filter unit. The analysis was conducted with a Dionex CarboPac PA-1 column (10 µm, 4 × 250 mm, Thermo Fisher Scientific). A 20 µL of the sample was injected and eluted with 18 mM NaOH at a flow rate of 1 mL min⁻¹. The monosaccharides were identified and calculated by comparison with the retention times and areas of eight monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, and fructose) with three points of the external standard method.

Orafti^® HSI and Fibruline^® Instant samples were dissolved in purified water and were subjected to high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), using ICS-MS 6000 (ThermoFisher). Separation was carried out on a Dionex Carbopac PA1 column (10 μm, 4.0 × 250 mm) (Thermo Scientific, Loughborough, UK), at 30 °C. A gradient analysis was performed using (A) 0.1 M sodium hydroxide and (B) 1.0 M sodium acetate/0.1 M sodium hydroxide eluents. Running conditions were 0–15 min: 100 mM NaOH/20 mM NaOAc, 15–70 min 100 mM NaOH/20–450 mM NaOAc, 70–70.1 min: 100 mM NaOH/20 mM NaOAc, 70.1–75 min: 100 mM NaOH/20 mM NaOAc), at a flow rate of 1 mL/min and an injection volume of 10 μL. Chromeleon 7.2 software (Thermo Scientific) was used to interpret the chromatograms. The relative percentage of DP was calculated based on the ratio of each peak area to the total sum of integrated peak areas for each sample. The DP of samples was identified based on in-house inulin hydrolysates originating from Jerusalem artichoke inulin samples that were used as reference standards.

GDP-d-glycero-4-keto-α-d-lyxo-heptose (22) was made from GDP-d-glycero-α-d-manno-heptose (5) using Cj1427 C4-dehydrogenase (from serotype
HS:2). Cj1427 (10 μM) was incubated with 4.0 mM GDP-d-glycero-α-d-manno-heptose (5) and 8.0 mM α-ketoglutarate in 4.0
mL of 50 mM HEPES, pH 8.0, for 18 h. The reaction mixture was subsequently
loaded onto a prepacked 9 mm × 25 mm Dionex CarboPac PA1 column
(Thermo Scientific) after the enzyme was removed using a 10 kDa molecular
weight cutoff filter (Pall Corporation). The column was washed with
water and then eluted using a linear gradient (0–80%) of 2
M ammonium acetate, pH 8.0, over four column volumes. Fractions of
1.0 mL were collected based on the UV spectra of the fractions (230–300
nm). The pooled fractions were analyzed using ESI-MS and then dried
under vacuum.