Ed40 electrochemical detector

Monosaccharide analysis. d-Glucose was measured enzymatically by the coupled GOD/POD assay, as described previously [20 (link)]. For the determination of d-galactose, the lactose/d-galactose test kit from Megazyme was used.
Oligosaccharide analysis. Capillary electrophoresis (CE) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex, Sunnyvale, CA, USA) were used for the qualitative and quantitative analysis of galacto-oligosaccharides. A capillary electrophoresis system with a UV-DAD detector (Agilent Technologies, Palo Alto, CA, USA) together with a fused silica capillary (internal diameter of 25 µm) equipped with a bubble cell detection window (bubble factor of five) was used for carbohydrate analysis. Carbohydrate samples were derivatized with 2-amino pyridine for CE analysis, as given in detail in [20 (link)]. HPAEC-PAD analysis was carried out on a Dionex DX-500 system consisting of a GP50 gradient pump (Dionex), an ED 40 electrochemical detector with a gold working electrode (Dionex), and an Ag/AgCl reference electrode (Dionex). Separations were performed at room temperature on a CarboPac PA-1 column (4 × 250 mm) connected to a CarboPac PA-1 guard column (Dionex).

Ionic content was detected using a DIONEX-DX500 ion chromatograph equipped with an AS40 autosampler and ED40 electrochemical detector (Dionex Corporation, Sunnyvale, CA, USA) as described by Ariz et al. (2011) (link). Frozen plant tissue (200 mg) was incubated in 1 ml of milli-Q water for 5 min at 80 °C in a water bath. The soluble ionic fraction was obtained by centrifugation at 16 000 g for 30 min. The supernatants, stored at –20 °C, were diluted 1:10 for injection. Ion Pac CG12A and Ion Pac CG12A were used as the stationary phase and 30% 100 mM NaOH and 70% milli-Q water were used as the mobile phase at 1.5 ml min^–1 flow rate for 15 min. Soluble cations (Na⁺, K⁺, Mg²⁺, Ca²⁺, and NH₄⁺) were determined using 20 mM methanosulfonic acid as the mobile phase for 13 min.

Extraction and measurement of starch was as previously described31 (link). AX oligosaccharide generation and fractionation: The husk and embryo were removed from 30–40 dry grains or seedlings and the remaining tissue was pooled, ground under liquid nitrogen and freeze-dried. Samples (100 mg) were prepared as described57 (link), then treated with 16 units of endo-xylanase M1 (EC 3.2.1.8) from Trichoderma viride (Megazyme)57 (link)58 (link). AX oligosaccharides were analysed by high-performance anion-exchange chromatography (HPAEC)58 (link) on a Carbopac PA-1 column (2 × 250 mm; Dionex) at 25 °C and a flow rate of 1 ml min⁻¹. Detection was with an ED40 electrochemical detector (Dionex) with an Au working electrode, and an Ag/AgCl reference electrode in pulsed amperometry mode. Xylose and xylan oligosaccharides (xylobiose to xylohexaose) were identified from the elution profile of standards (10 μM each; Megazyme). Peaks corresponding to AX oligosaccharides were identified through comparison with samples that had been digested with 2 units Aspergillus niger α-d-arabinofuranosidase (Megazyme) at 37 °C for 1 h.