Ics 5000 hplc system

Total fructan quantification in ground grain samples was performed using high performance anion exchange chromatography (HPAEC) analysis. An aliquot of the extract was hydrolyzed using a mild thermal-acid hydrolysis method (30 mM HCl in a heating block at 70 °C for 90 min). The total fructan level can then be calculated based on the released fructose and glucose molecules following hydrolysis [52 (link), 53 (link)]. WSC analysis was performed using an ICS-5000 HPLC system (Dionex) equipped with a CarboPac™ PA100 column (4 × 250 mm) and an electrochemical detector working in a pulsed amperometric mode using a gold working electrode and a combined pH-Ag/AgCl reference electrode. A standard quadruple-potential waveform for carbohydrates was used for all analyses (Supplementary Table 2). The elution program used for quantification of fructose, glucose, sucrose and raffinose is shown in Supplementary Table 3.

High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was performed using a Dionex ICS-5000 HPLC system equipped with an AS-AP autosampler in a sequential injection configuration using the Chromeleon software version 7. 10 μl of the samples were injected on a 3 × 250 mm Dionex Carbopac PA200 column (Thermo Scientific, Waltham, United States). 56 μM of the β-1,3/1,4-MLG tetrasaccharide or 45 μM of the β-1,3/1,4-MLG trisaccharide were loaded onto the column. The gradient was used as follows: 0–5 min, 10% B, 3.5% C (initial conditions); 5–12 min 10% B, linear gradient from 0–30% C; 12.0–12.1 min, 50% B, 50% C; 12.1–13.0 min, exponential gradient of B and C, back to initial conditions, 13–17 min initial conditions. Solvent A was ultrapure water, solvent B was 1 M sodium hydroxide and solvent C was 1 M sodium acetate.
Matrix Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) analysis of mixed-linkage glucans was performed with a Bruker Autoflex system (Bruker Daltonics) operated in reflectron mode. 10 mg/ml of the oligosaccharide were mixed 1:5 with 2,5-dihiydroxybenzoic acid in 1:1 H₂O:MeOH on a Bruker MTP 384 grounded steel MALDI plate. The samples were allowed to dry and directly analyzed.