Carbopac pa1 2 250 mm analytical column

Samples for carbohydrate and acid-insoluble lignin content analysis were prepared using a standard NREL two-stage acid hydrolysis protocol [28 ]. Acid hydrolysis generates soluble sugars and acid-insoluble lignin residues, where the later was dried overnight at 105 °C in an oven (Heratherm oven, Thermo Scientific, MA, USA) and weighed to obtain the acid-insoluble lignin (Klason lignin) content. The soluble sugars were analyzed for carbohydrate constituents by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex ICS-3000, Dionex, Sunnyvale, CA, USA). Separation of soluble sugars was achieved utilizing a CarboPac-PA1 2 × 250 mm analytical column equipped with a CarboPac PA1 2 × 50 mm guard column (both from Dionex, Sunnyvale, CA), operated at 30 °C, with Milli-Q water as a mobile phase with a flow rate of 0.250 mL/min. The total run time was 35 min. External calibration curves were established using the standard solutions of arabinose, galactose, glucose, mannose, and xylose. The standard solutions were prepared from their corresponding monosaccharide (> 99%) obtained from Sigma-Aldrich.

Reaction samples were vacuum-filtered using 0.22-μm, PVDF filter plates (Millipore, USA) with a Tecan liquid handler (500 mbar) (Tecan Trading AG, Switzerland). The flow-through was collected to Nunc™ 96-well polypropylene microplates (Thermo Fisher Scientific, USA), and covered with Nunc™ 96-well silicone cap mats. The presence of neutral and acidic sugars was detected using an ICS5000 HPAEC-PAD system (Dionex, USA) with a CarboPac PA1 (2 × 250 mm) analytical column (Dionex, USA). The HPAEC-PAD samples were eluted at 0.25 mL/min using NaOAc gradient (0–0.5 M) in 0.1 M NaOH. The same filtered samples were also analyzed by a Dionex Ultimate 3000 HPLC (Dionex, USA) equipped with a UV detector (DAD-3000) and a Shodex RI-101 differential refractive index detector. The products were separated using a Bio-Rad HPX-87H column at 65 °C, and eluted with 5 mM H₂SO₄ at 0.4 mL/min. Wavelengths were set at 210, 214, 260 and 280 nm. Chromatograms were analyzed using Chromeleon 7.2 (Dionex, USA).

Oligo- and monosaccharides were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS-3000 system operated by Chromeleon software version 7 (Dionex). Sugars were loaded onto a CarboPac PA1 2 × 250-mm analytical column (Dionex, Thermo Scientific) coupled to a CarboPac PA1 2 × 50-mm guard column kept at 30°C. Depending on the analytes, the following gradients were used. The system was run at a flow rate of 0.25 ml/min. For manno-oligosaccharides, the elution conditions were as follows: for 0 to 9 min, 0.1 M NaOH; for 9 to 35 min, 0.1 M NaOH with a 0.1 to 0.3 M sodium acetate (NaOAc) gradient; for 35 to 40 min, 0.1 M NaOH with 0.3 M NaOAc, and for 40 to 50 min 0.1 M NaOH. Commercial mannose and manno-oligosaccharides (DP, 2 to 6) were used as external standards.