Carbopac pa 1 guard column

Oxidized and non-oxidized cellooligosaccharides generated after LPMO action were analyzed by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) (ThermoFischer Scientific, IL) using a CarboPac™ PA1 column (2 × 250 mm) and CarboPac™ PA1 guard column (2 × 50 mm) at a 0.25 mL min⁻¹ flow rate as in [49 (link)]. Non-oxidized oligosaccharides were used as standards (Megazyme, Wicklow, Ireland).

A Dionex ICS 5000 chromatography HPLC system (Thermo Fisher Scientific, Switzerland) equipped with a CarboPac PA1 column (2 × 250 mm) in combination with a CarboPac PA1 guard column (2 × 25 mm) and a pulsed electrochemical detector in pulsed amperometric detection mode was used for the measurement of the sugar monomers according to a published method (23 (link)). The temperature of the column was set at 25°C with a flow rate of 1 mL/min. The monosaccharides were separated by a gradient using MilliQ water for eluent A and 200 mM NaOH solution for eluent B. The 37 min run had the following elution profile: 92% A and 8% B isocratically for the first 20 min, 100% B between 20.5 and 29 min, 92% A and 8% B at 29.5 min until the end. GlcN-HCl and Glc solutions with concentrations between 5 and 100 μM were used for the calibration, whereof 10 μL were injected into the system. The reported chitin and glycogen values are given on the basis of anhydrous monomer residues to represent the actual polysaccharide contents in the insects.

Samples were hydrolyzed using an adapted method based on a previously described procedure.^{26 (link)} In brief, samples were treated with concentrated H₂SO4 (1 h, 30 °C) and then hydrolyzed with 2.8 M H₂SO4 for 3 h at 100 °C. Samples were diluted 20 times and 10 μL was directly injected for analysis. High-performance anion exchange chromatography (HPAEC) was performed on a Dionex Ultimate 6000 system (Thermo Scientific, Sunnyvale, CA, USA) equipped with a CarboPac PA-1 column (2 mm × 250 mm ID) in combination with a CarboPac PA-1 guard column (2 mm × 50 mm ID) and pulsed amperometric detection (PAD). The system was controlled by Chromeleon 7.2.9 software (Thermo Scientific, Sunnyvale, CA, USA). Elution of monosaccharides was performed at a flow-rate of 0.25 mL min⁻¹ with a multi-step-gradient using the following eluents: A: 0.1 M NaOH, B: 1 M NaOAc in 0.1 M NaOH, C: 0.2 M NaOH, and D: MilliQ water. The gradient used was 16% A, 84% D (20 min), 45% A, 5% B, 50% D (5 min), and 60% A, 40% B (15 min). To regenerate the column, it was flushed for 12 min with 100% C by increasing the flow rate in the first 2 min to 0.35 mL min⁻¹. Finally, the column was equilibrated for 12 min with 16% A, 84% D by decreasing the flow rate in the first 2 min to 0.25 mL min⁻¹.

The oligosaccharides were also analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an ICS-3000 system (Dionex, Sunnyvale, CA, USA) and a CarboPac-PA1 analytical column (4 × 250 mm, Thermo Scientific, USA) fitted with a CarboPac PA1 guard column (4 × 50 mm, Thermo Scientific, USA). Before samples were injected, the column was washed with 200 mM NaOH for 10 min at a flow rate of 1 mL min⁻¹ and equilibrated for 5 min with 100 mM NaOH and 5% (v/v) 1 M NaOAc. The samples were eluted with a constant 100 mM NaOH solution and a gradient of 1 M NaOAc from 5% to 25% over 25 min. Pure DP3 (G4G3G, M3) and DP4 (G4G4G3G, M4) (1,3;1,4)-β-d-glucan oligosaccharide standards, and pure oligosaccharide standards derived from laminarin (laminaribiose L2, laminaritriose L3, and laminaritetraose L4) and cellulose (cellotriose C3, cellotetraose C4, cellopentaose C5, and cellohexaose C6) were obtained from Megazyme.

Separation of the oligosaccharides released from locust bean gum during the conversion described above was carried out using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Separation was performed at 30 °C on a CarboPac PA-1 column (4 mm × 250 mm) connected to a CarboPac PA-1 guard column (Dionex). For separation of manno-oligosaccharides, eluents A (150 mM NaOH) and B (100 mM NaOH and 0.5 M NaOAc) were mixed to form the following gradient: 100% A for 40 min, 100% B for 5 min, and then 100% A for another 15 min. The column was washed with 100% B for 10 min and re-equilibrated for 15 min with the starting conditions of the employed gradient. Manno-oligosaccharides standards are mannobiose, mannotriose, mannotetraose, mannopentose, mannohexaose and 63, 64-α-d-galactosyl-mannopentaose (O-GGM5) (Megazyme).

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).