Ics3000 chromatography system

The enzymatic digestibility of the dietary fiber samples was determined by calculating the enzymatic conversion (EC) after incubating samples with the Cellic CTec2 cellulase enzyme blend, as described by Chen and coworkers [32 (link)]. Cellulose was suspended (1.0% w/v) in a 50 mM sodium acetate buffer (pH 4.8) with 20 U Cellic CTec2 cellulase enzyme blend per gram cellulose and stirred at 900 rpm. After 1 h of incubation at 40 °C, the enzymes were denatured by heating the solution (5 min, 110 °C). The solid fraction was separated from the supernatant by centrifuging at 5000 g. The amount of glucose and cellobiose in the supernatant from cellulose hydrolysis was determined by high-performance-anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS3000 chromatography system (Sunnyvale, CA, USA). Saccharides were separated on a Dionex CarboPac PA-100 column (4 × 250 mm), equilibrated with 90 mM NaOH. The enzymatic conversion was calculated from the amount of glucose (m_g) and cellobiose (m_cb) in the supernatant, and the amount of starting substrate (m_c): $$EC=\frac{m_g+m_{cb}}{1.1m_c}$$

Carbohydrates were analyzed with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), performed on a Dionex ICS 3000 chromatography system (Sunnyvale, CA, USA). Analysis and detection were performed at 32°C and the flow rate was 250 μl per min. A 15 μl sample was injected on a Guard CarboPac PA 100 (2 × 50 mm) in series with an analytical CarboPac PA 100 (2 × 250 mm) equilibrated for 9 min with 90 mM CO₂-free NaOH. Sugars were eluted in 90 mM NaOH, with an increasing sodium acetate gradient: from 0 to 6 min, the sodium acetate concentration increased linearly from 0 to 10 mM; from 6 to 16 min from 10 to 100 mM; and from 16 to 26 min from 100 to 175 mM. The columns were then regenerated with 500 mM sodium acetate for 1 min and equilibrated with 90 mM NaOH for 9 min for the next run. Data were recorded and processed with Chromeleon software. In order to determine the total fructan content, 2.5 μl of 1.2 M HCl-solution was added to 50 μl of the watery extract (see above) and incubated for 90 min at 70°C. The hydrolysis was stopped by adding 2 μl of 1 M H₂CO₃. Deionized water was added up to a final volume of 1 ml, and the mixture was analyzed on a HPAEC-PAD. The fructan concentration and DP were calculated as described in Verspreet et al. (2012 (link)).

Dry matter (DM) content is defined as the oven-dry weight at 105 °C and was measured in triplicate using a National Renewable Energy Laboratory (NREL) standard method [51 ]. The composition of structural carbohydrates and lignin in the solids [52 ] and the sugars and degradation products in the liquid process streams [53 ] were analyzed by NREL standard methods. The HEX-treated materials were washed in a three-step procedure with 0.1 M NaOH, 0.05 M NaOH, and water prior to analysis to remove the hydrotropic agent and prevent redeposition of lignin onto the solids. The wavelength and extinction coefficient for the measurement and quantification of acid-soluble lignin was 240 nm and 25 L/g/cm, respectively [52 ]. All analyses were performed in triplicates.
Carbohydrates were measured by isocratic high-performance anion-exchange chromatography with pulsed amperometric detection, using an ICS-3000 chromatography system (Dionex, USA) that was equipped with a Carbo Pac PA1 analytical column (Dionex, USA). Measurements were performed at 30 °C using deionized water as the eluent at a flow rate of 1 mL/min.