Ultrahydrogel 500

Approximately 20 mg of the analyzed sample was weighed with accuracy of 0.1 mg and placed in a tube; next, 2 mL of 0.1 mol·L^-1 NaOH was added and the content was mixed until the compounds became diluted. A drop of phenolphthalein was added as a marker and subsequently neutralized with 2 mL of 0.1 mol·L^-1 HCl. Next, the sample was filtrated and injected into a system of chromatographic columns. The chromatographic analysis of the starch samples was carried out using gel chromatography (GPC).
The system consisted of two columns connected in a series (Ultrahydrogel 500, Ultrahydrogel 2000—Waters). A solution of 0.1 mol·L^-1 NaNO₃ and 0.02% NaN₃ was applied as an eluent. The flow rate was set to 0.6 mL·min^-1 and 100 μL of the sample volume was injected. Calibration was performed using the pullulan (Shodex) standards.
The following values were obtained for amylose: weighted molecular mass M_w = 35 kg·mol^-1, number molecular mass M_n = 23 kg·mol^-1, polydispersity 1.6, and for amylopectin respectively: M_w = 35 710 kg·mol^-1, M_n = 18 650 kg·mol^-1, polydispersity 2.

The apparent molecular weight of β-D-glucan phosphate and native β-D-glucan (NG) was determined by high-performance liquid chromatography (HPLC, Waters 1525, Waters, USA). Eight milligrams of lyophilised GP and NG powder was dissolved in 2.0 mL of distilled water and dimethylsulphoxide (DMSO), respectively, and passed through a 0.23-µm filter for HPLC analysis. HPLC was performed on two linked gel-filtration chromatographic columns of ultrahydrogel™ 500 (Waters, USA) (7.8×300 mm) eluted with 0.1 M NaNO₃ at a flow rate of 0.9 mL/min and detected by a refractive index detector. The PEAK molecular weight of GP was calculated by the calibration curve obtained by using various standard dextrans (Dextran T20, T40, T100, and T200).

Molecular characterization of the husk extract was performed using gel permeation chromatography (GPC). The system consists of two polymer-based columns—Ultrahydrogel-2000 and Ultrahydrogel-500 (Waters, USA) which are connected in a series with an RI detector (Knauer, Germany). An eluent was used in the form of NaNO₃ and NaN₃ solution in water, at a concentration of 0.1 mol·L⁻¹ and 0.02%, respectively. The eluent flow rate was set to 0.6 mL·min⁻¹ and 100 mL of the sample was injected. The extract used as a sample was prepared using 13 mg of husk in 1 mL of water. Pullulan standards (Shodex, Japan) were applied to perform the calibration procedure according to the previously described method [26 (link)]. All reagents and chemicals were purchased from Sigma Aldrich.
The protein content in the husk extract was determined via the Kjeldahl method according to the ISO standard (ISO 1871:200 [27 ]).

The homogeneity and Mw were analyzed by high-performance gel permeation chromatography (HPGPC) with a Waters Ultrahydrogel-500 column (7.8 mm × 300 mm)51 . The mobile phase was ultrapure water with 0.02% NaN₃ at a flow rate of 0.6 mL/min. The sample was dissolved in the mobile phase for 1 mg/mL, and the injection volume was 20 μL. A standard curve was drawn with glucose and the standard dextrans T-10, T-20, T-40, T-50, T-70, T-200, T-500 and T-2000 according to the time of the maximum peak and the logarithm of their respective Mw.

For further investigation, the molecular weight distribution concerning the soluble fractions of the hydrogel was analysed. These were isolated by placing the hydrogel in distilled water for 24 h, after which the soluble fraction was decanted. This procedure was repeated three times. The water was then evaporated, and the extracts thoroughly dried at 45 °C. The dried aqueous extracts were subjected to GPC analysis. The concentration of the samples used for analysis was 5 mg/mL. GPC analysis was performed at 25 °C with an eluent flow rate of 0.6 mL/min. A three-column system consisting of an Ultrahydrogel 2000 (Waters, Milford, MA, USA), Ultrahydrogel 500 (Waters) and Ultrahydrogel 120 (Knauer, Germany) was connected to an RI detector (Knauer, Berlin, Germany). A 0.1 mol/L aqueous solution of NaNO₃ with the addition of sodium azide was used as the eluent. Pullulan standards (Shodex, Yokohama, Japan) were used to calibrate the instrument.