Rid 20

The molecular weight of AMP was determined by HPLC-RID (LC-20, Shimadzu, Japan; RID-20, Shimadzu, Japan) equipped with an aqueous gel column (TSKgel GMPWXL, 7.5 mm × 300 mm, TOSOH, Tokyo, Japan). The parameter settings were as follows: the injection volume was 20 µL; the mobile phase was 0.1 mol/L NaNO₃ + 0.06% NaN₃ solution; the flow rate was 0.6 mL/min; the column temperature was 35 °C.

The molecular weight of HP was determined by high performance gel permeation chromatography (GPC) coupled with a refractive index detector (RID-20, Shimadzu, Kyoto, Japan). A guard column of TSK (35 mm × 4.6 mm i.d.), and columns of TSKgel G3000PWXL (300 mm × 7.8 mm i.d.) and TSKgel G4000PWXL (300 mm × 7.8 mm i.d.) (Tosoh Co., Ltd., Tokyo, Japan) were used. HP was dissolved by distilled water (20 mg/mL) and filtered through a 0.45 μm filter. The injection volume was 20 μL, and mobile phase was 0.05 M sodium nitrate solution containing 0.5 g/L of sodium azide as preservative. Elution was carried out at a flow rate of 0.6 mL/min at 35 °C. 1 mg/mL of pullulan polysaccharide (shodex Company, Yokohama, Japan) standard was used as markers [31 (link)].

The distributions of molecular weight and chain length of the AS were determined by size-exclusion chromatography (SEC), with which the molecules were separated based on size or hydrodynamic volume [22 (link)]. A solution of starch (20 mg) in 4 mL of DMSO (90% in distilled water, v/v) was heated at 100 °C for 15 min and then cooled to 25 °C under continuous stirring for 24 h. Subsequently, the mixture was diluted with 20 mL of 95% (v/v) ethanol and centrifuged at 3500× g for 10 min. The supernatant was discarded, and the pellet was reconstituted in 4 mL of deionized water, and the resulting suspension was stirred in a water bath at 100 °C for 15 min. The mixture was cooled to 25 °C and filtered through a 5-μm sieve, and then the filtrate was passed through a gel-permeation chromatography column equipped with refractive index detector (RID-20, Shimadzu, Kyoto, Japan). The mobile phase was an aqueous solution of 0.06% NaN₃ (ultrasonicated before injection), the flow rate was 0.2 mL/min and the SEC column (TSK gel GMPWXL, TOSOH, Tokyo, Japan) temperature was 60 °C. The ASTRA 6.1 software package (Wyatt Technology Inc., Goleta, CA, USA) was used to determine the molecular weight, from which the polydispersity index (D) was calculated based on the following equation:
where Mw is the weight average molecular weight, and Mn is the number average molecular weight.

The average Mw distribution and polydispersity (Mw/Mn) of the two RGPs were determined according to methods proposed in previous studies [28 (link),29 (link)]. The Mw of the RGPs was determined using a Shimadzu LC20 HPLC system equipped with a Shimadzu RID-20 differential refractive index detector (Shimadzu, Tokyo, Japan). The analysis conditions were as follows: the mobile phase was 0.1 mol/L NaNO₃ and 0.06% NaN₃; flow rate, 0.6 mL/min; the column temperature, 35 ± 0.1 °C; and injection volume, 20 μL.

A narrowly distributed polyethylene glycol (PEO) was used as the standard curve for the relative calibration method and as the standard sample group in the detection using a differential refractive index detector (RID-20, Shimadzu, Japan). The precipitate was washed twice with anhydrous ethanol, air dried, dissolved by adding a solution of 0.1 mol/l NaNO₃ and 0.06% NaN₃, reacted at 121°C for 20 min, and centrifuged at 5000 r/min for 10 min; subsequently, 20 μl of the sample was collected. The detection conditions were as follows: flow rate, 0.6 ml/min, and column temperature, 35°C.