Carbopac pa 20

Dionex Carbopac TM PA20 (3 × 150 mm) was used as the chromatographic column with the following mobile phases: A (H₂O), B (15 mM NaOH), and C (15 mM NaOH and 100 mM NaOAC). A flow rate of 0.3 mL/min and an injection volume of 5 μL was applied. The column temperature was 30^°C, and analysis was performed using a chemical detector.

Yam polysaccharide fractions (10.00 mg) were hydrolyzed with 10 mL of 3 M trifluoroacetic acid (TFA) at 120 °C for 3 h. After hydrolysis, excess TFA was removed by evaporating using a nitrogen blower and re-dissolved in 5 mL of deionized water. After that, the water solution (100 μL) was mixed with 900 μL of deionized water, and centrifuged at 12,000 rpm for 5 min. Then, 5 μL of the supernatant was analyzed using an ICS-5000 ion chromatographer (Thermo Fisher Technology Co., Ltd., Waltham, MA, USA) equipped with a Dionex CarbopacTMPA20 chromatographic column (150 mm × 3 mm) and a DC-2 electrochemical detector. The mobile phase: A was deionized water, B was a 15 mM NaOH solution and C was a 15 mM NaOH and 100 mM NaOAc solution; the elution gradients are detailed in Table 1. The rate of flow was 0.30 mL/min, and the column temperature was 30 °C.

Monosaccharide quantification of AIMs after two or three bleaching steps was performed according to the HPAE-PAD technology (High Pressure Anion-Exchange - Pulsed Amperometric Detection) on an ICS-3000, Dionex system equipped with a Dionex CarboPac^TM PA-20 (3 mm × 150 mm) analytical column. In short, lyophilized samples were hydrolyzed in 2 M trifluoroacetic acid at 105°C for 4 h (100 μg/100 μL), and the solution was neutralized with sodium hydroxide. Hydrolytic conditions deacetylate N-acetyl-glucosamine and N-acetyl-galactosamine, which are subsequently analyzed as glucosamine and galactosamine, respectively. Filtered samples (20 μL) were eluted at 0.4 mL/min (35°C) using the following sodium hydroxide gradient: pure water 99.2%/250 mM NaOH 0.8%: 0 ∼ 20 min; pure water 75%/250 mM NaOH 20%/NaOAc (1M)- NaOH (20 mM) 5%: 20 ∼ 37 min; pure water 40%/250 mM NaOH 20%/NaOAc (1M)-NaOH (20 mM) 40%: 37 ∼ 41 min. Each elution was followed by a wash and subsequent equilibration time. External sugar and uronic acids standards were used for calibration (7 points per curve): fucose, glucose, xylose, galactose, mannose, rhamnose, arabinose, glucosamine, galactosamine, galacturonic acid, and glucuronic acid (all provided by Sigma-Aldrich).

Sixteen monosaccharide standards were accurately weighed and configured as mixed standard samples as control. 5 mg of purified BMP sample was accurately weighed in an ampoule. 2 mL of 3 mol/L trifluoroacetic acid solution was added and the polysaccharides were hydrolyzed in an oven at 120°C for 3 h. After hydrolysis, the sample solution was transferred to a test tube, and nitrogen was used to evaporate it to dryness. The sample was re-dissolved with 5 mL of double-distilled water, and 50 μL was diluted 20 times and centrifuged at 12,000 rpm/min for 5 min. The supernatant was then removed. The ion chromatography was performed using a Dionex CarbopacTM PA20 (3*150 mm) column with mobile phases A. H₂O, B. 15 mM NaOH, C. 15 mM NaOH & 100 mM NaOAC and monitored by an electrochemical detector.

After hydrolyzing with 3 M trifluoroacetic acid at 120 °C for 3 h, the monosaccharide composition of CEP4 was determined by ion chromatography (IC) (ICS5000, Thermo Fisher). IC was performed in a Dionex CarbopacTM PA20 column (3 × 150) at 30 °C, eluting with H₂O, 250 mM NaOH, 50 mM NaOH, and 500 mM NaOAC, in turn, at a flow rate of 0.3 mL/min. An electrochemical detector (ECD) was used for detection.

The composition and content of monosaccharides in the Se−TPSs were determined by ion chromatography. Firstly, 5 mg of the sample was weighed accurately and placed in ampoules, adding 2 mL of 3 M trifluoroacetic acid. It was then hydrolyzed for 2 h at 120 °C. Once finished, the resultant was transferred into the tube and dried by nitrogen blowing. Subsequently, 10 mL of water was added for the vortex, of which 40 µL of the solution was taken and diluted to 1 mL. Then, the diluent was centrifuged at 12,000 rpm for 5 min, and the supernatant was injected for further analysis. Chromatographic column: Dionex Carbopac TMPA20 (3 × 150 mm). Moving phase: A: H₂O, B: 15 mM NaOH, C: 15 mM NaOH, and 100 mM NaOAC. Low rate: 0.3 mL/min. Injection volume: 5 µL. Column temperature: 30 °C. Detector: electrochemical detector.