Tsk gel g 6000 pwxl column

The molecular weight of polysaccharides was measured by high-performance gel permeation chromatography (HPGPC). The HPGPC system (Shimadzu LC-20A, Shimadzu Instrument Co., Ltd., Tokyo, Japan) was equipped with a TSK-GEL G-3000PWXL column (7.8 mm × 300 mm i.d., 7 μm, Tosoh Corporation, Tokyo, Japan) and a TSK-GEL G-6000 PWXL column (7.8 mm × 300 mm i.d.,13 μm, Tosoh Corporation, Tokyo, Japan) that were linked in series, eluted with 0.02 M potassium dihydrogen phosphate (KH₂PO₄) at a flow rate of 0.5 mL/min and detected by a Waters 2414 differential refractive index detector (Waters Co. Ltd., Milford, MA, USA). The column temperature was kept at 35 ± 1 °C. The polysaccharides were dissolved in 0.02 M KH₂PO₄ and filtered through a 0.22 µm filter membrane. An aliquot of 25 μL of the sample was injected into the system. The molecular weight of polysaccharides was determined by the calibration curve made from dextran standards with known molecular weights (4.66, 12.6, 63.3, 126, and 556 kDa).

The molecular weight distribution of these samples was determined by GPC on a Waters Series 1500 system, equipped with a TSKgel G3000PWXL column (7.8 mm × 300 mm, Tosoh, Japan) and a TSKgel G6000PWXL column (7.8 mm × 300 mm, Tosoh, Japan). The system was connected with a Wyatt MALS detector (DAWN HELEOS Ⅱ, Wyatt technology, Santa Barbara, CA, USA) and a Wyatt RI detector (Optilab Trex, Wyatt technology, Santa Barbara, CA, USA). Each polysaccharide fraction (10 ± 0.05 mg) was added to 90% dimethylsulfoxide (DMSO, v/v, 1 mL) overnight at 100 °C, followed by centrifugation. Then, the precipitate was washed twice with anhydrous ethanol (3 mL) to remove the residual DMSO. Afterwards, the dried precipitate was dissolved in 0.1 mol/L NaNO₃ (3 mL) at 121 °C for 20 min, and centrifuged (12,000 rpm, 10 min) for analysis. The sample (100 µL) was eluted off by NaNO₃ solution (0.1 mol/L) at a flow rate of 0.4 mL/min and the column oven was kept at 60 °C. The data was analyzed using ASTRA6.1 software (Wyatt Technology Corporation, Santa Barbara, CA, USA).

The fraction SEC-NPs was applied to a TSKgel G6000PWxl column (0.78 × 300 cm, Tosoh Bioscience, Japan) with a HPLC system (Quest 10 Plus, Bio-Rad, USA), eluted with the phosphate buffer (0.1 M, pH7.4) at a flow rate of 0.80 mL/min. The eluates were continuously monitored with a UV detector and a multi-angle laser light scattering detector (MALLS, DAWN HELEOS II, Wyatt Technology, CA, USA) to obtain the absorbance at 280 nm and light scattering intensity at 658 nm. The exclusion of single molecules in SEC-NPs was confirmed by calculating the geometric radius distribution of chromatographic peak of SEC-NPs.