Tskgel g3000pwxl column

The concentrations of carbohydrate, uronic acid, and monosaccharide composition were determined as previously reported [12 (link),13 (link),14 (link)]. Protein was determined using a Dumas nitrogen analyzer (NDA 701) [15 (link)]. The molecular weight was estimated with high-performance gel permeation chromatography (HPGPC) on a TSK-gel G-3000PWXL column (7.8 mm × 300 mm, TOSOH, Tokyo, Japan) coupled with a Shimadzu HPLC system.
The FT-IR spectrum was recorded with a KBr pellet among wave lengths 500 and 4000 cm⁻¹ on a NEXUS670 FT-IR spectrophotometer.
The ¹³C NMR spectrum was obtained on a Bruker AVIII spectrometer at 600 MHz. The sample (30 mg) was dissolved in D₂O (1 mL, 99.8%), and the spectra were recorded at 25 °C. Acetone was used as an internal standard.
WAP was added to a silicon plate with a thin-layer gold sputter-coated, and then synthesized hydro gel (CMH and NMH₃) was freeze-dried and covered with gold before the analysis of scanning electron microscope (SEM). The three-dimensional structure of WAP was characterized on XL 30 ESEM (Philips).

The change of molecular weight of citrus pectin after treatment with PpPel10a was analyzed by HPGPC using a TSK-gel G-3000PWXL column (7.8 × 300 mm; Tosoh, Tokyo, Japan) [43 (link)]. The column was calibrated with standard dextrans (1, 5, 12, 25 and 50 kDa) (Sigma-Aldrich, St. Louis, MO, USA). The unsaturated galacturonic acids released from citrus pectin by PpPel10a were analyzed by HPAEC with a pulsed amperometric detector [44 (link)]. The unsaturated galacturonic acids were identified by ESI-MS with the amaZon speed ion ETD trap (Bruker, Bremen, Germany) using negative electrospray as the ionization process [45 (link)].

A total of 3 mg/mL of polysaccharide was dissolved in Milli-Q water and subjected to separation by a multi-angle laser scattering system (MALLS) (DAWN HELEOS-II, Wyatt Technology Corporation, Santa Barbara, CA, USA), coupled with a TSK gel G3000PWXL column (7.5 × 600 mm, TOSOH Corporation, Japan) and a differential refractive detector (RI-10A, Shimazu Corporation, Kyoto, Japan), at 25 °C [31 (link)]. The mobile phase was a 0.15 mol/L NaNO₃ solution containing 0.02% NaN₃, with a flow rate of 0.6 mL/min. The value of 0.1380 mL/g was adopted as the refractive index increment dn/dc. ASTRA 5.3.4 software was used for the calculation of Mw and the radius of gyration (Rg).

HPLC—SEC analysis was used to estimate the molecular size distribution of OAg populations [34 (link),36 (link)]. Samples were run, without pre-treatment, on a TSK gel G3000 PWXL column (30 cm x_7.8 mm; particle size 7 um; cod. 808021) with a TSK gel PWXL guard column (4.0 cm _x 6.0 mm; particle size 12 um; cod. 808033) (Tosoh Bioscience, Tokyo, Japan). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2, at the flow rate of 0.5 ml/min (isocratic method for 30 min). OAg peaks were detected by differential refractive index (dRI). Void and bed volume calibration was performed with λ-DNA (λ-DNA molecular weight (MW) Marker III 0.12–21.2 kb; Roche) and sodium azide (Merck, New Jersey, USA), respectively. OAg average MW was estimated on standard dextrans (Sigma) calibration curve.

The purity and solution state of purified Simplagrin were analyzed using size exclusion chromatography with online multiangle light scattering (SEC-MALS-QELS-HPLC), refractive index, and ultraviolet (UV) detection. The instrument, a Waters Corporation (Milford, MA, USA) HPLC model 2695 and photodiode array detector model 2996 operated by Waters Corporation Empower software, connected in series to a Dawn EOS light scattering detector and Optilab DSP refractive index detector (Wyatt Technology, Santa Barbara, CA, USA), was used as directed by the manufacturer. Wyatt Technology's Astra V software suite was used for data analysis and processing. For separation, a TSK gel G3000PWxl column (7.8 mm×30 cm; 6 µm particle size) (Tosoh Bioscience, King of Prussia, PA, USA) was used with a TSK gel Guard PWxl column (6.0 mm×4.0 cm, 12 µm particle size). The column was equilibrated in mobile phase (1.04 mM KH₂PO₄, 2.97 mM Na₂HPO₄ • 7H₂O, 308 mM NaCl, 0.5 M urea, pH 7.4, 0.02% sodium azide) for at least 60 minutes at 0.5 mL/min prior to sample injection. SEC-MALS-HPLC analysis was performed on the Simplagrin using an isocratic elution at 0.5 mL/min in mobile phase. Bio-Rad gel filtration standards were run for size comparisons.

The Mw and homogeneity of SDF were determined via size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS, λ = 658 nm, Wyatt Technology Corporation, Santa Barbara, CA, USA). A TSKgel G5000PWXL column (30 cm × 7.8 mm ID, 10 µm; Tosoh Corporation, Tokyo, Japan) and a TSKgel G3000PWXL column (30 cm × 7.8 mm ID, 6 µm; Tosoh Corporation, Tokyo, Japan) were employed, along with a refractive index detector. The sample was prepared by ultra-pure water and filtered through a 0.22 μm filter before injection. The chromatographic conditions were as follows: column temperature, 25 °C; mobile phase, 0.1 mol/L NaNO₃; flow rate, 0.5 mL/min; and injection volume, 100 μL. The data were analyzed using Astra 7.3.2 software package, and the refractive index increment (dn/dc) value was determined to be 0.138 mL/g, in accordance with the literature [28 (link)].

The homogeneity and molecular weight of the purified RLPs were determined by high performance gel permeation chromatography (HPGPC), which was performed on a HPLC-1525 liquid chromatography instrument (Waters, Massachusetts, USA) with a TSK-GEL G5000 PWXL column (7.8 × 300 mm) series connected with a TSK-GEL G3000 PWXL column (7.8 × 300 mm) (Tosoh, Tokyo, Japan). The column was eluted with 0.02 M Na₂SO₄ with a flow rate of 0.6 mL/min and detected by a RID-2410 detector. The sample (2 mg) was dissolved in the mobile phase and centrifuged, and the volume of 20 μl sample was injected in each run. The molecular weight was estimated by reference to the calibration curve made from a standard dextran solution consisting T-400, T-300, T-200, T-100, T-40, T-20, T-10, and T-5 [24 (link)].