Tsk gel pwxl guard column

Vi, freeze dried as the sodium salt, was solubilized in water and H₂O₂ was added to give a final concentration of 2.5 mg/mL Vi and 5% (wt/v) H₂O₂ in water. The mixture was heated at 80±0.5°C for 2h. The mixture was then injected into a Hiscreen Capto Q [GE Healthcare] column (4.7 mL of resin loading up to 100 mg of fragmented Vi mixture) equilibrated with buffer A and populations of different average size were separated using a gradient step method. NaH₂PO₄ 20 mM pH 7.2 and NaH₂PO₄ 20 mM NaCl 1M pH 7.2 were used as buffer A and B respectively. Pools at average size Vi of 8.6 and 43 kDa were eluted at 25 and 37% of buffer B respectively. Each pool was desalted on a Sephadex G-25 column [GE Healthcare] equilibrated with water. The average size of the fragmented Vi pools was determined by HPLC-SEC equipped with a TSK gel 3000 PW_XL column and a TSK gel PW_XL guard column (Tosoh Bioscience). Dextrans (5, 25, 50, 80, 150 kDa) were used as standards (Sigma Aldrich). 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). HPAEC-PAD was used to measure Vi content [10 (link), 21 (link)]. ¹H NMR was used to verify Vi identity and confirm O-acetylation levels were >60% [10 (link), 21 (link)].

Concentration and mass average molecular weight (M_w) of γ-PGA was determined by gel permeation chromatography (GPC). The GPC was equipped with a TSKgel GMPWxl column (300 × 7.8 mm, Tosoh Bioscience GmbH, Griesheim, Germany) and a TSKgel PWxl guard column (40 × 6 mm, Tosoh Bioscience GmbH, Griesheim, Germany). The column was eluted with 30 mM KNO₃ at 40 °C at a flow rate of 1.0 mL min⁻¹. Peaks were detected with the RI detector of the integrated EcoSEC HLC-8320GPC system (Tosoh Bioscience GmbH, Griesheim, Germany). Data analysis was performed with the software WinGPC UniChrom (PSS Polymer Standards, Service GmbH, Mainz, Germany). Poly(ethylene oxide) standards with peak molecular weights (M_p) in the range of 56 to 1015 kDa (PSS Polymer Standards, Service GmbH, Mainz, Germany) were used to create a calibration curve for molecular weight. γ-PGA sodium salt (cosmetic grade; kindly provided by Henkel AG & Co. KGaA, Düsseldorf, Germany) was used to create a calibration curve to determine concentrations. Culture broth was diluted 1:50 with distilled water and filtered with 0.2 μm filters (Bulk GHP Acrodisc 13 mm syringe filter, GHP membrane, N° 4567, Pall Life Sciences, Dreieich, Germany). 30 μL of the standards and of the filtrated samples were injected.

OAg molecular weight (MW) distribution was evaluated by HPLC-SEC as previously described [30 (link), 48 (link)]. Samples were run, without pretreatment, on a TSK gel G3000 PWXL column (30 cm x 7.8 mm; particle size 7 μm; cod. 808021) with a TSK gel PWXL guard column (4.0 cm x 6.0 mm; particle size 12 μm; cod. 808033) (Tosoh Bioscience). 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). Void and bed volume calibration was performed with λ-DNA (λ-DNA MW Marker III 0.12–21.2 kb; Roche) and sodium azide (NaN₃; Merck), respectively. OAg peaks were detected by differential refractive index (dRI). OAg average MW was estimated on standard dextrans (Sigma) calibration curve.