Agilent 1200 series refractive index detector

Oligomers were separated according to their DP using a SECcurity GPC System (PSS Polymer Standards Service, Mainz, Germany) with an Agilent 1200 series refractive index detector (Agilent Technologies, Santa Clara, USA). Ten mg of chitosan hydrolyzates with F_A 0.19 and F_A 0.50 were filtered using a 0.22-µm syringe filter before being injected into the system. Separation was achieved with a HiLoad™ Superdex™ 30 prep-grade column (Pharmacia, Uppsala, Sweden) at a flow rate of 0.3 ml min⁻¹ of 0.15 M ammonium acetate buffer at a pH of 4.5. Data were recorded using the WinGPC UniChrom software (PSS Polymer Standards Service, Mainz, Germany).

Preparative separation of chitosan oligomers was achieved by size-exclusion chromatography using a SECcurity GPC System (PSS Polymer Standards Service, Mainz, Germany), three serial HiLoad 26/600 Superdex 30 prep grade columns (GE Healthcare Europe GmbH, Freiburg, Germany), and an Agilent 1200 series refractive index detector (Agilent Technologies, Santa Clara, USA). For data recording, the WinGPC UniChrom software (PSS Polymer Standards Service, Mainz, Germany) was used. Aliquots (100 mg each) of an oligomer mixture were dissolved in ammonium acetate buffer (0.15 M, pH 4.5) and separated using the same buffer as the mobile phase with a flow rate of 0.6 ml/min. The oligomers were collected in 3 ml fractions, analyzed via UHPLC-ELSD-LC-MS¹, and pooled accordingly. Twice the equimolar amount of HCl relative to the number of GlcN subunits was added to stabilize the oligomers. Ammonium acetate was removed by repeated freeze drying and dissolving in H₂O.

Samples analyzed by SEC-MALS were buffer-exchanged by dialysis into SEC-MALS running buffer containing 20 mm HEPES (pH 7.6), 200 mm NaCl, 5% glycerol, 0.5 mm TCEP, and 0.05% azide. The samples were applied to a Superose6 10/300 GL (Sigma–Aldrich) column, and in-line measurements were recorded for UV absorbance at 280 nm (using Agilent 1100 series UV detector, Agilent Technologies), static light scattering (DAWN HELEOS 8+light scattering detector, Wyatt Technology), and differential refractive index (Agilent 1200 series refractive index detector, Agilent Technologies) were recorded. System calibration was performed on a BSA standard (Sigma–Aldrich), first resuspended, and then buffer-exchanged into the SEC-MALS running buffer. SEC-MALS data were analyzed with ASTRA version 6.1 software (Wyatt Technology).