Spherisorb sax column

Heparin (Toronto Research Chemicals, North York, ON, Canada) was depolymerized with Heparinase I. Specifically, 1 g of Heparin was digested using 0.8 IU of Heparinase I in 20 mL of 50 mM NH₄CH₃CO₂, 5 mM CaCl₂, pH 7.0 buffer at 30 °C, until depolymerization was approximately 30% complete, as estimated using A₂₃₂. Digested fragments were separated via size exclusion chromatography on a 2.5 × 175 cm Bio-Rad Biogel P10 column with a flow rate of 0.2 mL/min. Heparin dp6 were further separated using SAX HPLC with a 4 × 250 mm Waters Spherisorb SAX column to produce pure DP6-C oligosaccharides. Selectively desulfated Heparin dp6 was purchased from Iduron (Alderly Edge, UK). Concentrations of all GAG samples were quantified using the carbazole assay [35 (link)].

Based on the different Mw of the oligosaccharides in dHfFS-2, dHfFS-2 was fractionated by repeated gel permeation chromatography (GPC) using Bio-Gel P6 column (fine, 2 × 180 cm, Bio-Rad). Briefly, dHfFS (~0.65 g) was dissolved in 3 mL of deionized water, subjected to the Bio-Gel P6 column equilibrated with 0.2 M NaCl, and then eluted with the same solution at a flow rate of 10 mL/h, and about 100 fractions (2 mL/tube) were collected. Each fraction was measured by the sulfuric acid-phenol method at 482 nm. According to the eluted profile, some fractions were analyzed by a Superdex Peptide 10/300 GL column (GE Healthcare Life Sciences), eluted with 0.2 M NaCl solution at the flow rate of 0.4 mL/min, monitored by a RID. The fractions that showed a single peak were combined and desalted by Sephadex G-10 or Bio-Gel P2 column and lyophilized, respectively. The obtained components were designated as F1, F2, F3, F4, and F5, respectively. The complex signals presented in the ¹H NMR of F3 were further purified using a Spherisorb SAX column (4.6 × 250 mm, Waters). The column was equilibrated and eluted with 0.05 M KH₂PO₄, pH 3.0, at a flow rate of 1.0 mL/min. The elution profile was measured by RID, and three peaks were collected, desalted, and freeze-dried. Two homogeneous oligosaccharides (F3-a and F3-b) were obtained for further structural analysis.

HPLC system from Shimadzu equipped with a Spherisorb SAX column (5 μm, 4.6 × 250 mm, Waters) fitted with a guard cartridge (Waters Spherisorb, 5 μm, 4.6 × 10 mm) was used for analysis of disaccharides after digestion of the GAGs fractions with chondroitin ABC and/or AC lyase (Sigma-Aldrich Co., St. Louis, MO, USA). Elution was performed at 1.5 mL/min in isocratic mode from 0 to 5 min with 50 mM NaCl (pH 4.0) and then in linear gradient from 5 to 25 min, starting with 50 mM NaCl (pH 4.0) and ending with 76% 50 mM NaCl (pH 4.0) and 24% 1.2 M NaCl (pH 4.0). Disaccharide peaks were detected at 232 nm and quantified by external calibration [21 (link)].