Xdb uv detector

Total sugar content was measured by the phenol-sulfuric acid method [29 (link)]. Protein content was determined according to the method of Bradford [30 (link)]. Sulfate content was assessed by the barium rhodizonic acid method [31 (link)]. Purity and molecular weight of polysaccharide were assessed by HPGPC on a Shodex OHpak SB-804 HQ column [19 (link)]. The molecular weight was estimated by reference to a calibration curve made by pullulan standards. Monosaccharide composition was assayed by a 1-phenyl-3-methyl-5-pyrazolone (PMP)-HPLC method using the Eclipse XDB-C18 column (4.6 × 250 mm, Agilent Technologies, Santa Clara, CA, USA) [21 (link)]. Sugar configuration was determined by reversed-phase HPLC [32 (link)] and the analysis was performed on an Agilent 1260 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) using an Agilent XDB-UV detector (250 nm).

The polysaccharide, F2-1 (5 mg), was hydrolyzed using 1 mL of 2 mol/L trifluoroacetic acid (TFA) at 105 °C for 6 h. TFA was removed by repeated co-evaporation with methanol and the hydrolysate was dried under reduced pressure. Uronic acid content was estimated by the carbazole–sulfuric-acid method [37 (link)]. Pyruvate acid content was determined by dinitrobenzene removal method [38 (link)]. The monosaccharide composition was analyzed using HPLC through pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) using an Agilent HPLC system fitted with an Agilent XDB-C₁₈ column (4.6 mm × 250 mm) and Agilent XDB-UV detector. The sulfate content of the polysaccharide was determined using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a CIC-100 ion chromatograph coupled with SH-AC-1 anion exchange column (4.6 mm × 250 mm, 13 µm) and conductivity detector [39 (link)]. The total sugar content was analyzed using the phenol–sulfuric-acid method using galactose as a standard [40 (link)], and protein content was measured using the Lowry method [41 (link)].

UP2-1 (5 mg) hydrolysis was performed with 2 mol/L trifluoroacetic acid (1 mL) at 105 °C for 6 h. After trifluoroacetic acid removal by repeated co-evaporation with methanol, the hydrolysate was dried under reduced pressure. The monosaccharide composition was then analyzed by HPLC through pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone using an Agilent HPLC system fitted with Agilent XDB-C18 (4.6 × 250 mm) and Agilent XDB-UV detector. The sulfate content of the polysaccharide was determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a CIC-100 ion chromatograph (Thermo Fisher Scientific, Walthsam, MA, USA) coupled with an SH-AC-1 anion exchange column (4.6 mm × 250 mm, 13 µm) (ShengHan Chromatograph Technology, Qingdao, China) and a conductivity detector [26 (link)].