G1315b dad detector

Liquid chromatography ultraviolet detection (LC-UV) analysis was performed using an Agilent 1290 series HPLC system equipped with an Agilent G1315B DAD detector. The UV detector was set at 245 nm, and separation was performed on an Acquity UPLC BEH C18 column (2 × 100 mm, 1.7 µm; Waters, MA, USA). Mobile phases were (A) 0.1% formic acid and (B) methanol. LC conditions were identical to those described in the previous section. The extract of the pigment with 1% SDS was passed through an ultrafiltration membrane (nominal molecular weight limit of 3 kDa), and 10-fold diluted samples were used for LC analysis.

The monosaccharide composition of BSP was determined using the 3-methyl-1-phenyl-5-pyrazolone (PMP)-labeling procedure as previously described (Liao et al., 2019 (link)). Briefly, BSP was hydrolyzed with 4 M trifluoroacetic acid at 110°C for 2 h, and subsequently labeled with PMP. PMP-labeled monosaccharides were identified on an Agilent 1100 HPLC system equipped with a ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm), and a G1315B DAD detector (Agilent, Waldbronn, Germany) at a wavelength of 245 nm. The mobile phase was A, acetonitrile and B, 0.1 M phosphate buffer (pH = 6.7) at a flow rate of 0.8 ml/min and a column temperature of 30°C. Mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose were used as standard. The molar ratios of the monosaccharides of BSP were measured on a Dionex ICS-3000 ion chromatography (IC) system with a CarboPac-PA20 column (3 mm × 15 mm), and integrated pulsed amperometry detector (Sunnyvale, CA, United States). The gradient elution conditions were as follows: 0–20 min, 99.2% H₂O (A), 0.8% 0.25 M NaOH (B); 21.1 min, 94.2% A, 0.8% B, 5% 1 M NaAc (C); 30.0 min, 79.2% A, 0.8% B, 20% C; 30.1–50.0 min, 20% A, 80% B. The flow rate was 0.5 ml/min and the injection volume was 20 μl.