Advance 3 500

NMR spectra were recorded on the Bruker Advance III 500 instrument (¹H: 500 MHz, ¹³C: 125 MHz; Bruker, Billerica, MA, USA), with chemical shift values expressed as ppm relative to tetramethylsilane (δ_H 0.00 ppm) or residual chloroform (δ_H 7.26 and δ_C 77.16 ppm) as a standard.

All chemicals were purchased from Sigma-Aldrich or Alfa Aesar unless otherwise stated and were used without further purification. The ¹H and ¹³C NMR spectra were recorded using a Bruker Advance III 500 MHz (11.4 T) spectrometer equipped with 5 mm PABBO probes and BVT-3000 temperature control unit. Chemical shifts δ are reported relative to TMS and were referenced using the residual proton solvent resonances. HPLC analyses and mass spectra were performed on a Waters HPLC-MS system equipped with a Waters 1525 binary pump. Analytical measurements were carried out on a Waters XTerra MS C18 (5 μm 4.6 × 100 mm) and on a Waters C18 XTerra Prep (5 μm 19 × 50 mm) for preparative purposes. Electrospray ionization mass spectra (ESI MS) were recorded using an SQD 3100 Mass Detector (Waters), operating in positive or negative ion mode, with 1% v/v formic acid in methanol as the carrier solvent.

For Fourier transform infrared spectroscopy (FT-IR) (Thermo Fisher Nicolet iS50, Waltham, MA, USA) analysis, the sample (2 mg) was first mixed with the dried KBr (w/w = 1:100), and then pressed into a flake for measurement. The spectrum was recorded in the range of 500–4000 cm⁻¹ at a resolution of 2 cm⁻¹. The molecular weight of the isolated chrysolaminarin was measured by high-performance gel permeation chromatography (HPGPC) (Agilent 1260, Santa Clara, CA, USA) equipped with a TSK-G3000 PW_XL column (7.5 mm × 300 mm) and a refractive index detector (Agilent RID-10A Series) [20 (link)]. The molecular weight was estimated by reference to a calibration curve made with T-series Dextran standards (1–670 kDa). The monosaccharide composition was determined using gas chromatography–mass spectrometry (GC–MS) as described by Xia et al. [15 (link)]. Identification of the derivatized monosaccharide was carried out according to the retention time and mass fragmentation patterns of the standards. For nuclear magnetic resonance (NMR) analysis (Bruker Advance III 500, Karlsruhe, Germany), the sample (10 mg) was dissolved in D₂O (deuterium oxide) in an NMR tube, and the ¹H (500 MHz) and ¹³C (125 MHz) spectra were recorded at 30 °C.