Lc 20 ad

Dried ANE samples were placed inside a furnace at 600°C for 6 h in order to obtain and quantify the ash content. Total sugars were quantified according to Rioux et al. (2007) (link). Total polyphenol content was determined spectrophotometrically following the method of Goñi et al. (2018) (link). The content of unidentified organic components was calculated by difference to the total organic amount. The molecular weight (M_w) distribution of carbohydrates from different samples was analyzed using high performance size exclusion chromatography-refraction index detector (HPSEC-RID). The HPSEC Shimadzu system consisted of a system controller CBM-20A, a solvent delivery module LC-20 AD, an online degasser DGU-20A5, an autosampler SIL-20ACHT, a refraction index detector (Varian Prostar 350 RID), and an LC workstation. HPSEC analysis was performed using 4 PL aquagel-OH MIXED-H columns in tandem (8 μm, 300 × 7.5 mm; Agilent). The mobile phase (0.1 M NaAc/0.1 M Na₂SO₄ buffer pH 7.8) was used as isocratic elution at room temperature. The flow rate and injection volume were set to 1 ml min⁻¹ and 40 μl, respectively. M_w values were calculated from the measured retention times through a calibration curve made with dextran standards.

The molecular weight (M_w) distribution of carbohydrates of the four brown seaweed extracts was detected and measured using high-performance size exclusion chromatography with a refraction index detector (HPSEC-RID). The HPSEC Shimadzu system consisted of a system controller CBM-20A, a solvent delivery module LC-20AD, an online degasser DGU-20A5, an autosampler SIL-20ACHT, a refraction index detector (Varian Prostar 350 RID) and an LC workstation. HPSEC analysis was performed using PL aquagel-OH MIXED-H columns (8 μm, 300 × 7⋅5 mm; Agilent). The mobile phase (0⋅1 M NaAc/0⋅1 M Na₂SO₄ buffer, pH 7⋅8) was used as the isocratic elution at room temperature. The flow rate and injection volume were set to 1 ml/min and 40 μl, respectively. A molecular weight calibration curve was constructed with the retention time values of known dextran standards (Sigma-Aldrich, MO, USA). For the analysis of the extracts, the measurable range was divided into four segments (>100, 50–100, 10–50 and <10 kDa). An average M_w for each extract within each range was determined, and relative peak area values were calculated using the LCsolution software (Shimadzu, Ireland).

The HPLC system (Shimadzu, Kyoto, Japan) consisted of a pump (LC-20AD), autosampler (SIL-30AC), column oven (CTO-20 A), and refractive index detector (RID-10 A), and was equipped with a Zorbox Eclipse AAA column (150 × 4.6 mm, 3.5 microns; Agilent Technologies, PA, CA, USA). Samples were fractionated using a binary nonlinear gradient with mobile phase A (0.1% acetic acid in deionized water) and mobile phase B (0.1% acetic acid in acetonitrile). The column temperature was 35 °C, flow rate was 0.5 mL/min, and UV detection wavelength was 254 nm. The percentages of the mobile phases and the time periods were as follows: 95% mobile phase A for 4 min, 65% mobile phase A for 48 min, 15% mobile phase A for 54 min, 100% mobile phase B for 56 min, 100% mobile phase B for 66 min, 95% mobile phase A for 75 min, and 95% mobile phase A for 80 min.