Zorbax ods column

Polysaccharide is a kind of macromolecular carbohydrate, which is consisted of at least 10 monosaccharides. The composed of monosaccharides is a critical element of polysaccharides, which is closely related to bioactivities of polysaccharides. The composition of ASP was analyzed by PMP‐label method as previously reported (Jin et al., 2018). In brief, the sample was hydrolyzed with trifluoroacetic acid (TFA) at 100°C for 6 hr. Then, the hydrolyzed production was reacted with PMP at 70°C for 30 min. The standards, including Glu, D‐Glu, Xyl, Rha, Man, Ara, Gal, Fuc, and lactose (internal reference), were treated in parallel with the sample. After several extractions, the aqueous phase was filtered into 0.45 μm membrane and separated by HPLC on an Agilent 1260 instrument (Agilent), which was equipped with Agilent ZORBAX ODS column (5 μm, 4.6 mm × 150 mm) plus a gradient elution program (Table 1). The A phase consisted of 0.1% methanoic acid and 50 mM ammonium acetate, while the B phase was acetonitrile and C phase was 0.1% methanoic acid. All the mobile phases were eluted in the column at a flow rate of 0.6 ml/min. The separation was recorded under 254 nm wavelength. The identifications were based on retention time with respect to standards. Quantification was carried out by integration of the chromatographic peak area.

Freeze-dried leaf samples were homogenized in 96% cold acetone with 0.3 mg mL⁻¹ calcium carbonate (CaCO₃) in a dim light. The pigments were separated by high-performance liquid chromatography (HPLC, Perkin Elmer, Waltham, MA, USA) with a diode array detector (DAD) and non-endcapped Zorbax ODS column (4.6 × 250 mm, 5 µm particle size, Agilent Technologies, Santa Clara, CA, USA). For pigment elution, the method described by Thayer and Björkman [55 (link)] was used with slight modification—100% solvent A (acetonitrile:methanol:water, 84:12:4) for the first 2 min followed by a 14 min linear gradient to 100% solvent B (methanol:ethyl acetate, 68:32), which continued isocratically for the next 9 min, with a flow rate of 1 mL min⁻¹. The pigments neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, β-carotene, chlorophyll a, and chlorophyll b were detected by absorbance at 440 nm and quantified against known standards (DHI Water and Environment, Hørsholm, Denmark).

Chlorophyll and carotenoids were extracted and analyzed as reported by Castagna et al.^{39 (link)}. Carotenoids included β-carotene, neoxanthin, lutein, violaxanthin, antheraxanthin and zeaxanthin. Leaf disks were homogenized under dimmed room light in 100% HPLC-grade acetone with 1 mM sodium ascorbate. The extract was filtered through 0.2-µm filters (Sartorius Stedim Biotech, Goettingen, Germany) and analyzed by a Spectra System P4000 HPLC equipped with a UV 6000 LP photodiode array detector (Thermo Fisher Scientific, Waltham, MA) using a Zorbax ODS column (5 μm particle size, 250 × 4.6 mm Ø, Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 1 mL min⁻¹. Acetonitrile/methanol (85/15) and methanol/ethyl acetate (68/32) were used as solvent A and solvent B, respectively, according to the following gradient: solvent A: 100% (0–15 min), 100–0% (15–17.5 min), 0% (17.5–32 min), followed by 5 min re-equilibration in the initial condition before the next injection. Commercial standards of chlorophyll a, chlorophyll b, lutein and β-carotene were used for external calibration curves. Pigments were detected at 445 nm and quantified by injecting known amounts of pure standards (Sigma-Aldrich, Milan, Italy).