Luna 5 μm silica 2 column

The dried algal material (233.4 g) was ground and sequentially macerated at room temperature in dichloromethane (DCM) and ethyl acetate (EtOAc) for extraction. Each solvent was renewed three times. The organic solvents were combined, filtered, and evaporated to obtain the crude extract (1.94 g). The extract was fractionated by gel filtration chromatography (Sephadex LH-20 column, n-hexane/DCM/methanol (7:2:1)) to afford fractions F1–F6. Size-exclusion chromatography in Sephadex LH-20 (n-hexane/DCM/methanol (3:1:1)) of the active fraction F6 (689 mg), followed by medium-pressure chromatography of fraction F6.4 (257.4 mg) with a Lobar LiChroprep Si 60 (40–63 μm) column (n-hexane/EtOAc (1:1)) and silica gel column (step gradient from CHCl₃/EtOAc (4:1) to 100% EtOAc) of fraction F6.4.3 (110 mg), allowed the isolation of cystomexicone B (1, 1.39 mg). The normal-phase HPLC of fraction F6.4.3-5 (51.86 mg) (Phenomenex, Luna 5 μm Silica (2) column, 100 Å, 250 × 10 mm; isocratic n-hexane/EtOAc (3:2), 10 min at 1 mL/min; gradient up to 100% EtOAc, 50 min at 2 mL/min; 100% EtOAc, 5 min, 2 mL/min) afforded the pure compounds gongolarone B (2, 1.75 mg, 34.1 min), 6Z-1′-methoxyamentadione (3, 3.10 mg, 37.1 min) and 1′-methoxyamentadione (4, 6.22 mg, 42.1 min). The physical properties and NMR data of compounds 1–4 were confirmed based on those previously reported [16 (link),34 (link)].

The analysis of tocopherol was conducted per the AOCS procedure Ce 8-89 [53 ]. Each oil extract (20 mg) was dissolved in 1.0 mL of hexane. The measurement was conducted with a Waters (Milford, MA, USA) model 2695 HPLC pumping system, a model 2475 fluorescence detector, and a Phenomenex Luna 5 μm Silica(2) column (150 mm × 4.6 mm). The mobile phase was 99% hexane and 1% isopropanol, with a pumping rate of 1.0 mL min⁻¹. The injection volume was 20 µL. Tocopherols were detected at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Concentrations were determined by relating sample peak areas to standard curves and converted to mg kg⁻¹ of butter sample.