Spherisorb s5 ods2

Each 0.5 mL sample (2.5 × 10⁶ cells mL⁻¹) was harvested by centrifugation (14,000× g, 2 min), and the supernatant was discarded. Xanthophyll pigments were extracted from the pellet using 0.5 mL of 90% (w/w) acetone for 1 min. After centrifugation at 14,000× g for 5 min (at 4 °C to prevent pigment degradation), the supernatant was filtered through a 0.2-μm nylon filter and was analyzed by using a Shimadzu Prominence high-performance liquid chromatography (HPLC) system (model LC-20AD) equipped with a Waters Spherisorb S5 ODS2 cartridge column (4.6 × 250 mm). Xanthophyll pigments were identified by retention time and absorption spectra with reference to pigment standards DHI C14 (DHI, Centralen, Denmark). Details of HPLC analysis have been described in Park et al. [56 (link)].

The organic acid content was determined according to the method reported by Flores et al. [46 (link)] with slight modifications. Extracts were diluted to 30% w/v with deionised water, filtered through a 0.45 nm filter and submitted to high-performance liquid chromatography (HPLC) analysis. Citric, malic, glutamic, succinic, oxalic and ascorbic acids were identified by comparing their retention times to those of the standards (Sigma, St. Louis, MO, USA). The results were expressed as mg/100 g of fresh sample. The HPLC analysis was performed on a HPLC Agilent 1100 Series system equipped with a diode array detector using a 20 μl sample injection loop. UV detection was performed at 210 nm. A reversed-phase column, Spherisorb S5 ODS2 (5 μm, 250 × 4.6 mm; Waters Corporation, Milford, MA, USA), was used, and the elution was carried out under isocratic conditions using water acidified with orthophosphoric acid (pH 2.1) as the mobile phase at a flow rate of 0.6 mL/min for 45 min.

Dichloromethane extract (100 mg) or incomptines A (IA, 2 mg) and B (IB, 2 mg) were dissolved in methanol (10 mL or 2 mL, respectively) and 20 µL of the sample was injected to HPLC. HPLC separations were performed on a Waters 2795 Alliance equipped with a Waters 996 detector photodiode array collecting data at 240 nm; a column Waters Spherisorb S5 ODS2 (4.5 × 250 mm, 5 µm) was used with a logarithmic gradient from 96% of aqueous acetic acid 2% and 4% of CH₃CN to 50% of aqueous acetic acid 2% and 96% of CH₃CN over a period of 60 min at a flow rate of 1 mL min⁻¹. All the solvents were HPLC grade.