Dgu 20a5r degassing unit

Semipreparative HPLC was carried out on a Shimadzu system consisting of DGU-20A5R degassing unit, LC-20AT solvent delivery pump, SIL-10AF autosampler, CBM-20A controller, CTO-20AC column oven, SPD-M20A diode array detector and FRC-10A fraction collector (all Shimadzu, Kyoto, Kinki, Japan). Stationary phase was a Luna C-10(2) column with 250 × 10 mm and 10 µm particle size (Phenomenex). Mobile phase consisted of water (A) and acetonitrile (B). Elution at 4 mL/min and 35 °C column temperature started at 40% B, rising to 100% B at 20.0 min. All compounds of interest eluted within this gradient, followed by varying plateaus of 100% B (0–5 min duration) for column cleaning and re-equilibration at 40% B. Injection volume was 200 µL.

Six coral nubbins were sampled in each experimental condition, after 1, 3, 7, 11 and 21 days. Samples were placed for 15 min under 400 µmol photons m⁻² s⁻¹ before being flash frozen in liquid nitrogen. They were then lyophilized, and pigments were extracted in 1.5 mL of MeOH (HPLC Grade, Merck, Darmstadt, Germany). Samples were then vortexed for 3 × 15 s with 0.5 mL glass beads (710–1180 µm; Sigma Aldrich, St. Louis, MO, USA) before being centrifuged at 16,000 × g for 20 min at 4 °C. Pigments were then separated by reverse-phase HPLC, using Shimadzu Prominence HPLC system, comprising a DGU-20A5R Degassing Unit, a LC-20AT Liquid chromatograph, a SIL-20AC Autosampler, a CTO-10ASVP Column Oven and a SPD-M20A Diode Array Detector (Shimadzu, Kyoto, Japan). The HPLC column (Nova Pak C18, 60A column, 150 mm length and 4 µm pore size) was eluted with a mobile phase gradient (1 mL min⁻¹) set to the program described in [49 (link)]. Absorbance chromatograms were detected at 430 nm and quantified with pigments standards purchased from DHI Lab (Horstholm, Denmark). Acquisition and data treatment were performed using the Waters Empower software (Waters, Milford, MA, USA).

IR spectra were recorded as ATR-FTIR spectra using a Perkin-Elmer UART TWO FT-IR-spectrometer. The UV/VIS spectra were recorded in high-purity solvents (UVASOl^®) using the JASCO double-beam photometer (V-630). NMR spectra were recorded in MeOD using a Bruker NEO-500 instrument operating at 500 and 125 MHz for ¹H and ¹³C{¹H} NMR, respectively. Spectra referencing was accomplished using the CD₂HOD solvent peak for ¹H and the CD₃OD solvent peak for ¹³C NMR spectra (δ = 3.31 and 49.0 for ¹H and ¹³C{¹H} signals, respectively). Multiplicities in ¹H NMR spectra were described using the common descriptors s (singlet), d (doublet), t (triplet), or m (multiplet). High-resolution mass spectra were obtained by EI-TOF (70 eV) using Waters Micromass instruments. Reversed-phase semi-preparative HPLC was performed on Shimadzu LC-20AP pump equipped with DGU-20A5R degassing unit, a Shimadzu SPD-M20A detector, a Shimadzu SIL-20ACHT auto-sampler and a Phenomenex Gemini C₁₈ column (10 × 250 mm, 10 μm). Data were recorded and analyzed using LabSolutions software. For column chromatography, Silica gel 60 (0.063–0.2 mm, Macherey-Nagel) was used as solid matrix. TLC was carried out on precoated Silica gel 60 plates (0.20 mm). Compounds were visualized under UV light and further by spraying with H₂SO₄–EtOH (1:9, v/v). All solvents used were of analytical grade.