Zorbax eclipse xdb c8 reverse phase column

Freeze-dried pellets were weighed and re-suspended at 2–3 mg dry biomass per mL of chilled ethanol before being sonicated with 1-s pulses using an ultrasonic homogeniser (Qsonica Q125) at 100% amplitude and filtered once extraction was confirmed. Extracts were filtered using 0.2 µm PTFE syringe filters and stored in − 80 °C until analysis. High Performance Liquid Chromatography (HPLC) was conducted using an Agilent Technologies 1290 Infinity, equipped with a binary pump with an integrated vacuum degasser, thermostatted column compartment modules, Infinity 1290 auto-sampler and PDA detector. Column separation was performed using a 4.6 mm × 150 mm Zorbax Eclipse XDB-C8 reverse-phase column (Agilent Technologies, Inc.) and guard column using a gradient of TBAA (tetrabutyl ammonium acetate): Methanol mix (30:70) (solvent A) and Methanol (Solvent B) as follows: 0–22 min, from 5 to 95% B; 22–29 min, 95% B; 29–31 min, 5% B; 31–40 min, column equilibration with 5% B. Column temperature was maintained at 55 °C. A complete pigment profile from 270 to 700 nm was recorded using PDA detector with 3.4 nm bandwidth.

Freeze-dried pellet samples were weighed and re-suspended in a solvent ratio of between 2–3 mg of sample (DW) per mL of ethanol and sonicated with an ultrasonic homogenizer (Qsonica Q125) at 100% amplitude for 3 × 3-s pulses before being stored at −20 °C overnight once blanching of pellets was confirmed using a centrifuge. They were then filtered using 0.2 µm PTFE 13 mm syringe filters and stored in −80 °C until analysis. High Performance Liquid Chromatography (HPLC) was conducted using an Agilent Technologies 1290 Infinity, equipped with a binary pump with integrated vacuum degasser, thermostatted column compartment modules, Infinity 1290 auto-sampler, and PDA detector. Column separation was performed using a 4.6 mm × 150 mm Zorbax Eclipse XDB-C8 reverse-phase column (Agilent Technologies, Santa Clara, CA, USA) and guard column using a gradient of TBAA (tert-Butyl acetoacetate): Methanol mix (30:70) (solvent A) and Methanol (Solvent B) as follows: 0–22 min, from 5 to 95% B; 22–29 min, 95% B; 29–31 min, 5% B; and 31–40 min, column equilibration with 5% B. Column temperature was maintained at 55 °C. A complete pigment profile from 270 to 700 nm was recorded using PDA detector with 3.4 nm bandwidth. Example chromatograms can be found in supplementary files.