Accela series hplc system

For white, 4h and 4h4d samples, cell pellets (80–170 mg wet weight) were transferred to extraction tubes containing 1 mm glass beads and extracted into 100% methanol using an ultrasonic probe (35 s; 50 W), and 1 mm glass beads. Pellets were repeat extracted with 1 ml methanol five times. Extracts were clarified and analyzed according to the method of Zapata et al. (2000) (link) by injecting onto a Waters Symmetry C8 reverse phase column (150 × 4.6 mm, 3.5 μm particle size) on a Thermo Accela Series HPLC system with chilled autosampler (4°C) and photodiode array detector. The HPLC was calibrated using a suite of standards purchased from DHI (Denmark). The standards were used to generate response factors used for quantification. For pigments where standards were not available (caloxanthin and nostoxanthin) the response factors of the most closely related standard, zeaxanthin, was used. Pigments were identified based on retention time, spectral match using photo-diode array and LC/MS (Supplementary Table S1). Probability (p) of significance between white light control and the 4h4d samples was determined using a Student’s t-test test with a two tailed distribution.