2.6 Quantification of fucoxanthin in P. tricornutum
Fucoxanthin was quantified using high-performance liquid chromatography
(HPLC), and the extraction was performed as reported in Paliwal et al[48]. Briefly, 0.5
mg cells were centrifuged and resuspended in 1 ml of 100% methanol. For
pigment extraction, the cell suspension briefly vortexed with glass
beads for 20 minutes. The supernatant was collected and analyzed by
HPLC-UV (Agilent Infinity series 1,260 HPLC, Agilent Technologies, Santa
Clara, CA, United States). Running condition were as follows, C30 column
(4.6 x 250 mm, 5 mm) was used to run the samples, at 35 °C with the
binary solvent system as the mobile phase, which included methanol as
primary solvent A and methyl tert-butyl ether (MTBE) as solvent B. The
run conditions were as follows: 2–20 % B for the first 10 minutes,
then 20 % B (10–12 minutes), 20–80 % B (12–30 minutes), 80 % B
(30–32 minutes), and 80–2 % B (32–35 minutes)[49]. Pigments were
measured at 437 nm and identified by comparing the retention duration of
DHI standards from Hrsholm, Denmark.