HPLC-DAD Analysis of Carotenoids

phytoene and other carotenoids were analysed in cell extracts using a High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD), as described in previous work [12 (link)]. Commercial standards of individual carotenoids, including all-trans β-carotene, all-trans α-carotene, lutein, zeaxanthin and phytoene were obtained from Sigma-Aldrich, UK. Standards were dissolved in methanol or acetone to make a solution of 1 mg/mL and dissolved to a series of working solutions (5, 10, 25 and 50 µg/mL) to generate standard curves. To detect various pigments in the extracts, the DAD scanned wavelengths of 280 nm (colourless phytoene), 355 nm (colourless phytofluene), 450 nm (all coloured carotenoids including β-carotene, α-carotene, lutein and zeaxanthin) and 663 nm (chlorophylls). Total chlorophylls and coloured carotenoids were evaluated by extracting pigments from the harvested algal biomass of a 1 mL culture using 1 mL of 80% (v/v) acetone and absorbance was measured at 664, 647 and 480 nm, according to previous work [12 (link)].