In the case of straight hexane extraction from microalgal paste,
the filtrate was transferred into a separating funnel to partition the
hexane from water. For hexane/isopropanol extraction from dried
microalgae, the filtrate was transferred into a separating funnel
and sufficient hexane and water (approximately 40 mL each) were
added to induce biphasic layering. After settling, the solvent mixture
partitioned into two distinct phases: a top dark-green hexane
layer containing most of the extracted lipids and a bottom lightgreen
aqueous-isopropanolic layer containing most of the co-extracted
non-lipid contaminants. The hexane phase from each
extraction was collected in a pre-weighed flask before it was
heated to dryness in the oven (60 C) to enable gravimetric quantification
of the lipid extract. The crude lipid was re-dissolved in
hexane (approximately 20 mL) and transferred into a sealed glass
vial for storage.