Carotenoids separation.
About 20 ml distilled petroleum ether was placed in a 250 ml separating funnel wrapped with black cloth.
The coloured acetone extract was carefully added to the petroleum ether through a funnel,
gently flowing down the wall of separating funnel.
The vacuum flask was rinsed with 1–2 ml acetone.
Then, 150 ml distilled water was added to the separating funnel (flowing down the funnel wall), the mixture was left
undisturbed for 5–10 min to allow for separation into organic and aqueous layers.
The aqueous layer, containing acetone, was discarded.
This step was repeated for 3–4 times with 100 ml
distilled
water
added
each
time until the residual acetone was removed. The petroleum ether extract in the separating funnel was collected,
through a funnel containing small amount of anhydrous sodium sulfate on a filter paper, into a 25 ml volumetric flask
covered with aluminum foil.
The separating funnel was rinsed with about 2 ml petroleum ether using a pipette. The eluent volume was
made up to 25 ml with petroleum ether.
The flask was capped and gently mixed.
For determination of the carotenoids, absorbance of the orange-coloured eluent was measured at 450 nm in a spectrophotometer (UV-1601, UV-Visible, Shimadzu, Tokyo, Japan).
All the preparative and extraction procedures were performed in dim light and/or excluded light as described.