2.4 Pre-concentration of Vitamin E
Products from enzymatic hydrolysis contained vitamin E (tocols), and
extraneous substances such as FFA and acylglycerols. Neutralization of
the product stream was carried out to separate and concentrate vitamin E
in the product.
Neutralization of PFAD was carried out using a modified version of the
method of Wang et al., (1998). Water in the product after reaction time
was separated using a separating funnel based on difference in mixture
densities. Lipid phase (30ml) was collected and then dissolved in 50 mL
of ethanol and then neutralized with 0.5 N sodium hydroxide to the
phenolphthalein end-point. Distilled water (50 mL) and 150mL hexane were
then added and the sample was shaken for 1 min. The sample was
subsequently allowed to stand for phase separation using a separation
funnel as well. Hexane layer was separated in a separator funnel. The
aqueous phase was re-extracted with an additional 100ml of hexane at
least four times. Hexane extracts were then combined, washed at least
three times with 150ml portions of distilled water to remove any
residual sodium hydroxide and soap and centrifuged at 3000\(\times\)g
for 2min to separate residual water prior to the transfer sample into a
500 ml round-bottom flask, and further heated at 50 under vacuum to
evaporate any water retained in the sample. The term
hydrolysed-neutralized PFAD (HNPFAD) is used to described the lipid
sample obtained from this process. The HPLC equipment was used to
determine the concentration of tocopherols (vitamin E). Vitamin E
concentration expressed in this study was the total concentration of all
tocopherol isomers in percentage.