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.