Another method implied for the separation of minor components is solvent extraction, after which the components of interest were further purified by distillation or chromatography (Gunawan et al, 2008). The pretreatment of the feedstock mainly consisted of hydrolysis and neutralization for the concentration of the target minor compound (Chu et al, 2002; Leng et al., 2008). However, esterification was also used to facilitate the separation in polar and non-polar components (Brown and Smith, 1964).

Gunawan et al. (2008) reported that the separation results of a modified Soxhlet extraction are comparable to those obtained from molecular distillation. The purpose of their work was to isolate and purify natural FASEs from SODD by a suitable method without degradation of the FASEs.

A modified Soxhlet extraction with hexane was first employed to separate SODD into two fractions based on differences in the polarities of the constituent compounds (Fig. 22.7). The resulting nonpolar lipid fraction (NPLF) was rich in hydrocarbons and FASEs, whereas the polar lipid fraction was rich in FFAs and acylglycerols. The NPLF was then fractionated by a modified silica gel column chromatography to yield FASE-rich fraction. FASEs with high purity were finally obtained by solvent extraction.

By combining a modified Soxhlet extraction, a modified silica gel column chromatography and water/acetone extractions, the FASE fraction with high purity (87%) and high total recovery (85%) could be obtained from SODD with an initial FASE content of 4%. According to the results, this separation process can yield the FASE fraction from SODD without degradation of the FASEs. The advantage of the process is that, starting with SODD, high-purity squalene and FASEs can be obtained. In addition, the polar fraction (PLF in Fig. 22.7) contains

most of the tocopherols and free physterols and can be further processed to obtain pure tocopherols and free phytosterols.

Leng et al. (2008) described a process to recover squalene from PFAD using commercial immobilized lipase. The PFAD was hydrolyzed and neutralized, and then squalene was concentrated after a second neutralization and extracted with hexane. In this study, an RSM (response surface methodology) was used to evaluate the effects of several variables (reaction time, water content and lipase concentration) on the enzymatic hydrolysis.

Chu et al. (2002) separated tocopherols from PFAD by extraction with hexane after pre-concentration using an enzymatic hydrolysis-neutralization method. Acylglycerols in PFAD were hydrolyzed using a commercial immobilized thermal- stable lipase to liberate FFA and was subsequently treated with alkali. Removal of the FFA salts resulted in concentration of tocopherols. Factors affecting the degree of hydrolysis were studied to reach a better understanding of the recovery of tocopherols from PFAD. It was observed that the FFA levels in PFAD remained unchanged, but the tocopherols concentration decreased when the reaction was prolonged to 7 h. This was explained by the possibility that tocopherols might have been oxidized due to the long period of heating at 65°C. Increase of water content in the reaction mixture from 20% to 50% increased both the FFA levels and tocopherols concentration significantly (p < 0.05). However, a further increase of water content in the mixture significantly (p < 0.05) decreased the FFA levels and the tocopherols concentration.

Winton and Smith (1964) describes a process for the separation of sterols and tocopherols that involves the treatment of DD with a strong acid to convert FFA into esters, followed by the liquid-liquid extraction with a polar and the nonpolar solvent. The obtained polar liquid solution contains mainly sterols and tocopherols and nonpolar liquid solution is rich in esters and TAG. However, under the concept of the invention, there must be a sufficient immiscibility not only of the solvents but also of the solutions formed after admixture of the solvents with the sludge to result in two liquid phases. The process comprised an additional step of separating the polar liquid solution or extract fraction into a sterols product and a tocopherol concentrate. This was obtained by concentrating the solution to the point of incipient precipitation of sterols or complete removal of the solvent by vacuum distillation, followed by crystallization and filtration.