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Separation and Purification Technology 60 (2008) 128–135

Separation and purification of squalene from

soybean oil deodorizer distillate
Setiyo Gunawan, Novy S. Kasim, Yi-Hsu Ju ∗
Department of Chemical Engineering, National Taiwan University of Science and Technology,
43 Keelung Road, Section 4, Taipei 106-07, Taiwan
Received 9 March 2007; received in revised form 1 August 2007; accepted 3 August 2007

Abstract
Depending on conditions in the refining process, soybean oil deodorizer distillate (SODD) in Taiwan typically contains about 45% free fatty
acids (FFAs) and 20% triacylglycerols (TAGs). Bioactive compounds such as tocopherols, free phytosterols, fatty acid steryl esters (FASEs) and
squalene also make up a significant portion of SODD. In the present work, a modified soxhlet extraction and silica gel column chromatography
were employed to isolate and purify squalene from SODD. The goal of this work was to obtain a quantitative analysis of the separation processes
and to assess the feasibility of this method to isolate the constituent compounds. Here, a modified soxhlet extraction was employed for the efficient
separation of FASEs and squalene into one fraction, and tocopherols, free phytosterols, TAGs and FFAs into another fraction. Starting with SODD
that contains 3.91% FASEs, 1.83% squalene, 6.40% tocopherols and 5.36% free phytosterols, it was possible to obtain the first fraction enriched
with FASEs (12.19%, recovery 94.32%) and squalene (6.29%, recovery 100%). The contents of FFAs, TAGs, tocopherols and free phytosterols
remaining in the second fraction were 35.05%, 3.49%, 2.39% and 0.41%, respectively. The corresponding recoveries of FFAs, TAGs, tocopherols
and free phytosterols in this fraction were 20.41%, 5.89%, 9.83% and 2.09%, respectively. The first fraction was subsequently introduced into a silica
gel column chromatography to isolate squalene. Squalene (95.90% purity and 93.09% recovery) was obtained in the second fraction after eluting
the column using 10.96 L hexane at 23 ◦ C. Although modified soxhlet extraction requires large amount of organic solvents that are flammable and
environmentally unfriendly, the solvents can be recovered easily and it requires less sophisticated equipment than molecular distillation, operates
under atmospheric pressure and lower temperature.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Fatty acid steryl esters; Free phytosterols; Modified soxhlet extraction; Soybean oil deodorizer distillate; Squalene

1. Introduction primarily from liver oil of some deep-sea shark (cartilaginous

In the refining of soybean oil, most bioactive compounds such
as squalene, fatty acid steryl esters (FASEs), tocopherols and
free phytosterols are concentrated in intermediate byproducts
and waste streams [1,2]. SODD is obtained as a byproduct of
the deodorization step during the refining of soybean oil. In addi-
tion to the bioactive compounds mentioned above, SODD also
contains aldehyde, ketones, pesticides, herbicides, polycyclic
hydrocarbons and FFAs [3].
Squalene is a hydrocarbon having applications in the prepara-
tion of cosmetics as a natural moisturizer and in the biosynthesis
of cholesterol [4], originally obtained for commercial purposes
∗ Corresponding author. Tel.: +886 2 27376612; fax: +886 2 27376644.
E-mail address: yhju@mail.ntust.edu.tw (Y.-H. Ju).
fishes). Due to environment concerns including the protec-
tion of marine life, the extraction of squalene from vegetable
sources is of great interest. FASEs are an important raw
material in the production of hormones and vitamins, and
they are more effective at lowering serum cholesterol lev-
els than free phytosterol. The presence of these physiological
effects has led to the development of several functional foods,
such as salad oil and dressing with added sterol and mar-
garine blended with FASEs. In particular, because FASEs and
oil (TAGs) completely dissolve each other, a lot of atten-
tion is being focused on the addition of FASEs in oil-related
foods [5].
Depending on the sources, deodorizer distillates usually have
significantly different characteristics, uses and value. They can
be a good raw material for the production of tocopherols,
phytosterols and fatty acids [6]. FFAs constitute 25–75% of

1383-5866/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2007.08.001
 S. Gunawan et al. / Separation and Purification Technology 60 (2008) 128–135 129

vegetable oil deodorizer distillate depending on the type of
raw material and the conditions of the oil refining process
[7]. Due to its high content, the efficient removal of FFAs
is crucial for the concentration of bioactive compounds in
SODD.
Molecular distillation at 250 ◦ C and 0.02 mmHg was
employed by several investigators [5,8,9] for the isolation of two
main groups of compounds. The FASE compounds are contained
in the first fraction, which is rich in diacylglycerols (DAGs)
and TAGs. The tocopherols are isolated in the second fraction,
which is rich in FFAs, free phytosterols and monoacylglycerols
(MAGs). Martins et al. [7] used molecular distillation at 160 ◦ C
and 7.5 × 10−4 mmHg for the removal of FFAs from SODD.
Bondioli et al. [10] esterified FFAs into their corresponding glyc-
erides and then applied a supercritical carbon dioxide extraction
to produce a squalene-enriched fraction (purity 90.0%, yield
91.1%).
Molecular distillation requires the operation to be conducted
under a high temperature and a high vacuum with expensive
equipment. The process that involves saponification usually
requires the use of a strong mineral acid to convert soap into
FFAs. Mineral acids can be dangerous in handling and can
induce discoloration or degradation of the distillate components
[11].
Gunawan et al. [12] applied repeated modified soxhlet extrac-
tions to isolate wax esters and FASEs from crude rice bran oil.
In this study, a modified soxhlet extraction was used to separate
SODD into two fractions based on differences in the polarity of
the constituent compounds. One fraction is rich in FASEs and
squalene, while the other fraction is rich in tocopherols, free
phytosterols, FFAs and acylglycerols. The effects of parameters
on the separation, such as the SODD to silica gel mass ratio,
the number of extractions, and the temperature were systemati-
cally investigated. A silica gel column chromatography was then
employed to purify the squalene from the squalene-enriched
fraction.
2.2. Extraction of nonpolar lipids from SODD
Foreign material was removed from SODD by using a 7 ␮m
Advantec filter paper (Toyo Roshi Kaisha Ltd., Tokyo, Japan).
The silica gel was kept in a furnace at 150 ◦ C for 1 h to remove its
water content. A soxhlet extractor, equipped with a temperature-
insulatory jacket, and connected to a condenser system was
employed in this study. A schematic drawing of the modified
soxhlet extractor is shown in Fig. 1. The SODD (10–20 g) was
dissolved in hexane (150 mL) and the solution was put into a
500 mL round-bottom flask at room temperature. The silica gel
(60 g) was added to this solution. The mixture was magnetically
stirred at 300 rpm for 1 h. Afterwards, the SODD was coated
onto silica gel by removing the hexane at 60 ◦ C and 160 mmHg.
Then, the SODD-loaded silica gel was packed into an extraction
thimble (9.4 cm × 3.3 cm i.d.), and the top surface was covered
with cotton to prevent spillage. A thermo couple was inserted
into the thimble and the thimble was placed inside the soxh-
let extractor (245 mL). The less-polar lipids were extracted with
hexane (350 mL), which was put in a 500 mL round-bottom flask
and was heated. The refrigerated circulating bath was used with
a refrigerant (ethyl alcohol), which flowed through the jacket.
The hexane vapor travelled up the distillation arm, and flooded
into the chamber housing the thimble. The condenser ensured
that any hexane vapor condenses and drips back down into the
chamber housing the thimble. The chamber containing the thim-

2. Materials and methods

2.1. Materials

SODD was donated by TTET Union Corporation (Tainan,

Taiwan). Thin-layer chromatograph (TLC) aluminum plates
(20 cm × 20 cm × 250 ␮m) were purchased from Machery-
Nagel (Schweiz, Germany). Silica gel (70–230 mesh) was
obtained from Silicycle (Quebec, Canada). Characteristics of
the gel according to the manufacturer were: particle size:
60–200 ␮m; pore size, 60 Å; pH 7; water content, 6%;
and specific surface area, 500 m2 /g. Standard nonacosane,
farnesene, cholesta-3,5-diene, squalene, fatty acids, ␣-,␦-,␥-
tocopherol, monooleylglycerol, diolein, triolein, and tripalmitin
were obtained from Sigma Chemicals Company (St. Louis,
MO). Standard ␤-sitosterol (practical grade) was obtained from
MP Biomedicals, LLC (Aurora, OH). All solvents and reagents
were either of HPLC grade or analytical reagent grade and were
obtained from commercial sources. Fig. 1. A schematic drawing of the modified soxhlet extractor.
130 S. Gunawan et al. / Separation and Purification Technology 60 (2008) 128–135
ble slowly filled with the hexane at controlled temperature (−6
to 65 ◦ C). At a certain controlled temperature, some compounds
will dissolve in the hexane. When the chamber was almost full, it
was automatically emptied by a siphon side arm, which returns
the hexane to the distillation flask. During each cycle, a portion of
the non-volatile compound dissolves in the hexane. After many
cycles the desired compounds were concentrated in the distilla-
tion flask. The solution in the distillation flask was then filtrated
and the hexane was removed to yield the hexane extractive. Squa-
lene and FASE were preferentially extracted into this hexane
phase, which is designated as the nonpolar lipid fraction (NPLF).
Lipids adsorbed on the silica gel were then extracted with ethyl
acetate for 3 h at 71 ± 1 ◦ C, and the extract was designated as
the polar lipid fraction (PLF).
2.3. Purification of squalene from nonpolar lipids
A silica gel column chromatography (300 mm × 45 mm i.d.
glass tube) equipped with a valve to control the flow rate of
eluent was employed in this study. The silica gel (60 g) was
kept in a furnace at 150 ◦ C for 1 h to remove its water content.
Next, slurry of silica gel in hexane was poured into the column
previously half-filled with hexane. The hexane was allowed to
flow slightly during packing. The top surface of the station-
ary phase was covered by cotton, and a thermo couple was put
above the cotton. The hexane level was lowered until it was
0.5 cm above the cotton. The exit of the silica gel column chro-
matography was plugged with cotton to retain the solid. About
3 g of NPLF (from the modified soxhlet extraction) was dis-
solved in hexane (100 mL) and the mixture was put into the
Fig. 2. Flow chart showing the separation and purification of squalene from SODD.
column at room temperature (23 ◦ C). The column was eluted
with hexane at 4.06 ± 0.18 mL/min. The first fraction, which
contains mostly hydrocarbons, was obtained after eluted with
3.17 L of hexane. The squalene-rich fraction was obtained after
eluting with 10.96 L of hexane. Fig. 2 shows a flow chart for the
separation and purification of squalene from SODD.
2.4. Determination of FFAs content in SODD
The FFAs content of the SODD as oleic acid was determined
by the AOCS official method (Official AOCS 1997, Method, Ca
5a-40) [13].
2.5. Analysis by TLC and HT-GC
The individual components in each fraction were identified
by using authentic standards for TLC and HT-GC. The TLC
plates were developed in pure hexane for squalene and FASEs
analysis and developed in a mixture of solvent (hexane/ethyl
acetate/acetic acid = 95/5/1, v/v) for aldehydes and ketones anal-
ysis. After air-drying, spots on each plate were visualized by
exposing the chromatogram to iodine vapor. The FASEs spot
was detected by spraying with a fresh solution of 50 mg ferric
chloride in a mixture of 90 mL water, 5 mL acetic acid, and 5 mL
sulfuric acid. After heating at 100 ◦ C for 3–5 min, it was indi-
cated by a red-violet color [14]. Spots for aldehydes and ketones
were detected by spraying with a fresh solution of 400 mg
2,4-dinitrophenylhydrazine in a mixture of 100 mL methanol
and 15 mL sulfuric acid. After heating the plates at 100 ◦ C for
5–10 min, the spots were indicated by a wine-red color [14].
 S. Gunawan et al. / Separation and Purification Technology 60 (2008) 128–135 131

The contents of hydrocarbons, FASEs, free phytosterols, Table 1

tocopherols and acylglycerols in each fraction were deter- SODD composition
mined by HT-GC. External standard calibration curves were Compounds Composition (wt.%)
obtained by using 0.2–20 mg of pure standard. The chro- This worka Hirota et al. [4]
matographic analysis was performed on a TLC plate and a
Shimadzu GC-17A (Kyoto, Japan) gas chromatograph equipped FFAs 45.38 ± 2.13 30.1
TAGs 18.45 ± 2.20 9.5
with a flame ionization detector. Separations were carried DAGs 4.85 ± 1.21 3.5
out on a DB-5HT (5%-phenyl)-methylpolysiloxane nonpolar Squalene 1.83 ± 0.05 N/Ab
column (15 m × 0.32 mm i.d.; Agilent Tech. Palo Alto, Cal- FASEs 3.91 ± 0.39 12.8
ifornia). Temperatures of the injector and the detector were Tocopherols 6.40 ± 0.85 10.4
both set at 370 ◦ C. The temperature of the column was started Free phytosterols 5.36 ± 0.19 10.3
Othersc 15.23 ± 0.16 19.3
at 80 ◦ C, and was increased to 365 ◦ C at rate of 15 ◦ C/min
and maintained at 365 ◦ C for 8 min. The split ratio was a Average of three independent measurements.
b Data not available.
1:50 using nitrogen as carrier gas with a linear velocity of c Hydrocarbons, aldehydes, ketones, pesticides, herbicides and the breakdown
30 cm/s at 80 ◦ C. A 20 mg sample was dissolved in 1 mL ethyl products of tocopherols and free phytosterols.
acetate, and a 1 ␮L sample was taken and injected into the
HT-GC.
3. Results and discussion

2.6. Statistical analysis 3.1. Modified soxhlet extraction

The reliability of the results was checked by a statistical anal-
ysis. The standard deviation of the measures (S) was calculated
considering the difference between the value of individual exper-
iment, x, and the mean value of three independent experiments,
x̄, using the formula
 extraction or a repeated modified soxhlet extraction resulted in
Σ(x − x̄)2
S=
n−1
where n represents the total number of experiments. The statis-
tical significance of effects, such as the SODD to silica gel mass
ratio, the number of extractions, and the extraction tempera-
ture upon separation was systematically checked by the P-value
method.
The SODD used in this study was brownish and semi-solid at
room temperature. Table 1 shows the composition of two kinds
of SODD, one was used in this study and the other was used in
Hirota et al.’s work.
Storage of the SODD coated on silica gel in a low temperature
environment (below −15 ◦ C for 5 h) prior to modified soxhlet
poor recovery of the FASEs in the NPLF (data not shown). Also,
using a solvent more polar than hexane during the extraction of
the NPLF did not result in a successful separation (data not
shown). Therefore, a single-stage modified soxhlet extraction
with the SODD coated on silica gel at room temperature, and
single nonpolar solvent (hexane) were used in this study.
The GC chromatograms in Fig. 3 show that after the modified
soxhlet extraction, most of the FFAs along with the tocopherols

Fig. 3. Results of the HT-GC and the TLC analyses. Operation conditions for the modified soxhlet extraction: SODD to silica gel mass ratio = 1:3, and extraction
temperature = −6 ◦ C. (A) The GC chromatograms of SODD, NPLF and PLF. Insert: enlargement of the region of tocopherols and free phytosterols. (B) The analysis
of the NPLF and the PLF by TLC developed in pure hexane.
132 S. Gunawan et al. / Separation and Purification Technology 60 (2008) 128–135

and free phytosterols were concentrated in the PLF. The FFAs
region in the chromatogram of the NPLF shows interferences by
minor components, which are hydrocarbons such as aliphatic,
sesquiterpene and triterpene (squalene) hydrocarbons. Steroidal
hydrocarbons existed among the tocopherols and free phytos-
terols in the region as shown inset in Fig. 3. These steroidal
hydrocarbons may have arisen from the oxidation of tocopherols
and free phytosterols during refining, and the enrichment of
hydrocarbons during the modified soxhlet extraction. TLC and
HT-GC analyses of the PLF showed the absence of squalene.
Significant amount of aldehydes and ketones were present, both
in the NPLF and the PLF, as were confirmed by the TLC analysis
by using 2,4-dinitrophenylhydrazine as a specific reagent (data
not shown).
3.2. Effects of the SODD to silica gel mass ratio
A higher SODD to silica gel mass ratio is more desirable in the
modified soxhlet extraction because it requires less adsorbent,
and can yield a higher amount of the NPLF in a lower number
of extractions. However, a SODD to silica gel mass ratio greater
than 1:3 did not result in a successful separation. Therefore,
the mass ratios employed in this study were 1:3, 1:4, 1:5 and
1:6. Squalene, a hydrocarbon, was chosen as the indicator for
the effective separation of the polar and nonpolar lipids. The
relation between the number of extractions needed to obtain
a 100% recovery of the squalene in NPLF and the SODD to
silica gel mass ratio is shown in Table 2. It can be seen that
the number of extractions increases by decreasing the SODD
to silica gel mass ratio. This is because as the SODD to silica
gel mass ratio decreases, more squalene molecules are adsorbed
onto the surface of the silica gel. Hence, it takes higher cycles
to extract all squalene that had been adsorbed onto the silica
gel.
Table 2 also shows that a lower SODD to silica gel mass
ratio results in a lower recovery of FFAs, TAGs, tocopherols
and free phytosterols in NPLF. The amount of NPLF obtained
per unit of SODD decreases because of the decreasing SODD
to silica gel mass ratio due to more molecules adsorbed onto the
silica gel as discussed above. The P-value method was applied
to determine the significance of these differences. At a SODD
to silica gel mass ratio of 1:3, the squalene and FASEs contents
were significantly lower (p < 0.05) while the recovery of FASEs
were significantly higher (p < 0.05) than those obtained while
using a lower SODD to silica mass ratio (1:4, 1:5 and 1:6) as
can be seen in Table 2. If it is desirable to concentrate squalene
and FASEs into NPLF, then a higher SODD to silica gel mass
ratio is preferred due to the higher recovery of the FASEs in
NPLF.
As the SODD to silica gel mass ratio increases from 1:6 to
1:3, the recoveries of tocopherols, free phytosterols, FFAs and
TAGs increase from 28.75% to 48.80%, 17.61% to 20.12%,
11.71% to 41.47% and 2.31% to 43.42%, respectively, while the
recovery of FASE increases from 64.03% to 97.6%. Tocopherols
contents were not significantly different (p > 0.05) among all the
SODD to silica gel mass ratios studied. However, the content
and recovery of the FFAs and TAGs, along with the recovery
of tocopherols and free phytosterols was significantly higher
(p < 0.05) at a SODD to silica gel mass ratio of 1:3, compared to
those obtained while using a SODD to silica mass ratio of 1:4,
1:5 or 1:6.
Nonpolar compounds such as squalene and FASEs were
weakly adsorbed onto the silica gel and were also displaced
by hexane during the extraction. On the other hand, the more
polar compounds, such as FFAs, and TAGs, as well as the toco-
pherols and free phytosterols, were more strongly adsorbed onto
the silica gel. A lower SODD to silica gel mass ratio yielded a
higher adsorption area available per unit mass of SODD, which

Table 2
The effects of the SODD to silica gel mass ratio on the composition of NPLF
SODD to silica gel mass ratio Compounds

Squalene FASEs FFAs TAGs Tocopherols Free phytosterols Othersa

1:3 4.03 ± 0.13b 7.17 ± 0.26 38.72 ± 0.44 28.85 ± 0.49 7.48 ± 0.23 4.48 ± 0.14 9.27 ± 1.06
100.00c 97.51 ± 0.74 41.47 ± 1.46 43.42 ± 0.96 48.80 ± 0.10 20.12 ± 0.04 57.94 ± 8.51
1:4 4.81 ± 0.06 7.80 ± 0.09 30.60 ± 0.07 15.19 ± 0.59 7.78 ± 0.09 3.51 ± 0.09 30.31 ± 0.66
100.0 80.79 ± 0.21 28.68 ± 0.59 16.02 ± 0.22 46.74 ± 1.32 19.40 ± 0.03 68.00 ± 1.90
1:5 5.61 ± 0.23 8.50 ± 0.21 30.37 ± 0.59 1.85 ± 0.22 7.94 ± 1.32 3.12 ± 0.03 42.61 ± 1.90
100.00 71.02 ± 1.12 20.94 ± 0.34 3.28 ± 0.32 35.60 ± 5.65 18.57 ± 0.56 72.73 ± 3.57
1:6 7.26 ± 0.24 9.32 ± 0.24 18.85 ± 0.27 3.30 ± 0.22 7.25 ± 1.45 4.81 ± 0.07 49.21 ± 2.02
100.00 64.03 ± 3.82 11.71 ± 0.23 2.31 ± 0.08 28.75 ± 4.82 17.61 ± 0.32 73.22 ± 5.45

SODD to silica gel mass ratio

1:3 1:4 1:5 1:6

NPLF/SODD (%) 47.28 ± 3.68 33.87 ± 0.12 31.58 ± 0.34 22.45 ± 0.75
Number of extractions (cycles) 40.00 ± 1.00 40.00 ± 1.00 70.00 ± 1.00 80.00 ± 1.00

The extraction temperature = 65 ◦ Cd .

a Hydrocarbons, aldehydes, ketones, pesticides, herbicides and the breakdown products of tocopherols and free phytosterols.
b Content (wt.%).
c Recovery (%).
d Each value represents the mean of three independent experiments.
 S. Gunawan et al. / Separation and Purification Technology 60 (2008) 128–135 133

resulted in a higher number of extractions, a poorer recovery
of the FASEs in NPLF, and less NPLF obtained. Moreover,
this result agrees with the previous observation that the pres-
ence of a hydroxyl group on the silica gel imparts a degree of
polarity to the surface, so, polar molecules such as water, alco-
hols, phenols and amines (which can form hydrogen bonds) and
unsaturated hydrocarbons (which can form ␲-complexes) are
adsorbed preferentially over nonpolar molecules such as satu-
rated hydrocarbons [15]. The amount of FFAs adsorbed per unit
area of silica gel was found to increase in proportional to the
surface area of the silica gel [16], which is again consistent with
the results shown in Table 2. In summary, the results show that
a lower SODD to silica gel mass ratio leads to a lower content
and recovery of FFAs in NPLF.
3.3. Effect of extraction temperature
A lower extraction temperature is favorable because it mini-
mizes the degradation of the bioactive compounds in the SODD.
Table 3 shows the relation between the extraction temperature
and the number of extractions required to achieve 100% recovery
of the squalene in NPLF. The lowest temperature investigated
in this study was −6 ◦ C due to the capacity of the cooler used in
this study. The highest temperature used was 65 ◦ C, which was
limited by the boiling point of hexane (68 ◦ C). Our study shows
that the number of extraction required to achieve 100% recovery
of the squalene in NPLF increases from 40 to 88 cycles as the
extraction temperature decreases from 65 ◦ C to −6 ◦ C.
It can be seen from Table 3 that the amount of the NPLF
obtained, in term of a percentage of the SODD, increases with
the extraction temperature. While the contents of squalene and
FASEs were significantly different (p < 0.05) at −6 ◦ C from
those at other temperatures, the recovery of FASEs was not
significantly different (p > 0.05). The important criteria for a suc-
cessful separation are to recover most of the FASEs in NPLF. As
seen in Table 3, within the temperature range studied, the lower
the extraction temperature, the recovery of the FASEs varies
according to the extraction temperature. An extraction temper-
ature of −6 ◦ C was chosen in this study even though at this
temperature the squalene content in NPLF obtained was lower
than that at 3 ◦ C. Both the contents and the recoveries of toco-
pherols, free phytosterols, FFAs and TAGs were significantly
lower (p < 0.05) at −6 ◦ C than those obtained at 7, 55 and 65 ◦ C.
Therefore, −6 ◦ C seems to be a better choice, since we prefer
most of the tocopherols, free phytosterols, FFAs and TAGs to
exist in PLF. While it is true that modified soxhlet extraction
resulted in a lower number of extractions, and a higher NPLF to
SODD mass ratio while operated under higher extraction tem-
perature, it is less efficient at removing FFAs, TAGs, tocopherols
and free phytosterols from NPLF.
Since adsorption is usually an exothermic process, an
increase in extraction temperature will result in a decrease of
the polar components adsorbed onto the silica gel. A decrease
in extraction temperature yields a better separation of polar and
nonpolar components, as can be seen in Table 3. These results
agree with previous observations that the ultimate capacity of
silica gel is generally higher at low temperature [15]. In another
study, Chu et al. [17] found that a lower temperature led to
higher vitamin E uptake at equilibrium, indicating that vitamin
E adsorption by silica is an exothermic process. The binding
strength between silica and acid is higher than that between sil-
ica and ester, which is still higher that that between silica and
alcohol [16]. Also, Moreira et al. [18] has shown that separa-
tion of free phytosterols and tocopherols is usually performed
through a fractional crystallization because free phytosterols
tend to precipitate at a low temperature.

Table 3
The effects of the extraction temperature on the composition of NPLF
Extraction temperature (◦ C) Compounds

Squalene FASEs FFAs TAGs Tocopherols Free phytosterols Othersa

65 4.03 ± 0.13b 7.17 ± 0.26 38.72 ± 0.44 28.85 ± 0.49 7.48 ± 0.23 4.48 ± 0.14 9.27 ± 1.06
100.00c 97.51 ± 0.74 41.47 ± 1.46 43.42 ± 0.96 48.80 ± 0.10 20.12 ± 0.04 57.94 ± 8.51
55 5.16 ± 0.19 8.95 ± 0.39 43.32 ± 3.37 20.03 ± 1.43 5.82 ± 0.31 2.27 ± 0.15 8.98 ± 1.11
100.0 95.11 ± 1.82 36.28 ± 3.93 23.54 ± 0.93 29.71 ± 2.59 7.96 ± 0.79 70.37 ± 9.55
7 5.77 ± 0.33 9.29 ± 0.91 44.64 ± 4.61 18.09 ± 1.84 6.08 ± 1.89 0.78 ± 0.31 15.37 ± 4.24
100.00 94.99 ± 0.69 34.08 ± 2.68 26.92 ± 3.46 24.99 ± 5.80 3.12 ± 0.51 66.46 ± 9.67
3 6.83 ± 0.24 9.46 ± 0.96 46.57 ± 9.22 0.55 ± 0.52 5.69 ± 1.99 0.49 ± 0.59 30.40 ± 9.94
100.00 91.47 ± 0.65 22.62 ± 8.51 1.18 ± 1.25 18.30 ± 1.43 2.06 ± 2.59 78.07 ± 9.85
−6 6.29 ± 0.05 12.19 ± 0.30 35.05 ± 2.77 3.49 ± 1.80 2.39 ± 0.51 0.41 ± 0.14 40.19 ± 0.06
100.00 94.32 ± 3.01 20.41 ± 1.46 5.89 ± 3.08 9.83 ± 2.18 2.09 ± 0.65 75.91 ± 1.78

Extraction temperature (◦ C)
65 55 7 3 −6

NPLF/SODD (%) 47.28 ± 3.68 34.76 ± 0.93 35.00 ± 2.23 26.32 ± 4.76 28.84 ± 0.21
Number of extractions (cycles) 40.00 ± 1.00 40.00 ± 1.00 66.64 ± 1.00 72.00 ± 1.00 88.00 ± 1.00

SODD to silica gel mass ratio = 1:3 (w/w)d .

a Hydrocarbons, aldehydes, ketones, pesticides, herbicides and the breakdown product of tocopherols and free phytosterols.
b Content (wt.%).
c Recovery (%).
d Each value represents the mean of three different experiments.
134 S. Gunawan et al. / Separation and Purification Technology 60 (2008) 128–135

Table 4
A comparison of the fractionation of SODD obtained by modified soxhlet extraction and molecular distillation
Compounds Separation method

Modified soxhlet extractiona Molecular distillationb

Nonpolar lipid fraction Polar lipid fraction Low bp fraction High bp fraction

Squalene 6.29c 100.00d NDe 0.00 N/Af N/A

FASEs 12.19 94.32 0.30 5.80 ND 0.00 45.4 100.73
FFAs 35.05 20.41 55.42 79.62 41.40 95.75 1.4 1.32
TAGs 3.49 5.89 22.71 94.55 ND 0.00 32.1 95.96
Tocopherols 2.39 9.83 8.88 90.17 14.80 99.05 0.70 1.90
Free phytosterols 0.41 2.09 7.67 98.09 12.80 86.41 4.00 11.03
Othersg 40.19 75.91 5.03 23.42 25.40 91.60 4.80 7.06
Fraction/SODD (%) 28.84 71.16 69.60 28.40

The composition of SODD is given in Table 1.

a Each value represents the mean of three independent experiments. Operating conditions: SODD to silica gel mass ratio = 1:3; T = −6 ◦ C; and number of

extractions = 88 cycles.
b Hirota et al. [4]. Operating conditions: T = 250 ◦ C at 0.02 mmHg.
c Content (wt.%).
d Recovery (%).
e Not detected.
f Data not included in study.
g Hydrocarbons, aldehydes, ketones, pesticides, herbicides and the breakdown products of tocopherols and free phytosterols.

Our results suggest that under the following operation con-
ditions: SODD to silica gel mass ratio = 1:3 (w/w); extraction
temperature = −6 ◦ C; number of extractions = 88 cycles, the
modified soxhlet extraction operated on SODD could yield a
3.4- and 3.3-fold increase in squalene and FASEs content in
NPLF and a 1.3-fold increase in tocopherols content and 1.4-fold
increase in free phytosterols content in the PLF. These results are
comparable with those obtained from the molecular distillation,
as shown in Table 4. A 3.5-fold increase in FASEs content could
be concentrated in a high boiling point fraction, and a 1.4-fold
tocopherols increase and 1.2-fold free phytosterols increase in
contents, could be concentrated in a low boiling point fraction.
less sophisticated equipment. By combining a modified soxhlet
extraction with a silica gel column chromatography, we were
successful in obtaining squalene with a high purity (95.90%)
and a high recovery (93.09%) from SODD with an initial squa-
lene content of 1.83%. Although modified soxhlet extraction
and silica gel column chromatography require large amounts of
organic solvents, the solvents can be recovered easily. Currently,
a modified version of the silica gel column chromatography is
being developed in our laboratory. Preliminary results show that
the set-up could drastically reduce both the amount of solvent
and the time required to obtain squalene during elution without
compromising both purity and recovery.

3.4. Silica gel column chromatography Acknowledgement

The first fraction obtained from the silica gel column
chromatography of NPLF is mainly comprised of aliphatic,
sesquiterpene and steroidal hydrocarbons. Squalene (95.90%
purity and 93.09% recovery) was obtained in the second fraction
after eluting the column with 10.96 L of hexane. The continued
elution of the column with 33.13 L of hexane yielded a third
fraction rich in FASE (84.04% purity and 63.17% recovery).
The washing fraction, which recovered most of the components
that were adsorbed onto the silica gel, was collected by eluting
the column with ethyl acetate.
4. Conclusions
A modified soxhlet extraction was applied on SODD to obtain
one fraction enriched with FASEs (12.19%, recovery 94.32%)
and squalene (6.29%, recovery 100%), while tocopherols, free
phytosterols, TAGs and FFAs were enriched in another fraction.
The advantages of the modified soxhlet extraction employed
in this study over molecular distillation are that it operates
under atmospheric pressure and lower temperature, and requires
This work was supported by a grant (NSC94-2214-E011-
004) provided by the National Science Council of Taiwan.
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