Ultrasonics Sonochemistry 15 (2008) 938–942

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultsonch

Quantitative determination of oil content in small quantity of oilseed rape

by ultrasound-assisted extraction combined with gas chromatography
Fang Wei a,b,*, Gui-Zhen Gao a, Xin-Fa Wang a, Xu-Yan Dong a, Ping-Ping Li a,
Wei Hua a, Xu Wang c, Xiao-Ming Wu a, Hong Chen a,b
a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062, PR China
b
Key Laboratory for Genetic Improvement of Oil Crops, Ministry of Agriculture, PR China
c
Department of Chemistry, Fudan University, Shanghai 200433, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Accurately quantitative determination of oil content in oilseed rape plays an important role in varieties
Received 6 December 2007 breeding for improving oil content in seeds. However, large quantity of oilseeds were needed in order to
Received in revised form 14 April 2008 obtain accuracy and precision results by using standard Soxhlet extraction method, which may be a
Accepted 15 April 2008
handicap in analysis of small, rare and precious samples in plant breeding. In the present work, ultra-
Available online 22 April 2008
sound-assisted extraction was evaluated as a simpler and more effective alternative to conventional
extraction method for the isolation of oil from small quantity of oilseed rape (<20 mg). The oil of oilseed
Keywords:
rape samples was extracted by ultrasound-assisted method, and then the fatty acids and total oil content
Ultrasound-assisted extraction
Oilseed rape
of the seeds were qualitatively and quantitatively determined by gas chromatography (GC). Extraction
Quantitative determination efficiency of total oil obtained by ultrasound-assisted extraction through an orthogonal experiment (L9
Fatty acids and total oil (34)) were investigated to get the best extraction conditions. Statistical analysis showed that the variable
Orthogonal experiment with the largest effect was the ultrasound-assisted extraction time which was followed by the ultra-
sound-assisted extraction power, and the liquid:solid ratio. A liquid:solid ratio of 1:4 (L:g), an ultra-
sound-assisted extraction time of 60 min and an ultrasound-assisted extraction power of 500 W were
found to be optimal for oil extraction from oilseed rape. By comparing with the conventional method,
it was found that the ultrasound-assisted extraction of oil from oilseed rape was about five times faster
than the traditional extraction method. By the use of ultrasound-assisted extraction combined with GC
analysis, the fatty acids and total oil content in small quantity of seeds (<20 mg) were successfully qual-
itatively determined and the results are in agreement with that obtained by traditional standard method.
Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction Nowadays much attention has been paid to the study of fatty
acids and total oil content of oilseed rape in plant breeding pro-
Oilseed rape (Brassica napus L. and Brassica rapus L.) has been grams for quality improvement breeding.
grown worldwide as one of the most important oil crops for both Soxhlet extraction and refluxing extraction [5,6] are usually
edible and industrial uses [1–3]. Oilseed rape oils nowadays are a performed to extract oils from oilseed rape. Although these extrac-
great source of maintaining oil consumption in families and the tion techniques usually offer satisfied results, they also show some
utilization of the oil as bio-fuel has also been commercialized [4], handicaps, such as time-consuming, high costs and high labor in-
as a substitute for traditional fossil diesel fuel, to reduce green- put. In addition, large numbers of oilseed samples were needed
house gas emissions. For the edible rapeseed oil, to increase oleic by the use of these methods in order to obtain high level of accu-
acid, linoleic acid contents and to decrease saturated fatty acid, racy and precision results, which may be a handicap in the analysis
linolenic acid contents in the oil is a major goal for the coming of small, rare and precious samples in plant breeding. Therefore, it
years in order to make it as the healthiest edible oil. For the indus- is necessary to exploit a better method for the extraction and anal-
trial purpose, to breed high erucic acid rapeseed and high mid- ysis of fatty acids and total oil content of oilseed rape in the re-
length fatty acids cultivars has been done. search of quality improvement breeding.
Ultrasonic solvent extraction method provides a high efficient
contact between sample matrix and solvent. Acoustic cavitation,
* Corresponding author. Address: Oil Crops Research Institute, Chinese Academy
mechanical function and thermal function have direct effect on
of Agricultural Sciences, Wuhan, Hubei 430062, PR China. Tel.: +86 27 86711552;
fax: +86 27 86822291. the efficiency of ultrasonic extraction [7,8]. Cavitation is the most
E-mail address: willasa@163.com (F. Wei). significant factor. Under the ultrasound irradiation, micro-bubbles

1350-4177/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2008.04.003
 F. Wei et al. / Ultrasonics Sonochemistry 15 (2008) 938–942 939

will be created when the negative pressure is high enough. Once 25 psi. The temperatures of injection port and detector (FID) were
created, the bubbles will grow during the period of negative pres- maintained at 250 and 260 °C and the temperature programming
sure and compress during the period of positive pressure. The for the column was applied as follows, 210 °C (1 min), then to
expansion and compression can cause constant pulsating or violent 230 °C at 10 °C/min (22 min).
collapsing of micro-bubbles. When the collapse occurred near solid The computer software used for statistics analysis was STATIS-
surface, it can damage the cell walls to facilitate the release of con- TICA, version 6.0, by Statsoft Inc.
tents. Meanwhile, promotion of solvent penetration into cell walls
by mechanical function and decrease of solvent viscosity by ther- 2.3. Sample preparation
mal function can also improve mass transfer [9–11]. These factors,
taken together, enhance the efficiency of ultrasonic extraction A small quantity of dry oilseed rape samples (<20 mg) were
greatly. quantified and then crushed to a particle size less than 0.5 mm.
Ultrasonic treatment is a simple and rapid method in the frac- After that, the samples were extracted by petroleum ether with
tionation of plant materials [12] and well established in the pro- an ultrasound cleaning bath. The effect of the extraction conditions
cessing of plant materials, particularly, for extracting low such as liquid:solid ratio, ultrasound time and ultrasound power
molecular substances [13–15]. The mechanochemical effect of on the extraction yields, which was defined as the relative mass
ultrasound is believed to accelerate the extraction of organic com- of total oil determined and dry oilseed (total oil as % (w/w) dry oil-
pounds from plant materials due to disruption of cell walls and en- seed), were evaluated and all experiments were duplicated. After
hanced mass transfer of the cell contents [16]. Organic compounds extraction, petroleum ether was vaporized to dryness by a gentle
can be highly efficiently extracted with ultrasound without obser- stream of nitrogen and then a certain volume of fresh petroleum
vation of substantial changes in their structural, molecular proper- ether was added to the extracted sample. After addition of internal
ties and biological activities [17,18]. The effect of ultrasound on standard (0.04 mL of 5 g/L, methylheptadecanoate (C17:0)), 1 mL
vegetal tissues during solvent extraction has been described [19] KOH–methanol solution (0.4 M) was added to the extracted sam-
and the ultrasonically assisted extraction of bioactive principles ple for fatty acid methyl esters (FAMEs) preparation, and the ex-
from herbs reviewed [12]. tract was esterified in an ultrasound cleaning bath for 20 min.
The aim of this work was to establish a simple, low cost and After addition of 2 mL distilled water, the sample was shaken
effective ultrasonic solvent extraction method combined with gas and then centrifuged (10 min, 4500 rpm). The upper part was ta-
chromatography (GC) for quantitative determination of fatty acids ken for GC analysis. The peaks were identified based on their reten-
and oil content in small quantity of oilseed rape (<20 mg). At first, tion times using authentic standard fatty acids methyl esters and
the effect of liquid:solid ratio, ultrasound time and ultrasound all samples were run in duplicate. The relative peak areas (analyte
power on the ultrasound-assisted extraction of total oils from area/IS area) were used for quantification of the contents of the
rapeseeds were evaluated through an orthogonal experiment (L9 fatty acids, with considering response factors. The total fatty acids
(34)) in order to find an optimal use of ultrasound for the extraction content of oilseed rapes have been converted to the total oil con-
of oils. In the second part, the ultrasound effect on production of tents by converting the forms of the fatty acid methyl esters to
total oils from rapeseeds compared with the conventional method their relative forms of the fatty acid glycerides.
was studied. Furthermore, by the use of ultrasound-assisted
extraction combined with gas chromatography, the fatty acids 3. Results and discussion
and total oil content of different varieties of oilseed rapes were
successfully qualitatively determined. 3.1. The optimization of the ultrasonic-assisted extraction conditions

3.1.1. The effect of liquid:solid ratio on the extraction efficiency of oils

2. Experimental When the ultrasound power was set at 500w, the oilseed rape
samples were extracted for 60 min by petroleum ether with differ-
2.1. Reagents and materials ent liquid:solid ratios (L:g), respectively. Fig. 1 shows the effect of
liquid:solid ratio on the extraction efficiency of total oils. As shown
The oilseed rape samples were obtained from mature fruits in Fig. 1, the extraction yield increased with increasing the liquid:
growing in the test field of the Oil Crops Research Institute, Chinese solid ratio up to 1:4, above which there was no significant increase
Academy of Agricultural Sciences and were freeze-dried to con-
stant weight before the experiment. Petroleum ether, methanol
and potassium hydroxide (KOH) were of analytical grade and ob-
tained from Shanghai Chemical Co., Ltd. (Shanghai, China). Methyl
heptadecanoate (used as internal standard) and oilseed rapeseed
fatty acid methyl ester standard mixes were obtained from Sigma
(MO, USA).

2.2. Instrument and analytical conditions

The ultrasound-assisted extraction was performed in an ultra-

sound cleaning bath KQ-500DB (Ultrasound instrument Co., Ltd,
Kunshan, China). The working frequency and power were 40 kHz
and 500 W, respectively. The internal dimensions of the ultrasonic
cleaning bath were 50 cm  30 cm  15 cm with the volume of
22.5 L. The experiment temperature was 25 °C at the beginning
and did not exceed 35 °C in any experiment.
The esters were separated by GC (Agilent 6890N, USA) fitted
with a capillary column (FFAP, 30 m, 0.25 mm i.d., 0.50 lm film
thickness). Nitrogen was used as carrier gas at inlet pressure of Fig. 1. The effect of liquid:solid ratio (L:g) on the extraction efficiency of total oils.
940 F. Wei et al. / Ultrasonics Sonochemistry 15 (2008) 938–942

in the extraction yield. This result was predictable because raising

the liquid:solid ratio leads to an enhanced mass transfer procedure,
which increases the extraction yield. However, when the solvent
saturates with the extracted compound the concentration gradient
becomes null and the phenomena stops. In ultrasound extraction
of oils from oilseed rape, liquid:solid ratios for values above 1:4
slightly increased the amount of oils that was extracted. In consid-
eration of economy and environment factors, continued increasing
of the liquid:solid ratio was not desirable.

3.1.2. The effect of ultrasound time on the extraction efficiency of oils

The ultrasound time on the extraction efficiency of oils was
evaluated when the ultrasound power was set at 500 W and the li-
quid:solid ratio (L:g) was set at 1:4. Fig. 2 shows the effect of ultra-
sound time on the extraction efficiency of total oils. When the
ultrasound time increased from 10 to 60 min, the extraction yield
of oils was increased due to the mass transfer of oils from cellular
material to petroleum ether by diffusion and osmosis. During this Fig. 3. The effect of ultrasound power on the extraction efficiency of total oils.
period, the major action of ultrasound is acoustic cavitation, which
disrupted the cell walls and accelerated the washing out of the cell
the extraction process was shown in Table 2. Table 3 shows the
contents. As the time expanding, more and more cells were broken,
analysis of variance (ANOVA) results for the calculated models. It
and oils inside the seed material were released gradually. However,
can be seen that the ultrasound time plays an important role in
the extraction efficiency had no noticeable enhancement when the
the extraction of oil from oilseed rape samples, followed by the
ultrasound time increasing from 60 min to 90 min. Thus, 60 min
ultrasound power, and the liquid:solid ratio. The optimum combi-
was selected as the optimum ultrasound time.
nation of variables was A1B2C3, i.e. liquid:solid ratio 1:4, extraction
time 60 min and extraction power 500 W.
3.1.3. The effect of ultrasound power on the extraction efficiency of oils
The following regression equation, which is an empirical rela-
When the liquid:solid ratio (L:g) was set at 1:4, the oilseed rape
tionship between the oil extraction yield and the experimental lev-
samples were extracted for 60 min with different extraction
els of each test factor, was obtained by the use of STATISTICA,
power, respectively. Fig. 3 shows the effect of ultrasound power
version 6.0.
on the extraction efficiency of total oils. As shown in Fig. 3, the
extraction yield increased with increasing the ultrasound power,
which is because of the increased extraction power leading to an
Table 1
enhanced mass transfer procedure. However, when the extraction
Independent variable values of the process and their corresponding levels
power was set above 400 W, no significant increase in the extrac-
tion yield was observed which can be ascribed to that the balance Levels Independent variables
of the extraction system has been reached. Liquid:solid ratio (L:g) Ultrasound time (min) Ultrasound power (w)
A B C
3.1.4. Orthogonal experiment 1 1:4 30 200
On the basis of the single factor experiments, the combination 2 3:8 60 300
3 1:2 90 500
effect of liquid:solid ratio, ultrasound time and ultrasound power
on the extraction of total oils from oilseed rape samples were eval-
uated through an orthogonal experiment (L9 (34)) in order to deter-
mine the optimum combination of variables. The effect of the Table 2
independent variables liquid:solid ratio (A), ultrasound time (B) Orthogonal experiment (L9 (34)) for the ultrasound-assisted extraction of total oils
and ultrasound power (C) at three variation levels (Table 1) in
Treat Variable levels Determined total oil contents (%)
A B C
1 1 1 1 28.66
2 1 2 2 41.24
3 1 3 3 37.25
4 2 1 2 27.05
5 2 2 3 47.26
6 2 3 1 28.51
7 3 1 3 30.74
8 3 2 1 35.59
9 3 3 2 27.78

Table 3
Analysis of variance (ANOVA) table for the experiments

Source of variance Degree of freedom Sum of square Mean value F Value

Liquid:solid ratio 2 26.73 13.36 7.24
Ultrasound time 2 256.23 128.11 69.37
Ultrasound power 2 105.70 52.85 28.62
Pooled error 2 3.70 1.85
Total 8 392.36
Fig. 2. The effect of ultrasound time on the extraction efficiency of total oils.
 F. Wei et al. / Ultrasonics Sonochemistry 15 (2008) 938–942 941

y ¼ 0:461972a þ 45:441361b  8:031972c  0:442229a2 sults summarized in Table 4. Eight fatty acids were identified in No.
2 2 1–No. 3 oilseed rape samples, in which erucic acid (C22:1) was
 11:078895b þ 2:931105c
determined to be the pre-dominant fatty acid and oleic acid
On the basis of the experiment, the optimal solution of the equation (C18:1) was determined to be the second most abundant in these
is a = 1, b = 2, c = 3 and y = 47.95, i.e. the maximum value of the samples (the GC chromatogram of No. 1 sample shown in Fig. 4).
extraction yield is 47.95% under the optimum combination of vari- Seven fatty acids were identified in No. 4 and No. 5 oilseed rape
ables (A1B2C3). varieties, in which oleic acid (C18:1) was determined to be the
pre-dominant fatty acid and linoleic acid (C18:2) was determined
3.2. Experimental verification to be the second most abundant in these samples. Furthermore,
palmitic (C16:0), stearic (C18:0), linolenic (C18:3), arachidic
The oils have been extracted from the homogeneous oilseed (C20:0) and eicosenoic acids (C20:1) were also determined in all
rape samples under the optimum extraction conditions, and the oilseed rape varieties.
experimental mean value of the total oils content (46.55%) for five The total fatty acids content of five oilseed rape cultivars have
determinations was in agreement with the calculated maximum also been converted to the total oil contents by converting the
value (47.95%). The relative standard deviation (RSD) was below forms of the fatty acid methyl esters to their relative forms of the
7.1% for five measurements, which showed that this method has fatty acid glycerides (with the results shown in Table 4). It can
good reproducibility for real sample analysis. be seen that the total oil contents of different oilseed rape varieties
differ from each other, and No. 4 and No. 5 oilseed rape varieties
3.3. Comparison with the conventional method were high and low oil content oilseeds, respectively. Furthermore,
the results showed that the total oil contents of No. 4 and No. 5 oil-
For comparison with the ultrasound-assisted extraction meth- seed rapeseeds were 46.77% and 25.86%, respectively, which are in
od, refluxing extraction method was also carried out for the extrac- agreement with the results (46.82% and 27.08%) obtained by tradi-
tion of oil from oilseed rape samples with the extraction time and tional standard method.
oil extraction yield 5 h and 39.06%, respectively. The results
showed that ultrasound-assisted extraction method was about five 4. Conclusions
times faster than the traditional method and the application of
ultrasound during the extraction of oil from oilseed rape samples The application of ultrasound for the extraction of oils from oil-
had positive effect on the extraction yield. seed rape samples offers important advantages over traditional
extraction method, namely: shorter extraction time (60 min
3.4. Analysis of real sample against 5 h for refluxing extraction method), cleaner features and
low cost (as no great volume of the organic solvent is involved).
Fatty acids and total oil content of five oilseed rape cultivars Furthermore, classical extraction procedure would be hard to per-
have been analyzed by the optimum method with the detection re- form due to very small sample amounts available for the analysis,

Table 4
Fatty acids compositiona (fatty esters as % (w/w) total fatty acid esters), fatty acids contenta,b (fatty esters as % (w/w) dry oilseed) and total oil contenta (total oil as % (w/w) dry
oilseed) of five oilseed rape cultivars

No. Oils C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:1
1 38.53 3.56 (1.33)b 1.26 (0.47) 21.30 (7.97) 12.71 (4.75) 7.03 (2.63) 0.51 (0.19) 13.58 (5.08) 40.56 (15.17)
2 41.38 3.74 (1.50) 1.23 (0.49) 26.17 (10.51) 14.10 (5.66) 6.75 (2.71) 0.35 (0.14) 14.55 (5.84) 33.46 (13.44)
3 35.52 3.71 (1.29) 1.23 (0.43) 21.67 (7.50) 13.03 (4.51) 6.95 (2.41) 0.21 (0.073) 13.15 (4.56) 40.23 (13.94)
4 46.77 4.05 (1.89) 2.15 (1.00) 69.96 (32.57) 15.58 (7.25) 7.41 (3.45) 0.61 (0.28) 0.86 (0.40) ND
5 25.86 6.04 (1.56) 2.29 (0.59) 58.85 (15.18) 23.49 (6.06) 9.31 (2.40) 0.25 (0.065) 0.35 (0.090) ND
a
Values are average of three individual samples each analyzed in duplicate.
b
Fatty acids content.

Fig. 4. GC chromatogram obtained by the ultrasound-assisted extraction of No. 1 sample (C17:0 was used as IS).
942 F. Wei et al. / Ultrasonics Sonochemistry 15 (2008) 938–942

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