Industrial Crops and Products 76 (2015) 472–478

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Supercritical CO2 extraction of hemp (Cannabis sativa L.) seed oil

Krunoslav Aladić a , Kristjan Jarni b , Tina Barbir c , Senka Vidović d , Jelena Vladić d,∗ ,
Mate Bilić e , Stela Jokić e
a
Croatian Veterinary Institute, Branch - Veterinary Institute Vinkovci, Josipa Kozarca 24, 32100 Vinkovci, Croatia
b
Biotechnical Faculty, University of Ljubljana, Večna pot 83, 1000 Ljubljana, Slovenia
c
Croatian Veterinary Institute Zagreb, Savska 143, 10000 Zagreb, Croatia
d
University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
e
Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhaca 20, 31000 Osijek, Croatia

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

Article history: In this work, hemp (Cannabis sativa L.) seed oil was produced by extraction with supercritical CO2 under
Received 12 March 2015 different extraction conditions (temperature, pressure and time). The objective was to evaluate the influ-
Received in revised form 2 June 2015 ence of extraction conditions on concentration of tocopherols, fatty acids and pigments in hemp seed oil.
Accepted 13 July 2015
The composition of hemp seed oil obtained with supercritical CO2 was compared with the hemp oil
Available online 1 August 2015
extracted by n-hexane using Soxhlet method and with oil obtained by pressing using screw expeller.
Using supercritical CO2 extraction the extracts higher in concentration of tocopherol were produced.
Keywords:
The amount of ␣- tocopherol in supercritical extracts ranged from 37.09 to 110.61 mg L−1 , depending
Hemp seed oil
Supercritical CO2 extraction
on the applied process conditions, while -tocopherol content was significantly higher (2–3 times). The
Tocopherols content of pigments in the hemp oil obtained by supercritical CO2 had been changed significantly during
Fatty acids the extraction time from 9.79 to 178.76 mg kg−1 for total chlorophyll content and 8.15 to 57.66 mg kg−1
Pigments for total carotene content. By selecting the relevant process conditions of supercritical extraction it is
possible to obtain hemp seed oil with physical or nutritional properties of interest to the food industry.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction Supercritical carbon dioxide (CO2 ) extraction is valuable tech-

Hemp has been grown and has had a share in the industrial pro-
duction in Croatia for a significant length of time. Hemp (Cannabis
sativa L.) is fully usable in the production of a large range of prod-
ucts but its use has decreased in previous decades because of great
similarities with Indian hemp (Cannabis indica L.). The main reason
that hemp was banned in the past is the presence of psychoactive
substance ␦-9-tetrahydrocannabinol (THC) (Oomah et al., 2002).
According to EU legislation production of hemp is permitted if
the THC content is less than 0.2% and Kriese et al. (2004) found
51 varieties with levels less than 0.2%. Hemp is certainly a mul-
tifunctional plant and the most important products which can be
produced from hemp are: oil, proteins, which can be used in food
and feed production, and fibers used in the paper and textile indus-
try (Callaway, 2004; Bertoli et al., 2010). Hemp seeds are high value
with approximately 25–35% lipids, 20–25% proteins, 20–30% car-
bohydrate, 10–15% insoluble fibers and numerous of natural source
minerals (Oomah et al., 2002; Deferne and Pate, 1996).
∗ Corresponding author.
E-mail address: vladicjelena@gmail.com (J. Vladić).
nology in seeds, fruit and vegetable processing and preservation
(Rawson et al., 2012). Supercritical CO2 extraction as a green
technology is certainly alternative method to replace or to comple-
ment conventional industrial process such as pressing and solvent
extraction. Supercritical fluid extraction (SFE) technique has many
advantages over traditional methods, especially in preservation
of thermosensitive compounds using low temperatures, which
results reduced energy consumption (Moslavac et al., 2014). Super-
critical fluids, like supercritical CO2 , are characterized by high mass
transfer rates, liquid like density, and variable selectivity (achiev-
able by variation of temperature and pressure). By variation of
supercritical CO2 selectivity extracts of desirable content and con-
centration of certain compounds can be produced. One of very
important characteristics of this extraction technique is produc-
tion of extracts without residues of extraction solvent - “solvent
free extracts”.
Food industry is always looking for processes that can minimize
the environmental impact, decrease toxic residues, use by-products
more efficiently and also obtain high-quality products with good
nutritional and organoleptic properties. SFE is still relatively new
in the extraction of hemp oil and other edible oils mainly due to
very high investment costs of SFE equipment (Jokić et al., 2014a).

http://dx.doi.org/10.1016/j.indcrop.2015.07.016
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 K. Aladić et al. / Industrial Crops and Products 76 (2015) 472–478
But nowadays, according to global trends, “green” products and
technologies are needed to replace conventional ones.
Several authors used supercritical CO2 extraction to obtain
hemp oil. Da Porto et al. (2012a) followed fatty acid composition
and oxidation stability of obtained oil at different process param-
eters. Also Da Porto et al. (2012b) optimized supercritical CO2
extraction parameters in the production of hemp oil using response
surface methodology. Kriese et al. (2004) investigated fatty acid
composition and tocopherol content in 51 different genotypes of
hemp. Tomita et al. (2013) followed different process parameters
on solubility of hemp oil in supercritical CO2 extraction. In this
study, supercritical CO2 , was used as a solvent in the extraction
of hemp seed oil at different process conditions of temperature,
pressure and time in order to determine fatty acids, tocopherols
and pigment content (chlorophyll a and b and total carotene) of
extracted oil. According to our knowledge there are no previ-
ous reports on pigment content of hemp oil collected at different
extraction period with supercritical CO2 . The composition of hemp
seed oil obtained by supercritical CO2 was compared with the hemp
seed oil extracted by other extraction techniques.
2. Material and methods
2.1. Chemicals
The purity of CO2 used for extraction was 99.97% (w/w) (Messer,
Osijek, Croatia). For preparation of calibration curves standard
compounds ␣-tocopherol (Dr. Ehrenstorfer Cat No. 1792430),
␤-tocopherol (Supelco Cat No. 46401-U), -tocopherol (Supelco
Cat No. 47785) and ı-tocopherol (Supelco Cat No. 47784) were
used. Standard compounds for determination of fatty acids were
provided from SupelcoTM 37. Component FAME Mix (Bellefonte,
Pennsylvania, SAD) were used. Potassium hydroxide was supplied
by Kemika (Zagreb, Croatia). n-hexane was provided from Merck
KGaA (Darmstadt, Germany). All other chemicals and reagents were
of analytical reagent grade.
2.2. Material
Hemp seeds (Cannabis sativa L.) genotype Fedora 17 were
obtained from family farm Organica Vita (Vraneševci, Croatia) in
2013. Moisture content of the hemp seeds was determined accord-
ing to AOAC Official Method 925.40 (2000). Samples were cleaned
from impurities and grounded using laboratory mill (IKA Basic A11,
Germany).
2.3. Determination of particle size distribution of hemp seeds
with sieving
Hemp seeds were sieved using a vertical vibratory sieve shaker
(Labortechnik Gmbh, Ilmenau, Germany) for 20 min. About 200 g
loading were used at each sieving. The raw material size distribu-
tion was determined using a nest of 9 sieves of aperture sizes 1.4,
0.8, 0.63, 0.5, 0.4, 0.315, 0.2, 0.1 and 0.05 mm. The mass of fragments
remaining on each sieve after sieving was used to calculate the dis-
tribution of fragments, which was then normalized in respect of
the total mass. For evaluation of sieve analysis results the Rosin-
Rammler-Bennet (RRB) distribution (Allen, 1981) was chosen. The
percentage by mass of particles (R) greater than screen size (d) is
given as:
  n 
d by saponification 0.5 g of sample in 50 ml of potassium hydroxide,
R = 100 exp −
d0
where d0 represents the particle size corresponding to the 36.8th
percentile of the cumulative probability distribution (size con-
473
stant), and n controls the shape of the distribution (uniformity
coefficient). The function of the sum of sieve residue (R) was fitted
to the experimental data by changing the representative particle
size d0 and the uniformity coefficient n, minimizing the sum of the
mean square error using STATISTICA 8.0 software (Stat Soft Inc.,
USA).
2.4. Organic solvent extraction
The initial oil content in hemp seeds was measured by auto-
matic extraction systems Soxterm by Gerdhart with n-hexane. 5 g of
ground hemp seeds was extracted with 120 ml solvent, until totally
depleted according to HRN ISO 6492 (2001). The whole process took
2 h and 45 min, at 180 ◦ C. The measurement was done in triplicate.
2.5. Cold pressing
The cold pressed hemp oil was obtained by pressing the 1 kg
of hemp seeds using following parameters: temperature of head
presses of 60 ◦ C, frequency of 20 Hz and using nozzle of ID 6 mm.
The pressing of the seeds were performed in a screw expeller SPU
20 (Senta, Serbia) with capacity 20–25 kgh−1 . The measurement
was done in duplicate.
2.6. Supercritical CO2 extraction
The experiment was performed in SFE system explained in detail
elsewhere (Jokić et al., 2014b; Moslavac et al., 2014). The grounded
hemp seeds of 100 g were placed into extractor vessel. The extracts
were collected in previously weighed glass tubes. The amount of
extract obtained at regular intervals of time was established by
weight using a balance with a precision of ±0.0001 g. Separator
conditions were 15 bar and 25 ◦ C.
The extraction was performed at different extraction conditions
of pressure (300 and 400 bar) and temperature (40 and 60 ◦ C) until
the extraction yield became constant. During extraction process,
every half hour, sample of extracted oil were collected for further
analysis.CO2 mass flow rate of 1.94 kg/h were kept constant. Each
extraction experiment was conducted two times and the average
value of two replication is given in Fig. 2.
2.7. Determination of chlorophyll a, chlorophyll b, and total
carotene content
For determination of chlorophyll a and b, and total carotene
content, the modified method of Dere et al. (1998) was used.
The weighted sample, having been added diethyl ether (50 ml for
each gram), was dissolved in an ultrasonic bath for one minute.
It was then homogenized for 30 s with homogenizer, and again
in the ultrasonic bath for one minute. The homogenate was cen-
trifuged for 10 min at 3000 rpm. The supernatant was separated
and the absorbances were measured at 400–700 nm in the UV spec-
trophotometer. Chlorophyll a showed the maximum absorbance at
660.0 nm, chlorophyll b at 642.5 nm, and total carotene at 470 nm.
All analyses were repeated three times. The amount of these
pigments was calculated according to the formulas given in our
previous published paper (Aladić et al., 2014).
2.8. Determination of tocopherols
Preparation of samples for GC–MS analysis has been provided
(1) and then by extraction unsaponifiable components using diethyl
ether as extraction solvent.
For analysis of tocopherols Agilent 7890 A GC equipped with
Agilent 5975 MSD has been used. For this analysis GC–MS was fitted
474 K. Aladić et al. / Industrial Crops and Products 76 (2015) 472–478
Fig. 1. Collected hemp seed oil during the extraction process at temperature of 40 ◦ C and pressure 300 bar.
with HP-5MS (Agilent J&W 19091S-433) column (30m × 0.25 mm
ID, 0.25 ␮m). The temperature of injection port was 250 ◦ C, split-
less injection. The temperature of transfer line was 280 ◦ C. The
initial oven temperature was set at 200 ◦ C for 3 min, and then
programmed as 8 ◦ C/min to 280 ◦ C. The carrier gas was He. MS
conditions were: scan (45 to 450 amu), threshold 100 MS quad
150 ◦ C, MS source 250 ◦ C. Injected sample volume was 1 ␮l. The
identification of components was carried out based on computer
matching with NIST 2008 MS library. Quantification of compounds
was provided using calibration curves. Standard compounds were
dissolved in n-hexane to prepare six different concentration of each
standard compound. R2 for each calibration curve was 0.999. All
analyses were performed in triplicate.
2.9. Determination of fatty acids composition
Preparation of fatty acid methyl esters was carried out accord-
ing to EN ISO 5509:2000 standard. Prepared fatty acid methyl
esters were analyzed by gas chromatography according to EN
ISO 5508:1995. Gas chromatograph 7890B (Agilent Technolo-
gies, Lake Forest, USA) with a capillary column HP88 100 m long
with a diameter of 0.25 mm and the thickness of the station-
ary phase 0.20 microns (Agilent Technologies, Lake Forest, USA), a
split-splitless injector (temperature 250 ◦ C) and a flame-ionization
detector (temperature 280 ◦ C) was used. A sample (1 ␮L) was
injected with a split ratio of 1:50. Start column temperature was
120 ◦ C with holding time for 1 min. The oven temperature was
increased with a rate of 10 ◦ C/min to 175 ◦ C/min, holding for
10 min, then at a rate of 5 ◦ C/min was heated to 210 ◦ C, hold-
ing for 5 min, then again at a rate of 5 ◦ C/min was heated up to
230 ◦ C holding for 5 min. Carrier gas was helium (99.9999%) at con-
stant flow rate of 2 ml/min. The hydrogen flow was 40 ml/min, air
flow was 450 ml/min, and the makeup gas flow (nitrogen) was
30 ml/min.
Fatty acid methyl esters were identified by comparison with
retention times of 37 fatty acid methyl ester standard compound
analyzed at the same conditions. With samples and standards, for
every analysis, certified reference material (CRM), was prepared
and analyzed at the same conditions. The result is expressed as per-
centage (%) individual fatty acids to total fatty acids determined.
The detection limit method was 0.1%. Values determined in the
validation process for parameter truthfulness were compared with
the criterion of the Guidelines for the implementation of analytical
methods and interpretation of results (N.N 2/2005), that to prove
the truth of the proportion by weight > 10 mg/kg may vary from
−20% to +10% as compared to the certified value.
2.10. Statistical analysis
Regression analysis was used to predict the amount of extracted
substance (fatty acids and tocopherols) during the extraction pro-
cess. The simple regression analysis was carried out with the model:
Y i = (b0 + b1 Xi ) + i (2)
where parameter b1 is the gradient of the straight line fitted to the
data, b0 is the intercept of that line and i is residual. Overall fitting
of the model was tested with the analysis of variance (ANOVA) with
IBM SPSS for Windows software.
3. Results and discussion
To examine the influence of different extraction parameters
(pressure, temperature and time) on extraction yield and com-
position, solvent flow rate and particle size of grounded material
were kept constant during the experiment. Solvent mass flow rate
was 1.94 kg/h, and the average particle size by sieving was deter-
mined to be 0.382 mm ±0.15. The initial oil content in hemp seeds
was 33.34 ± 0.23% and moisture content was 8.09 ± 0.09%. During
each supercritical extraction process, every half hour, sample of
extracted oil were collected. Fig. 1. shows one example (photo-
graph) of collected hemp seed oil during the extraction process at
temperature of 40 ◦ C and pressure 300 bar. It was very interesting
to notice how the color of collected oil changes from the beginning
to the end of extraction process. At very beginning the oil color
was almost completely yellow, and during the extraction process it
received more dark green color. According to Cert et al. (2000) the
hemp oil dark green color comes from the most important pigments
in hemp oil: chlorophyll a and b. Furthermore, carotenoids are also
presented in the hemp oil. The literature about the pigments in
hemp oil is very scarce. During the extraction process the reduction
in the amount of collected oil was observed. At the same time the
increase in green coloration occurs. According to observed color
and pigment analysis the content of total chlorophyll pigments
at the very beginning of extraction was very small, while in the
last collected fraction the amount of chlorophyll pigment increased
(Table 1). The highest chlorophyll content was determined in hemp
oil obtained at higher pressure (400 bar) after 180 min of extraction
(178.76 mg kg−1 ). Furthermore, the total carotene content was in
the range from 8.15 to 57.66 mg kg−1 depending on applied extrac-
tion parameters. According to the results given in Table 1, it can
be concluded that extraction time had significant influence on pig-
ment content in hemp oil.
 K. Aladić et al. / Industrial Crops and Products 76 (2015) 472–478 475

Table 1
Pigment content of hemp oil obtained by supercritical CO2 during the extraction process.

Protocols ↓ Extraction time → 30 60 90 120 150 180 210

40 ◦ C Total chlorophyll content (mg kg−1 ) 12.89 49.65 56.35 84.99 91.28 136.14 146.80
300 bar
Total carotene content (mg kg−1 ) 8.15 15.50 16.37 26.67 45.07 47.32 57.66

40 ◦ C Total chlorophyll content (mg kg−1 ) 9.79 42.83 103.44 138.90 163.75 178.76 p.e.
400 bar
Total carotene content (mg kg−1 ) 10.02 14.33 38.37 50.09 53.18 54.38 p.e.

60 ◦ C Total chlorophyll content (mg kg−1 ) 48.98 62.38 71.28 74.01 82.69 94.49 118.06
300 bar
Total carotene content (mg kg−1 ) 21.07 22.95 23.54 28.71 30.85 37.54 39.39

p.e.: process ended

3.1. The effect of temperature and pressure on hemp oil 2012a; Kriese et al., 2004; Oomah et al., 2002). The main fatty
extraction yield and composition acid was linoleic acid. The concentration of obtained palmitic acid
was significantly higher at a higher temperature than at a lower,
The effect of temperature (40 and 60 ◦ C) and pressure (300 and t(8) = −4.72, p < 0.01. On the contrary, extraction of oleic acid is
400 bar) on the extraction yield, as well as on the content of fatty significantly better at a lower temperature than at a higher one,
acids and tocopherols, of hemp seed oil obtained by supercritical t(8) = 2.91, p < 0.05. In the case of other three fatty acids significant
CO2 was examined. The pressure and solvent mass flow rate in influence of extraction temperature on their extraction yield was
this experiments were kept as constants, at 300 bar and 1.94 kg/h, not found. Regarding to tocopherols composition, the temperature
respectively. The results of extraction yield are shown in Fig. 2, had statistically significant influence on both tocopherol form. ␣-
where the yield was plotted versus time of extraction. From extrac- tocopherol in hemp oil obtained at 40 ◦ C was 110.613 mg L−1 , while
tion curves it can be seen that almost all hemp oil was extracted at 60 ◦ C that concentration was 37.086 mg L−1 . The same results
from the seeds using supercritical CO2 (by soxhlet standard the ini- were obtained with ␥-tocopherol which concentration was signif-
tial oil content was 33.34%). Generally, hemp seeds contain 28–35% icantly reduced at higher temperatures (from 158.264 mg L−1 at
oil, depending on the variety, climate, geographical area and year 40 ◦ C to 95.397 mg L−1 at 60 ◦ C).
of cultivation (Matthäus & Brühl, 2008). Due to high oil content in Concentration of fatty acids in obtained supercritical extracts
the hemp seeds, it is not efficient to apply pressure values lower was not affected by pressure, while it had influence on tocopherol
than 30 MPa, because of the long duration of the extraction process
and extremely low extraction yields (King, 1997).
From Fig. 2a it can be seen that temperature did not have sig-
nificant influence on the extraction yield. Eggers (1996) and Salgın
et al., 2006 reported that the increase in the extraction yield as a
function of increasing temperature, at a constant pressure, were
recorded only at the value of pressure of 400 bar and at higher
pressures. Furthermore, during the analysis of pressure impact on
extraction yield (Fig. 2b) the temperature and solvent flow rate has
been maintained constant. As expected, the yield of hemp seed oil
increased with the increase in extraction pressure, in a manner to
seed oil solubility. At a pressure lower than 300 bar the seed oil
have a very low solubility, while by increasing of pressure up to
700 bar solubility increased significantly (King, 1997). The simi-
lar results related to extraction pressure effect of hemp seed oil
extraction curves were found in study published by Da Porto et al.
(2012b). They published that with increasing pressure increase the
total obtained oil.
When considering the influence of temperature on extraction of
some compounds it can be noticed that extraction temperature had
statistically significant influence, especially on tocopherol content.
In Tables 2,3 concentration for just five the most abundant fatty
acids was given, while other acids, such as C16:1 palmitoleic, C18:0
stearic, C20:0 arachidic and C20:2n6 eicosadienoic, were found in
very small amounts and therefore they are not presented in this
paper. From our preliminary experiments we noticed that extrac-
tion time had no influence on fatty acid content. The fatty acid
content during extraction process stayed unchanged. So, the aver-
age value of each determined fatty acid in hemp oil collected during
supercritical CO2 extraction (each half hour) are presented in these
tables.
The most frequent form of tocopherols in hemp oil was -
tocopherol followed by ␣-tocopherol. Other two tocopherols (ˇ-
and ı-) were not detected. Almost identical results related to fatty
Fig. 2. Effect of temperature (a) and pressure (b) on the extraction yield of hemp
acid composition were found also by other authors (Da Porto et al., seed oil.
476 K. Aladić et al. / Industrial Crops and Products 76 (2015) 472–478

Table 2
The effect of different temperatures on the extraction of fatty acids and tocopherols.

40 ◦ C 60 ◦ C t-test

Average STDEV Average STDEV

Fatty acids
Palmitic acid (%) 6.94 0.61 7.37 0.64 −4.72**
-linolenic acid (%) 3.15 0.32 3.00 0.13 1.94 n.s.
˛-linolenic acid (%) 16.30 0.47 16.55 0.06 −1.41 n.s.
Oleic acid (%) 13.15 0.57 12.82 0.26 2.91*
Linoleic acid (%) 58.18 0.60 58.45 0.17 −1.56 n.s.

Tocopherols
˛-tocopherol (mg/L) 110.61 45.02 37.09 68.89 3.02*
-tocopherol (mg/L) 158.26 46.80 95.40 92.40 2.95*

(n.s.: not significant at P > 0.05; *0.01 < P < 0.05; **0.001 < P <0.01;***P < 0.001).

Table 3
The effect of different pressures on the extraction of fatty acids and tocopherols.

300 Bar 400 Bar t-test

Average STDEV Average STDEV

Fatty acids
Palmitic acid (%) 7.12 0.61 7.02 0.37 0.59 n.s.
-linolenic acid (%) 3.05 0.30 3.19 0.28 −2.28 n.s.
˛–linolenic acid (%) 16.46 0.44 16.18 0.57 2.13 n.s.
Oleic acid (%) 12.98 0.56 13.24 0.58 −1.88 n.s.
Linoleic acid (%) 58.38 0.56 58.09 0.60 1.97 n.s.

Tocopherols
˛-tocopherol (mg/L) 110.61 45.02 31.43 58.19 3.69**
-tocopherol (mg/L) 181.60 44.85 163.66 42.02 1.92 n.s.

(n.s.: not significant at P > 0.05; *0.01 < P< 0.05; **0.001 < P <0.01; ***P < 0.001).

Table 4
The effect of extraction time on amount of extracted fatty acids at different temperatures and pressures.

Fatty acids → Palmitic -linolenic ˛-linolenic Oleic Linoleic

Protocols ↓ R 2
F R 2
F R2
F R2
F R2 F
◦
40 C 300 Bar 0.85 32.80** 0.90 54.40*** 0.91 57.30*** 0.88 43.70** 0.90 51.90***
40 ◦ C 400 Bar 0.64 6.98 n.s. 0.87 26.10** 0.87 26.30** 0.75 12.00* 0.89 32.50**
60 ◦ C 300 Bar 0.99 517.00*** 0.92 81.30*** 0.23 2.04 n.s. 0.95 127.00*** 0.88 51.50***

F: F-ratio; R: goodness of fit of the model.

(n.s.: not significant at P > 0.05; *0.01 < P < 0.05; **0.001 < P <0.01; ***P < 0.001).

content (Table 3). Very similar results regarding concentration of
tocopherols were published by Kriese et al. (2004). Furthermore,
Teh and Birch (2013), Uluata and Özdemir (2012), Gruszka and
Kruk (2007) and Oomah et al. (2002) published much higher con-
centration of tocopherols than in our study, probably because of
different hemp seed genotype. It seems that higher pressure and
higher temperature in the process of supercritical CO2 extraction
have negative impact on tocopherol extraction. Namely, content of
both ␣- and ␥-tocopherols were significantly higher at a lower tem-
perature, except for -tocopherol where the effect of the change of
pressure were not confirmed.
stance. In most cases, this part is more than 80% (R2 values). The
only exceptions are ␣–linolenic acid in the extraction process at
a higher temperature and palmitic acid at the low temperature
and higher pressure (p > 0.05). From the obtained results it can be
concluded that our regression model overall predicts amount of
substance significantly well.
The effect of time on the amount of extracted substance was
confirmed also in the tocopherols composition (Table 5). The effect
of extraction time on the extracted of -tocopherol was confirmed
in the process at a lower temperature both at the lower and at
high pressure. The effect of extraction time on the ␣-tocopherol
was confirmed only for higher temperature. In comparison with

3.2. The effect of extraction time on fatty acid and tocopherol

content in hemp oil
Table 5
The effect of extraction time on amount of extracted tocopherols at different tem-
The composition of fatty acids described in Table 3 was ana- peratures and pressures.
lyzed in every obtained extract collected during the extraction time,
Tocopherols→ ˛-tocopherol -tocopherol
and there were no differences in the detected fatty acid composi-
tions. Main detected fatty acid was linoleic acid in concentration Protocols ↓ R2
F R2 F
of 58.04%, followed by ␣–linolenic acid 16.55%, oleic acid 12.85%, 40 ◦ C 300 Bar 0.44 4.70 n.s. 0.74 16.95**
palmitic acid 7.25% and ␥–linolenic 3.03%. 40 ◦ C 400 Bar 0.75 9.14 n.s. 0.83 14.21*
Statistic results given in Table 4 show that regardless of the 60 ◦ C 300 Bar 0.53 7.96* 0.42 4.99 n.s.
protocol used, the length of the experimental process (extraction F: F-ratio; R: goodness of fit of the model.
time) can account for major part of variability in extracted sub- (n.s.: not significant at P > 0.05; *0.01 < P< 0.05; **0.001 < P <0.01; ***P < 0.001).
 K. Aladić et al. / Industrial Crops and Products 76 (2015) 472–478
Table 6
Fatty acid, tocopherol and pigment content in hemp seed oil obtained by different extraction methods.
Fatty acids Cold pressing
Palmitic acid (%) 7.31
-linolenic acid (%) 3.20
˛-linolenic acid (%) 16.24
Oleic acid (%) 13.00
Linoleic acid (%) 58.25
Tocopherols
˛-tocopherol (mg L−1 ) n.d.
-tocopherol (mg L−1 ) 137.46
Pigments
Total chlorophyll content (mg kg−1 ) 157.23
Total carotene content (mg kg−1 ) 39.39
n.d.: not detected.
the fatty acids, the model explain a little less variability what means
that there must be other variables that have an influence also.
In our previous studies (Jokić et al., 2013, 2012) the obtained oil
from soybeans by supercritical CO2 were investigated and soybean
oil fractions were collected at different extraction time intervals.
It was noticed that extraction temperature and pressure do not
have influenced on the fatty acids compositions, while fatty acids
composition differed according to different fractions collected at
different time intervals. Furthermore, by selecting the relevant
process conditions of supercritical extraction it was possible to
obtain soybean oil with different mass concentrations of toco-
pherols which is the case also with hemp oil extracted in this study.
3.3. Comparation of results for hemp oil obtained with other
extraction methods
The composition of hemp seed oil obtained with supercriti-
cal CO2 was compared with the oil extracted by n-hexane using
Soxhlet apparatus and with oil obtained by pressing using screw
expeller. Table 6 show fatty acids, tocopherol and pigment content
of hemp seed oil obtained using different extraction methods where
it can be concluded that fatty acid content of hemp oil obtained
by all three different extraction methods were very similar, while
obtained hemp oil by supercritical CO2 had higher tocopherol con-
tent compared to cold pressing. Oil obtained by organic solvent did
not have tocopherols according to high temperatures during Soxh-
let extraction process. It can be seen that the hemp oil obtained
by supercritical CO2 has the lowest total pigment content followed
by oil obtained with method by Soxhlet while oil obtained by cold
pressing have the highest total pigment content. The highest pig-
ment content obtained by cold pressing can be attributed to the
intensive mechanical destruction of hemp seed cells and destruc-
tion of chloroplasts (Hannides et al., 2014).
4. Conclusion
Supercritical extraction and fractionation of natural compounds
is one of the early and most studied applications in the field
of supercritical fluids. Different process conditions for supercrit-
ical extraction had statistically significant influence on different
mass concentrations of tocopherols, while fatty acid profile stayed
almost unchanged. Compared to other extraction methods, using
supercritical CO2 the lowest concentrations of pigments were
obtained. By collecting hemp oil during supercritical CO2 extraction
process it is possible to control the amount of extracted pigments.
Hemp oil was extracted almost completely by supercritical CO2
and the obtained oil had higher tocopherol content compared to
cold pressing. Low temperatures of extraction, reduced energy con-
sumption, high product quality and absence of solvent in extracts
477
Soxhlet Supercritical CO2 extraction
(n-
hexane)
7.41 6.92
3.18 3.16
16.10 16.29
13.27 13.17
57.94 58.19
n.d. 189.98
n.d. 134.06
101.99 90.65
28.19 34.21
are numerous advantages than supercritical fluid extraction tech-
nique can provide as opposed to traditional methods of extraction.
Acknowledgement
This work has been fully supported by Croatian Science Foun-
dation under the project 1321.
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