Accepted Manuscript

Title: Hemp (Cannabis sativa L.) seed oil extraction with

pressurized n-propane and supercritical carbon dioxide

Authors: Daniel Ribeiro Grijó, Gryele Karen Piva, Ignacio

Vieitez Osorio, Lúcio Cardozo-Filho

PII: S0896-8446(18)30463-7
DOI: https://doi.org/10.1016/j.supflu.2018.09.004
Reference: SUPFLU 4366

To appear in: J. of Supercritical Fluids

Received date: 11-7-2018

Revised date: 4-9-2018
Accepted date: 5-9-2018

Please cite this article as: Grijó DR, Piva GK, Osorio IV, Cardozo-Filho
L, Hemp (Cannabis sativa L.) seed oil extraction with pressurized n-propane
and supercritical carbon dioxide, The Journal of Supercritical Fluids (2018),
https://doi.org/10.1016/j.supflu.2018.09.004

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 Hemp (Cannabis sativa L.) seed oil extraction with pressurized n-

propane and supercritical carbon dioxide

Daniel Ribeiro Grijó a, Gryele Karen Piva a, Ignacio Vieitez Osorio b, Lúcio Cardozo-

Filho a,b,*

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a
Department of Chemical Engineering, Universidade Estadual de Maringá, Maringá,

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PR, Brazil

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b
Departamento de Ciencia y Tecnología de los Alimentos, Facultad de Química,

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Universidad de la República, Montevideo, Uruguay
c

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Center for Research, Centro Universitário da Fundação de Ensino Octávio Bastos
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(UNIFEOB), São João da Boa Vista, SP, Brazil
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*
Corresponding author: lucio.cardozo@gmail.com
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Graphical abstract
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 45 Hemp dehulled seeds
+
36 Pressurized fluid
(scCO2 or n-Propane)

27
Yield (%)

18 40 ºC and 30 MPa
40 ºC and 40 MPa
9 60 ºC and 30 MPa

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60 ºC and 40 MPa
0

IP
0 18 36 54 72 Hemp Seeds Oil
q (g of CO2 /g dehulled seeds) (free organic solvent)

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SC
45

36

27 U
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18
40 ºC and 6 MPa
40 ºC and 10 MPa
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9 60 ºC and 6 MPa
60 ºC and 10 MPa
50 ºC and 8 MPa
0
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0.0 0.7 1.4 2.1

q (g of n-propane/g of dehulled seeds)
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Highlights

• Extraction of oil from hemp seed using scCO2 and pressurized n-propane with

high yield;
• High antioxidant contents in oil extracted using pressurized n-propane;

• New experimental data of phase transition to the system n-propane (1) / hemp seed

oil (2) were measured;

• Hemp seed oil with ratio of 6/3 between 2.5 and 5.0 and solvent free, ratio

ideal for the consumption of fatty acids.

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Abstract

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Hemp (Cannabis sativa L.) seeds are composed of a high oil content rich in

polyunsaturated fatty acids and antioxidants such as tocopherols and carotenes, resulting

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in an interesting nutritional aspect. Oil from hemp seeds extracted using supercritical
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carbon dioxide (scCO2) as the solvent preserves the qualities described above. However,
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the high operating pressures and the low solubility of the oil in scCO2 significantly
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increase operating costs. Generally, the use of pressurized n-propane as a solvent for
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extraction of vegetable oils is an alternative to overcome these challenges without

changing their nutritional qualities. The main objective of this work was to study the
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extraction of the oil from dehulled hemp seeds using scCO2, pressurized n-propane and

organic solvents at atmosphere conditions, to compare some physicochemical properties

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of the extracts. The extraction conditions using scCO2 as the solvent were 40 and 60 °C

and 30 and 40 MPa. These operating conditions of extraction are similar to those available
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in the literature. For pressurized n-propane used as a solvent, temperatures of 40, 50 and

60 ºC and pressures of 6, 8 and 10 MPa were used. The values of the extraction pressures

with n-propane were defined from the knowledge of the phase behavior measured for the

system n-propane (1) / hemp seed oil (2). The values of the mass percentage yields were
similar for both pressurized solvents. However, the solubility of the oil in n-propane was

higher than in scCO2. The kinetics of the extractions were satisfactorily using the Sovová

modeling.

Keywords: Hemp seeds; Pressurized n-propane; Supercritical carbon dioxide; Mass

transfer model; Phase equilibria.

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1. Introduction

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Hemp varieties are cultivars of the species Cannabis sativa L. [1] with low levels

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of the psychoactive substance 9-tetrahydrocannabinol (9-THC) [2]. The seeds of hemp

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are completely free from 9-THC [3] and can be used for the production of food [4],

cosmetics [5], biodiesel [6] and polymers [7].

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The literature showed that the ideal ratio for the consumption of fatty acids omega-
A
6 (6) and omega-3 (3) must be between 2.5 and 5.0 [8]. Hemp seeds have a high
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content of linoleic (6) and -linolenic (3) fatty acids. A study comparing fatty acid

contents in oilseeds presents 6 and 3 ratio values of 2.5 and 2.7 for two different
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varieties of hemp [9]. Hemp seeds also have high levels of natural antioxidants such as
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tocopherols and carotenes [10].

The oil of hemp seeds extracted with supercritical carbon dioxide (scCO 2) has
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physical and nutritional properties superior to those of such extracted with organic
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solvents at atmosphere conditions (conventional technique). However, the extraction

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pressures are too high to obtain comparable mass yields to those obtained with organic

solvents [10]. The extraction of vegetable oils using pressurized n-propane is an

alternative to reduce the extraction pressure and to preserve the physical and nutritional

properties of the oil. The critical pressure and temperature values of n-propane are,
respectively, 4.25 MPa and 96.70 °C, and of carbon dioxide are 7.38 MPa and 31.06 °C

[11].

The purpose of this work was to evaluate the various physicochemical properties

of hemp seed oil obtained using supercritical carbon dioxide (scCO2), pressurized n-

propane and conventional technique. The chemical profile of the fatty acids, β-carotene

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and total tocopherols of the extracts were determined by both solvents. The Sovová model

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[12] was applied to model the experimental kinetic curves of extraction. The phase

transition pressures for the system n-propane (1) / hemp seed oil (2) were determined

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using the synthetic visual method for the temperatures and mass fractions used in the

extraction.

2. Materials and methods U

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2.1. Chemicals
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Table 1 presents some information on the chemicals used in this work. All

chemicals were used without further treatment. Purities were provided by the
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manufacturers.
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2.2. Plant material

Hemp seeds were purchased from the company Indigo Herbs (United Kingdom)
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in the dehulled form. The seeds were milled using a manual crusher due to the high lipid
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of the dehulled hemp seeds. The density of the dehulled seed was determined in triplicate
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using a pycnometer (Quantachrome Instruments, 440-C Stainless Steel) of helium gas at

20 psi.
2.3. Percentage composition

The percentage composition of the seeds was analyzed in lipids, crude fiber,

proteins, water, ashes and carbohydrates by difference. All measurements were

performed in triplicate.

2.3.1. Lipids

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The lipid content was determined using a mixture of hexane and isopropanol (3:

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2) according to the methodology proposed by Hara & Norman [13]. Organic solvents

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mixture used have low toxicity [13] and solubility similar to the commonly used n-hexane

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[14]. Initially, about 1 g of milled sample was weighed into a tube, and 20 mL of the

solvent mixture was added. Extraction was performed at room temperature (20 ºC) with

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vigorous magnetic stirring for 1 h. The resulting extract was centrifuged (Hermle, model
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Z 200A) at 3000 rpm for 15 minutes. The specific rotor diameter of the centrifuge is 12
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cm so the equivalent G force is 1250. The supernatant was transferred to a new test tube.
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To the remaining original solution was added a further 5 mL of the solvent mixture. The

described procedure was repeated until the oil of the initial solid sample was depleted.
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The solvent mixture of the accumulated supernatants was removed using a gentle nitrogen
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flow at 40 °C until the weight of the extract remained constant.

2.3.2. Crude fiber

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The crude fiber percentage of the dehulled and defatted seeds was determined

using an AOCS Ba 6a-05 technique [15]. Briefly, the crude fiber percentage of the
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samples was determined by conducting an acid digestion (aqueous solution of 1.25%

sulfuric acid), followed by a basic digestion (aqueous solution of 1.25% sodium

hydroxide) and calcination at 105 ºC for 4 h.

2.3.3. Proteins

Protein analyses were performed by Kjeldahl method [16] using a nitrogen distiller

(Tecnal, model TE 036/1). The percentage of protein in the sample was calculated using

the protein conversion factor of 6.25.

2.3.4. Moisture

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The percentage moisture content of the milled dehulled seeds was determined using

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the official method 925.40 of AOAC [17]. Approximately 2 g of samples were dried in

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an oven at 105 °C until constant weight.

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2.3.5. Ashes

The ash content was determined using the official method 923.03 of AOAC [17].

Approximately 3 g of samples were incinerated directly into a muffle furnace (Jung 2011,

model 0712) at 550 °C for 8 h.

2.4. Extractions with pressurized fluids

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Seed oil extractions were conducted using high purity carbon dioxide (CO2) and

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n-propane. The experimental extraction apparatus is shown in Fig. 1. Basically, the

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experimental apparatus has a fluid pressurizing system with a syringe type pump (Isco,

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model 260D), a stainless-steel extractor with a capacity of 58 cm3 (1.95 cm of internal

diameter and 19.4 cm of high), thermostatic baths to promote refrigeration of the

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pressurized fluid in the syringe pump (Julabo, model F25-ME) and in the extraction vessel
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(Quimis, model Q214M2). The depressurizing is performed by a micrometric valve with
A
heating to allow the control of the flow and to minimize the Joule and Thomson effect
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[18].

The operational conditions of seed oil extraction using scCO2 as solvent were 40
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and 60 ºC for the temperature, 30 and 40 MPa for the pressure and 1.15 mL/min for the
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volumetric flow. Already, for the n-propane solvent the operating conditions were 40, 50

and 60 ºC for the temperature, 6, 8 and 10 MPa for the pressure and 2.45 mL/min for the
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volumetric flow. The operational conditions of extraction using scCO2 were selected from
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the work of [10]. However, the operating conditions of extraction using n-propane as

solvent were selected from phase transition measurements for the system n-propane (1) /
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hemp seed oil (2), at temperatures 40, 50, 60 ºC and mass fractions of n-propane from 0.6

to 0.8. The phase transition pressures observed under these conditions were lower than

the pressures employed in the extraction process, it is indicating that the operating

conditions of extraction were conducted in the region of only one phase. The dehulled
and milled seed masses used for the each determination of experimental kinetic extraction

curves were 4.0 g for scCO2 and 16.5 g for n-propane. The extractor volume was filled

with glass beads for extractions with scCO2. The period of each run of extraction process

took 240 min to the scCO2 and 30 min to the n-propane.

2.4.1 Mathematical modeling

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The modeling of the experimental data of the oil extractions of the hemp seeds

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was carried out using the model of Sovová [12]. This model considers that the oil is

divided into a fraction of easy access and a fraction of harder access. The easily accessible

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fraction of oil is related to the convective mass transfer mechanism and is therefore

obtained at a Constant Extraction Rate (CER). The mass extracted as a function of time

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𝑚(𝑡) for 𝑡  𝑡𝐶𝐸𝑅 (Eq. (1)) is related to the dimensionless factor Z (Eq. (2)):
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𝑚(𝑡) = ṁ𝐹 • 𝑌𝑆 • 𝑡 • [1 − exp(−𝑍)] (1)
A
𝐾𝐹 • 𝑚𝑆 • 𝜌𝐹
𝑍= (2)
ṁ𝐹 • (1 − 𝜀𝐵𝐸𝐷 ) • 𝜌𝑆
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where ṁ𝐹 is the mass flow rate of the solvent, 𝑌𝑆 is the solubility of the oil in the solvent,
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𝑚𝑆 is the sample mass on an oil-free basis, 𝑞0 is the ratio of the mass of available oil to

the sample mass on an oil-free basis, 𝜌𝐹 and 𝜌𝑆 are the fluid and solid densities
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respectively, 𝐾𝐹 is the coefficient of mass transfer by convection and 𝜀𝐵𝐸𝐷 is the porosity
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of the bed.
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The extraction of the oil fraction of harder access is controlled by the diffusion

mechanism in the solid and causes a Fall in Extraction Rate (FER). The mass extracted
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as a function of time 𝑚(𝑡) for 𝑡𝐶𝐸𝑅  t  𝑡𝐹𝐸𝑅 (Eq. (3))Error! Reference source not

found. is related to the dimensionless factors Z and W (Eq. (4)):

𝑍 ∙ 𝑌𝑆 1 𝑊 • ṁ𝐹
𝑚(𝑡) = ṁ𝐹 • 𝑌𝑆 • {𝑡 − 𝑡𝐶𝐸𝑅 • exp { • ln [ [exp ( ) • (𝑡 − 𝑡𝐶𝐸𝑅 ) − 𝑟]] − 𝑍}} (3)
𝑊 ∙ 𝑞0 (1 − 𝑟) 𝑚𝑆
 𝐾𝑆 • 𝑚𝑆
𝑊= (4)
ṁ𝐹 • (1 − 𝜀𝐵𝐸𝐷 )

where 𝐾𝑆 is the diffusion mass transfer coefficient and 𝑟 corresponds to the fraction of

available oil in harder access.

The porosity of the bed was obtained using Eq. (5).

𝑚 𝑚𝑆
𝜌𝐵𝐸𝐷 (𝑉 𝑆 ) [
(ℎ. 𝜋. 𝐷 2 )⁄4
]
𝐵𝐸𝐷
𝜀𝐵𝐸𝐷 =1− =1− =1− (5)

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𝜌𝑆 𝜌𝑆 𝜌𝑆

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where 𝜌𝐵𝐸𝐷 is the density of bed, 𝑉𝐵𝐸𝐷 is the effective volume of bed, ℎ is the effective

height of bed and 𝐷 is diameter of bed.

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The mass extracted as a function of time 𝑚(𝑡) for t ≥ 𝑡𝐿𝐸𝑅 (Eq. (6)) will occur

only by diffusion. In this case there is a Low Extraction Rate (LER).

𝑚(𝑡) = 𝑚𝑆 {𝑞0 −
𝑌𝑆
• ln {1 + [exp (
𝑊 • 𝑞0
) − 1] • exp ( U
𝑊 • ṁ𝐹 • (𝑡𝐶𝐸𝑅 − 𝑡)
) • 𝑟}} (6)
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𝑊 𝑌𝑆 𝑚𝑆
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The periods delimiting the extraction modeling are described in Eqs. (7), (8).
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(1 − 𝑟) • 𝑚𝑆 • 𝑞0
𝑡𝐶𝐸𝑅 = (7)
𝑌𝑆 • 𝑍 • ṁ𝐹
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𝑚𝑆 𝑊 • 𝑞0
𝑡𝐹𝐸𝑅 = 𝑡𝐶𝐸𝑅 + • ln [𝑟 + (1 − 𝑟) • exp ( )] (8)
𝑊 • ṁ𝐹 𝑌𝑆

The parameters 𝑟, 𝑍 and 𝑊 were calculated by minimizing the objective function

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given by Eq. (9):

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𝑛 𝑒𝑥𝑝 𝑁
𝑐𝑎𝑙𝑐 𝑒𝑥𝑝 2
𝑂𝐹 = ∑ ∑(𝑚𝑖,𝑗 − 𝑚𝑖,𝑗 ) (9)
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𝑖=1 𝑗=1

where 𝑛 𝑒𝑥𝑝 is the number of experiments performed, 𝑁 is the number of experimental

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𝑐𝑎𝑙𝑐 𝑒𝑥𝑝
data of each experiment, 𝑚𝑖,𝑗 and 𝑚𝑖,𝑗 are the calculated and experimental masses,

respectively.

2.5. Phase equilibria

Samples of the oil extracted with n-propane under 50 ºC and 8.0 MPa were used
to evaluate the phase transitions for the system n-propane (1) / hemp seed oil (2) in the

mass fractions of n-propane of 0.6, 0.7 and 0.8. The values of the selected mass fractions

are close to the mass ratios used in the extraction system. The experiments were

performed in triplicate using the static synthetic method with visual determination of the

phase transition points. Full details of the experimental procedure for the determination

of phase transition points are described in a previous work of our research group [19]. In

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Fig. 2 is shown a simple scheme of the apparatus used in these experiments.

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2.6. Physicochemical properties of extracts

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The physicochemical properties like as fatty acid composition, acid index and total

tocopherols and β-carotene concentration, were evaluated for each extraction condition.

2.6.1 Fatty acids U

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The extracted triglycerides were previously derivatized to the corresponding
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methyl esters according to the IUPAC method 2.301 [20]. Analysis of the esters formed
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was performed using a gas chromatograph equipped (Shimadzu GC-2010) with a flame
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ionization detector (FID) and Supelco SP 2330 capillary column (30 m × 0.25 mm × 0.2

μm). High purity nitrogen at 70 kPa was used as the carrier gas with a split ratio at 1:80.
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The initial oven temperature was 160 °C followed by heating at 4 °C/min to 230 °C and

then maintained at constant temperature for 10 min. The injector and detector
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temperatures were maintained at 260 and 340 °C respectively. A mixture of standard

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methyl esters was used to identify the respective fatty acids.

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2.6.2 Total tocopherols

The total tocopherols (, ,  and ) were quantified by HPLC using as mobile

phase acetonitrile, methanol, isopropyl and water under conditions described in a

previous work [21]. The samples diluted in isopropyl were quantified in a chromatograph
(Shimadizu 20A HPLC) coupled to a fluorescence detector (Shimadzu RF 20A XS) with

excitation and emission wavelength at 290 and 330 nm, respectively. A Macherey-Nagel

C18 (250 × 4.6 mm, 100 μm) column packed at 40 °C was used.

2.5.3 -carotene

The concentrations of β-carotene in the extracts were determined using a UV Vis

spectrophotometer (Thermo Fisher Scientific, Genesys 10-S) at a wavelength of 450 nm.

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The calibration curve was obtained with standard β-carotene solutions diluted in n-hexane

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at concentrations of 1–100 mg/L.

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2.6.4 Acidity index

The acidity index was determined using the official method 940.28 of AOAC [17].

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The results were expressed in milligrams of potassium hydroxide per milligram of the oil
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(mg KOH / mg oil). The analyses were conducted in triplicate using approximately 200
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mg of each extracted oil.
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3. Results and discussions

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3.1. Percentage composition

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Table 2 shows the percentage composition data described on the packaging of the

product purchased and those determined in this work.

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In general, the typical nutritional compositions provided by the manufacturer and

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literature reports [22] are comparable with the values determined in this work.
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3.2. Extraction kinetics

Fig. 3 shows the experimental and calculated kinetic curves for the extractions of

the dehulled hemp seeds using the scCO2 and pressurized n-propane for the operating

conditions used. It is noted that the percentage yields using both solvents were closed.
The use of dehulled seeds increased the percentage mass yields obtained under same

scCO2 conditions described in the literature [10]. The percentage mass yield obtained

with pressurized fluids (CO2 and n-propane) was lower than the percentage yields

obtained with conventional technique (Table 2).

Table 3 shows the operating conditions used to determine the experimental

extraction curves and the calculated parameters for the Sovová model. All different

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experimental conditions investigated were fit well by Sovová model with low values of

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the AARD (%) (Fig. 3). The value of the dehulled seed density measured experimentally

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was 1.88 ± 0.50 g/mL.

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From the data in Table 3, it can be observed, in general, for both solvents, high

yield values were obtained from the high values of pressure, density (𝜌𝐹 ), solubility (𝑌𝑆 )
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N
and convective mass transfer coefficient (𝐾𝐹 ). It can be seen that temperature did not have
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a significant influence on the extraction yield, at a constant pressure. The same result was

literature reported [10]. The absolute values of 𝐾𝐹 and 𝐾𝑆 indicate that the diffusional
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mechanism is fewer representative than the convective mechanism for both solvents used
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on pressurized extractions from dehulled seeds. The 𝐾𝐹 and 𝐾𝑆 values here obtained have

the same order of magnitude of the literature reports to extraction process of vegetable oil
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using pressurized fluid (scCO2 and n-propane) [23,24]. The extracts obtained with

pressurized n-propane presented 𝑌𝑆 and 𝐾𝑆 higher than those obtained using scCO2.
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Therefore, smaller amounts of n-propane were required to obtain similar values of

percentage mass yields. In Fig. 3, the representation form of the kinetic curves of
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extraction in dimensionless unit indicates this result. The dimensionless values of q (g of

solvent / g of dehulled seeds) employed to compare yields using the pressurized solvents

represent better the effects of the bulk density, flow rate, and time extraction. Scale-up

process concept gets more ease and quickly.

3.3. Phase equilibria

Table 4 shows the composition, temperature, pressure at the phase transition and

equilibria type measured for the system n-propane (1) / hemp seed oil (2). Due to the

complexity of the system different types of phase transitions were observed for the studied

conditions.

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The values of the phase transition pressures of the system n-propane (1) / hemp

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seed oil (2) presented a behavior similar to those reported for the system n-propane (1) /

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soybean oil (2) under the same temperature and mass fraction conditions [25]. Both oils

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have linoleic acid (55 %) as the main component but the oil of hemp seeds has a higher

content of polyunsaturated fatty acids [9]. As shown in Fig. 4, the liquid-liquid (LL),

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liquid-liquid-vapor (LLV) and liquid-vapor (LV) phase transition pressures for hemp seed
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oil were lower than for soybean oil. The complex behavior of phase transition of the
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system, for the studied conditions, suggests that the multiphase region should be avoided.
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The experimental values of solubility obtained by static method are higher than
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the values of solubility calculated using the Sovová model showed in Table 3. The static

methods have a great difficulty achieving equilibrium. The density of the supercritical
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solvent-rich phase is higher than the density of the solute-rich liquid phase thus leading

to different solubility values among dynamic and static methods applied [18].
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3.4. Physicochemical properties of extracts

The comparative analysis of the composition of fatty acids, total tocopherols and -
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carotene, as well as the acid index, of extracts obtained with conventional technique,

scCO2 and pressurized n-propane is presented in Table 5.

 Duncan's statistical tests showed that no significant variation was observed in the

composition of fatty acids with the different forms of extraction used. On average, 8.8 %

saturated, 13.3 % mono-unsaturated and 76.0 % polyunsaturated contents were observed.

The 6/3 ratio values were similar for both solvents for all operating conditions used.

Values of 6/3 determined, approximately 3, indicate a good nutritional index [8] and

are close to those reported in the literature [9]. The extracts obtained with pressurized n-

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propane presented higher contents of total tocopherols and -carotene, as well as lower

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acidity indexes. The exact amount of total tocopherol and -carotene in the sample can

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be obtained by multiplying the extraction yield and the specific concentration. Therefore,

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it is a promising alternative to obtain the oil at mild conditions of temperature and pressure

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without losing quality. The extractions under constant pressure for both pressurized
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solvents presented higher total tocopherols contents using high extraction temperatures.
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Conclusion
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The composition and yield of dehulled hemp seed oil obtained in this study with
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supercritical CO2 and conventional technique (mixture of hexane and isopropanol at

atmospheric conditions) were similar to results of reported in literature [10]. However,

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higher total tocopherol contents in dehulled hemp oil were obtained in this work. The
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typical nutritional compositions of various hemp seeds products, literature reported [24],
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show that the dehulled seeds total tocopherol content is highest than on whole seeds. The

nutritional index of the fatty acids and the percentage mass yield were similar for both
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solvents used in the extraction of the oil from the hemp seeds. However, pressurized n-

propane presented some advantages over supercritical carbon dioxide to obtain the oil of

the hemp seeds. Lower extraction pressures and solvent amounts were required using

pressurized n-propane as the solvent. This indicates operating costs lower than those with
supercritical carbon dioxide. In addition, the extracts obtained with pressurized n-propane

presented higher concentrations of antioxidants and lower acidity and humidity indexes,

it showed to be a high quality food product. Therefore, from the results obtained it is

concluded that pressurized n-propane is an interesting alternative solvent to obtain the oil

of hemp seeds in relation to supercritical carbon dioxide and conventional technique. The

phase behavior of the n-propane and oil system is complex due to the high asymmetry of

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the system. The knowledge of phase behavior got it possible to avoid the regions of

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multiphase mass transfer in the conduction of the extraction experiments. The mass

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transfer model proposed enabled the fit the experimental extractions kinetic curves for

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both employed solvents with low values of the AARD (%).

Acknowledgement
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We thank the Coordination of Improvement of Higher Education Personnel
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(CAPES-Brazil) for the granting of scholarships.

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References
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[1] C. Linnaei, Species Plantarum, first ed., Holmiae, Stockholm, 1753.

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[2] E.P.M. de Meijer, H.J. van der Kamp, F.A. Van Eeuwijk, Characterisation of

Cannabis accessions with regard to cannabinoid content in relation to other plant

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characters, Euphytica 62 (1992) 187–200. https://doi.org/10.1007/BF00041753.

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E PT
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Figure captions

Fig. 1. Experimental extraction apparatus: cylinder of solvent (C), globe valve (Vg), dual
A

syringe pump (P), pump control (X), thermostatic bath with cooled circulator (TB-1),

automatic valve for solvent inlet (V1), automatic valve for solvent outlet (V2), manometer

(M), extractor vessel (E), ball valve 90 degree (Vb), micrometric valve (Vm), resistance

(R), micrometer valve thermometer (T) and heated thermostatic bath (TB-2).
Fig. 2. Phase equilibrium apparatus: cylinder of solvent (C), globe valve (Vg), syringe

pump (P), pump control (X), thermostatic bath with cooled circulator (TB-1), pump inlet

valve (V1), pump outlet valve (V2), pressure sensor (PS), pressure gauge (PG),

depressurizing valve (Vd), equilibria cell (E), rear valve (Vr), front valve (Vf), resistance

under controlled (R), forcer (F, piston), front sapphire window (Sf), perpendicular

sapphire window (Sp), magnetic stirrer (MS), thermocouple (T), syringe (S).

T
IP
Fig. 3. Kinetic curves of extractions: □ 1 (40 ºC; 30 MPa for scCO2 and 6 MPa for n-

propane), ■ 2 (40 ºC; 40 MPa for scCO2 and 10 MPa for n-propane), ▲ 3 (60 ºC; 30 MPa

R
for scCO2 and 6 MPa for n-propane), △ 4 (60 ºC; 40 MPa for scCO2 and 10 MPa for n-

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propane), ○ 5 (50 ºC; 8 MPa), models, maximum yield obtained with

organic solvents by conventional extraction method.

U
N
Fig.4. Phase equilibria data of the system n-propane (1) / hemp seed oil (2): (60 ºC –
A
LL), (60 ºC – LLV), (50 ºC – LV) and (40 ºC – LV).
M

Fig. 1
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E PT
CC
A
Fig. 2

T
R IP
SC
U
N
Fig. 3
A

52 52
M

39 39
ED
Yield (%)

26 26
PT

13 13
E
CC

0 0
0 18 36 54 72 0.0 0.7 1.4 2.1
q q
(g of CO2 / g of dehulled seeds) (g of n-propane / g of dehulled seeds)
A
Fig. 4

3.0

2.5

Pressure (MPa)
2.0

1.5

T
IP
1.0
0.5 0.6 0.7 0.8 0.9

R
Mass fraction of n-propane (w1)

SC
U
N
A
M
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E PT
CC
A
Table 1. Chemicals employed in this work.

IUPAC Molecular Molar mass Supplier Minimum

Chemical
Nomenclature formula (g∙mol-1) (Country) purity (%)
Linde
n-Propane Propane C3H8 44.10 99.5
(Brazil)
Linde
Carbon dioxide Carbon dioxide CO2 44.01 99.95
(Brazil)
White Martins 99.999
Helium Helium He 4.00
(Brazil)
Linde
Nitrogen Nitrogen N2 28.01 99.995

T
(Uruguay)
Sigma Aldrich

IP
Hexane Hexane C3H8O 86.18 99.9
(Uruguay)
Sigma
Isopropyl 2-Propanol C3H14 60.10 99.9
(Uruguay)

R
Sigma Aldrich
Methanol Methanol CH4O 32.04 99.9
(Uruguay)

SC
Sigma
Acetonitrile Acetonitrile C2H6N 41.05 99.5
(Uruguay)
Sartorius Ultra-
Water Water H2O 18.02

Sulfuric Acid Sulfuric Acid H2SO4 U 98.08

(Uruguay)
Alphatec
(Brazil)
pure

95
N
Alphatec
Sodium hydroxide Sodium hydroxide NaOH 40.00 98
(Brazil)
A
†
C16:0 ME hexadecanoate C17H34O2 270.46
†
C16:17 ME hexadec-9(Z)-enoate C17H32O2 268.44
M

†
C18:0 ME octadecanoate C19H38O2 298.51
†
C18:19 ME octadec-9(Z)-enoate C19H36O2 296.50 Sigma Aldrich
†
Mixture
C18:2 ME octadeca-9(Z),12(Z)-dienoate C19H34O2 294.48 (Uruguay)
ED

†
C18:33 ME octadeca-9(Z),12(Z),15(Z)-trienoate C19H32O2 292.46
† (Z)
C20:19 ME icos-11 -enoate C21H40O2 324.55
† (Z)
C22:19 ME docos-13 -enoate C23H4O2 352.60
-Tocopherol 2(R),5,7,8-tetramethyl- ‡
PT

C29H50O2 430.72 96.0

-Tocopherol (R)
2 ,5,8-trimethyl- ‡
C28H48O2 416.69 Sigma Aldrich -
-Tocopherol 2(R),7,8-trimethyl- ‡ C28H48O2 416.69 (Uruguay) -
-Tocopherol (R) ‡
E

2 ,8-dimethyl- C27H46O2 402.66 -

1,3,3-trimethyl-2-[( ) - - § ⊥
CC

tetramethyl-18-(2,6,6-
Sigma Aldrich
-carotene trimethylcyclohexen-1-yl) C40H56 536.89 97.0
(Brazil)
octadeca-1,3,5,7,9,11,13,15,17-
nonaenyl] ║
†
Methyl
A

‡
2-[4(R),8(R),12-trimethyltridecyl]-3,4-dihydrochromen-6-ol
§ ⊥ ║
= 1(E),3(E),5(E),7(E),9(E),11(E),13(E),15(E),17(E) ; = 3,7,12,16 ; = cyclohexene
Table 2. Percentage composition

Analysis * Product (%) This work (%)

Lipids 49.1 52.0 ± 0.4
Crude fiber - 2.7 ± 0.8
Moisture - 5.4 ± 0.0
Ashes - 4.9 ± 0.2
Proteins 31.1 29.4 ± 1.0
Carbohydrates 4.1 5.7 ± 2.6
*Organic Hulled Hemp Seeds, www.indigo-herbs.co.uk (accessed 6 June 2018)

T
R IP
SC
U
N
A
M
ED
E PT
CC
A
 Table 3. Operational conditions of the experimental kinetic curves and parameters

calculated for the Sovová model.

scCO2 Pressurized n-propane

Run 1 2 3 4 1 2 3 4 5
𝑇 (ºC) 40 40 60 60 40 40 60 60 50
𝑃 (MPa) 30 40 30 40 6 10 6 10 8
𝜌𝐹 (g•mL ) -1
0.911 0.956 0.830 0.890 0.484 0.496 0.451 0.467 0.476

T
ṁ𝐹 (g•min-1) 2.393 2.315 2.310 2.234 1.480 1.053 1.019 1.098 1.232
𝑌𝑆 (g•g-1) 1.5•10-2 2.0•10-2 1.3•10-2 1.9•10-2 3.0•10-1 7.1•10-1 6.3•10-1 5.7•10-1 5.1•10-1

IP
𝐾𝐹 (min-1) 3.1•10-2 9.4•10-2 6.2•10-2 3.9•10-2 4.8•10-1 1.8 2.9•10-1 3.0•10-1 1.5•10-1
𝐾𝑆 (min-1) 3.7•10-4 5.4•10-4 3.4•10-4 6.9•10-4 2.5•10-2 3.7•10-2 2.6•10-2 3.7•10-2 2.9•10-2

R
Yield (%) † 37.35 40.88 37.05 43.13 32.67 37.07 33.85 37.81 35.42

SC
‡
𝐴𝐴𝑅𝐷 (%) 7.32 1.11 2.74 1.79 5.13 0.90 1.12 3.10 2.11
†
𝑌𝑖𝑒𝑙𝑑 (%) = [𝑔 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑜𝑖𝑙 ⁄𝑔 𝑜𝑓 𝑖𝑛𝑖𝑐𝑖𝑎𝑙 𝑑𝑒ℎ𝑢𝑙𝑙𝑒𝑑 𝑠𝑒𝑒𝑑𝑠 ] • 100
100 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑦𝑖𝑒𝑙𝑑 𝑖𝑛 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑡𝑖𝑚𝑒− 𝑐𝑎𝑙𝑐𝑢𝑙𝑒𝑑 𝑦𝑖𝑒𝑙𝑑 𝑖𝑛 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑡𝑖𝑚𝑒 2
‡
𝐴𝐴𝑅𝐷 (%) = • ∑( )
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑛𝑎𝑙𝑖𝑠𝑖𝑠 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑦𝑖𝑒𝑙𝑑 𝑖𝑛 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑡𝑖𝑚𝑒

U
N
A
M
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CC
A
Table 4. Phase equilibria data of the system n-propane (1) / hemp seed oil (2).

TSET (ºC) TEXP (ºC) PEXP (MPa) Equilibria Type

w1 = 0.6
40 40.43 ± 0.45 1.35 ± 0.03 LV
50 50.23 ± 0.57 1.69 ± 0.02 LV
60 61.47 ± 0.23 2.36 ± 0.02 LLV
60 61.57 ± 0.15 2.55 ± 0.03 LL
w1 = 0.7

T
40 40.57 ± 0.15 1.31 ± 0.00 LV
50 50.17 ± 0.06 1.64 ± 0.01 LV

IP
60 60.27 ± 0.47 2.18 ± 0.01 LLV
60 59.90 ± 0.42 2.54 ± 0.03 LL

R
w1 = 0.8
40 40.47 ± 0.51 1.23 ± 0.03 LV

SC
50 50.13 ± 0.15 1.53 ± 0.03 LV
60 60.63 ± 0.15 2.05 ± 0.03 LLV
60 60.70 ± 0.10 2.15 ± 0.02 LL

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N
A
M
ED
E PT
CC
A
 R I
27

SC
Table 5. Physicochemical properties of the extracts obtained. Analyzes were done in duplicate with p 0.05 according to Duncan’s post-hoc test.

U
*
Physico- Conventional Supercritical carbon dioxide Pressurized n-propane

N
chemical organic 40 ºC 40 ºC 60 ºC 60 ºC 40 ºC 40 ºC 60 ºC 60 ºC 50 ºC
Properties solvents 30 MPa 40 MPa 30 MPa 40 MPa 6 MPa 10 MPa 6 MPa 10 MPa 8 MPa

A
a a a a a a a a b b
C16:0 (%) 6.15 ± 0.07 6.25 ± 0.03 6.14 ± 0.08 6.20 ± 0.04 6.28 ± 0.07 6.09 ± 0.01 6.07 ± 0.03 6.08 ± 0.01 5.76 ± 0.11 5.85 ± 0.07
C16:1 7 (%) a
0.09 ± 0.00 a
0.09 ± 0.01 a
0.09 ± 0.00 a
0.09 ± 0.00 a
0.10 ± 0.01 a
0.11 ± 0.02 a
0.11 ± 0.00 a
0.11 ± 0.00 a
0.11 ± 0.02 a
0.10 ± 0.01

M
b b b b b a a a a a
C18:0 (%) 2.60 ± 0.03 2.52 ± 0.02 2.61 ± 0.03 2.53 ± 0.03 2.58 ± 0.02 2.83 ± 0.03 2.83 ± 0.05 2.85 ± 0.04 2.82 ± 0.03 2.82 ± 0.05
C18:1 9 (%) b
12.75 ± 0.03 c
12.54 ± 0.04 c
12.64 ± 0.03 c
12.50 ± 0.02 c
12.59 ± 0.03 a
13.16 ± 0.05 a
13.03 ± 0.03 a
12.99 ± 0.06 a
12.96 ± 0.03 a
13.08 ± 0.02
C18:2 6 (%) a a a a a b b b b b

C18:3 3 (%)
ED a
57.84 ± 0.13
18.31 ± 0.04 a
57.98 ± 0.06
18.54 ± 0.05 a
57.97 ± 0.10
18.43 ± 0.04 a
57.99 ± 0.08
18.42 ± 0.06 a
57.64 ± 0.11
18.33 ± 0.04 a
57.11 ± 0.05
18.53 ± 0.03 a
57.08 ± 0.08
18.66 ± 0.09 a
56.99 ± 0.04
18.55 ± 0.05 a
57.26 ± 0.09
18.63 ± 0.07 a
57.17 ± 0.12
18.65 ± 0.08
C20:1 9 (%) a
0.31 ± 0.01 a
0.29 ± 0.00 a
0.31 ± 0.01 a
0.31 ± 0.03 a
0.31 ± 0.01 a
0.35 ± 0.02 a
0.35 ± 0.02 a
0.33 ± 0.04 a
0.37 ± 0.02 a
0.38 ± 0.01
C22:1 9 (%) b
0.02 ± 0.00 b
0.01 ± 0.00 b
0.02 ± 0.01 b
0.02 ± 0.00 b
0.02 ± 0.01 a
0.21 ± 0.01 a
0.23 ± 0.00 a
0.22 ± 0.01 a
0.24 ± 0.02 a
0.24 ± 0.01
PT

a a a a a a a a a a
Total fatty acids (%) 98.07 ± 0.23 98.22 ± 0.02 98.21 ± 0.19 98.06 ± 0.14 97.85 ± 0.18 98.39 ± 0.02 98.36 ± 0.08 98.12 ± 0.02 98.15 ± 0.15 98.29 ± 0.43
6/3 ratio a
3.16 ± 0.02 a
3.13 ± 0.01 a
3.15 ± 0.02 a
3.15 ± 0.02 a
3.14 ± 0.02 a
3.08 ± 0.01 b
3.06 ± 0.01 b
3.07 ± 0.01 b
3.07 ± 0.01 b
3.06 ± 0.03
E

Total tocopherols (mg • L-1) c

746.4 ± 0.8 e
237.8 ± 6.8 f
192.3 ± 5.7 b
877.3 ± 14.7 d
545.3 ± 21.0 a
935.5 ± 12.2 a
935.7 ± 2.5 a
950.6 ± 15.0 a
973.7 ± 8.9 a
952.7 ± 12.7
-carotene (mg • L )
-1 b
12.65 ± 0.23 d
7.0 ± 0.6 d
6.22 ± 0.21 d
6.89 ± 0.17 c
9.42 ± 0.18 a
16.84 ± 0.09 d
7.83 ± 0.22 d
7.36 ± 0.81 d
6.40 ± 0.03 d
6.42 ± 0.75
CC

mg KOH • mg oil -1 b
7.31 ± 0.44 b
7.4 ± 0.1 5.21 ± 0.08c b
7.13 ± 0.14 a
11.21 ± 0.28 c
4.95 ± 0.01 c
5.06 ± 0.00 c
5.00 ± 0.03 c
4.90 ± 0.17 d
4.57 ± 0.12
*
Mixture of hexane and isopropanol (3:2 v/v) at atmosphere conditions.
A