J. Ocean Univ.

China (Oceanic and Coastal Sea Research)

DOI 10.1007/s11802-012-1948-0
ISSN 1672-5182, 2012 11 (4): 562-568
http://www.ouc.edu.cn/xbywb/
E-mail: xbywb@ouc.edu.cn

Extraction of Astaxanthin from Euphausia pacific Using

Subcritical 1, 1, 1, 2-tetrafluoroethane
HAN Yuqian*, MA Qinchuan, WANG Lan, and XUE Changhu
Department of Food Science and Engineering, Ocean University of China, Qingdao 266003, P. R. China
(Received March 2, 2012; revised April 7, 2012; accepted June 21, 2012)
Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2012
Abstract Euphausia pacific is an important source of natural astaxanthin. Studies were carried out to assess the extractability of
astaxanthin from E. pacific using subcritical 1, 1, 1, 2-tetrafluoroethane (R134a). To examine the effects of multiple process variables
on the extraction yield, astaxanthin was extracted under various conditions of pressure (30 150 bar), temperature (303 343 K), time
(10 50 min), flow rate (2 10 g min1), moisture content (5.5% 63.61%), and particle size (0.25 0.109 mm). The results showed
that the extraction yield increased with temperature, pressure, time and flow rate, but decreased with moisture content and particle
size. A maximum yield of 87.74% was obtained under conditions of 100 bar, 333 K, and 30 min with a flow rate of 6 g min1 and a
moisture content of 5.5%. The substantial astaxanthin yield obtained under low-pressure conditions demonstrates that subcritical
R134a is a good alternative to CO2 for extraction of astaxanthin from E. pacific.
Key words

subcritical R134a; astaxanthin extraction; Euphausia pacific

1 Introduction
Euphausia pacific is an euphausiid of the subphylum
Arthropoda Crustacean, which is widely distributed in the
Pacific Ocean, Yellow Sea and the adjacent areas of Taiwan Strait. Due to the small individual size, E. pacific is
commonly used as an animal feed and a food additive.
As an astaxanthin-producing creature, E. pacific is
thought to contain high levels of astaxanthin. Astaxanthin
(3, 3'-dihydroxy-, -carotene-4, 4'-dione) is one of the
most effective carotenoids, with antioxidant activity 10
and 500 times higher than that of any other carotenoids
and vitamin E, respectively (Shimidzu et al., 1996). Astaxanthin is mainly used as a dyeing agent in the diets of
aquaculture of salmon and other fish species, as well as in
the cosmetic and pharmaceutical industries (Higuera-Ciapara et al., 2006).
Conventional astaxanthin isolation methods based on
solvent extraction from natural matrices are considered
time-consuming, expensive and potentially hazardous,
which often involve multiple extraction steps and require
a large amount of organic solvents (Rockville, 1992).
Another disadvantage of solvent extraction is that the
stability of astaxanthin generally decreases due to oxidation if antioxidants are not used. In addition, more legal
regulations have been enforced to restrict the use of toxic
organic solvents, leading to an increasing number of
* Corresponding author. Tel/Fax: 0086-532-82031629
E-mail: hanyuqian@ouc.edu.cn

studies which aim to find a simple, fast, and efficient alternative method for extracting astaxanthin from natural
matrices (Lpez et al., 2004; Careri et al., 2001).
The extraction method currently in use employs supercritical carbon dioxide (scCO2), which is inert, nontoxic,
nonflammable, inexpensive and ideal for the application
in food industry. However, this method typically requires
a high pressure (up to 300 bar) for satisfactory yield of
astaxanthin extraction (Nobre et al., 2006). Considering
the economic and environmental needs, it is necessary to
explore an alternative supercritical fluid extraction (SFE)
solvent to enable the operation under less intense conditions.
Due to gradual destruction of the ozone layer, people
have been looking for an alternative to the refrigerant
Freon-12 (R12). The chemical 1, 1, 1, 2-tetrafluoroethane
(R134a) has been identified as a potential replacement of
R12 as it is a non-ozone-depleting substance with a low
toxicity and no flammability. Together the increasing
commercial availability, permanent dipole moment (2.05 D)
and reasonable critical properties (374.25 K, 4.06 MPa) of
R134a have led to an evaluation of its use as an alternative to scCO2 for extraction of polar analytes (Corr, 2002).
Normally, R134a is gaseous and liquefies under relatively
low pressures. It is ideal for continuous extraction processes, as the target material can rapidly be separated via
depressurisation of the solvent. With a modest pressure
and low operating temperature, continuous depressurisation / pressurisation cycles need no substantial energy,
thus reducing the operating cost and associated greenhouse gas emissions (Lapkin et al., 2006).

HAN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (4): 562-568

Recent studies regarding the equilibrium properties of

R134a have demonstrated that R134a is a thermodynamically and physically ideal solvent for extraction. The
supercritical properties of R134a, supercritical mixture of
CO2 and R134a and the vapour-liquid equilibrium of the
CO2-R134a binary mixture were studied (Abbott and
Eardly, 1998Abbott et al., 1999). It is found that a
mixture of 30 mol% R134a in CO2 is extremely useful for
extraction purposes. Roth (1996) has indicated that in the
high-pressure and low compressibility region, the solvating powers of six refrigerants (HFC: R32, R125, R134a,
and R152a; HCFC: R22 and R123) are greater than that
of CO2. A high extraction efficiency can be achieved under low pressure with subcritical fluid extraction technology, which overcomes the shortcomings of SFE. Consequently, the subcritical fluid extraction method has
gained substantial attention and frequently been studied.
R134a has become the first choice of subcritical fluid, as
it is easy to reach the critical conditions due to the desired
properties such as low toxicity, inertness, non-flammability and zero ozone depression potential (ODP). Currently, research of R134a largely focuses on its application in extraction of valuable substances, such as artemisinin, palm oil, and lipid (Lapkin et al., 2006;
Mustapa et al., 2009; Simes and Catchpole, 2002).
This study evaluated the use of R134a as a potential
alternative to CO2 for subcritical extraction of astaxanthin
from E. pacific. The objective was to investigate the effects of multiple process variables, such as time, pressure,

563

temperature, R134a flow rate, particle size and moisture

content, on the extraction yield of astaxanthin.

2 Materials and Methods

2.1 Materials
E. pacific captured from the East China Sea was supplied by Daishan Tongqu Aquatic Food Co., Ltd (Shanghai, China). The crustacean was lyophilised at 213 K for
48 h in a freeze-drier with a moisture content of 5.5%.
The lyophilised tissues were mechanically ground to obtain a homogeneous powder.
E. pacific tissue samples were freeze-dried, and, depending on the duration of the drying period, different
moisture contents could be achieved. The lyophilised E.
pacific was ground mechanically, and the particle size of
samples was controlled by grinding time and the rotating
speed of the crusher. To prevent degradation, the samples
were stored at room temperature in a sealed aluminum
bag before use.
A cylinder of liquefied R134a with a purity of 99.9%
was purchased from Xinjie Refrigeration Equipment Co.,
Ltd. (Qingdao, China), and petroleum ether was purchased
from Tianjin Siyou Co., Ltd. (HPLC grade, Tianjin, China).
2.2 Instruments and Apparatus
The schematic diagram of the apparatus used for subcritical R134a extraction is shown in Fig.1. This home-

Fig.1 Schematic diagram of the R134a extraction apparatus. A, R134a cylinder; B, filter; C, cooler; D, high pressure
pump; E, heat exchanger; F, extraction cell; G, heating bath; H, back pressure regulator; I, collecto; and J, rotameter.

made device mainly consisted of a high-pressure pump

with a maximum pressure of 350 bar and a R134a flow
rate of approximately 0.4 L h1 (Hangzhou Zhijiang Petrochemical Equipment Co., Ltd.). The apparatus was composed of a 50 cm3 extraction vessel and a separator with a
capacity of 50 cm3, operating under pressures up to 300 bar.
Liquid R134a solvent was pressurised by a high pressure
metering pump with jacketed heads for cooling. The flow
rate was adjustable between 0 and 0.4 L h1. A cooling
system was used for condensation of R134a, and two
heating baths used for controlling the temperature of the
jacketed extractor and separator. The pressure in the extractor and the separator was regulated using back pres-

sure valves.

2.3 Solvent Extraction of Astaxanthin

Soxhlet extraction was performed to determine the total
amount of extractable astaxanthin in E. pacific. The astaxanthin was extracted repeatedly from 1.00 g of sample
using 20 mL of dichloromethane, and the extraction was
terminated after 3 h or when the orange colour of the
sample began to fade. The solvent extract was filtered
through a 0.45-m nylon membrane filter, and then
washed with a 4% (w : w) sodium chloride solution, with
the dichloromethane layer separated and evaporated to
dryness. The residue was dissolved in petroleum ether (bp

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313 333 K) to 50 mL and vortexed for 30 s. The absorbance of the appropriately diluted extract was measured at
468 nm using a Spectronic 21 spectrophotometer (SaCarotenoid yield (g astaxanthin / sample) =

where A is the absorbance, V is the volume of extract, 0.2

is the A468 of 1 g mL1 of standard astaxanthin, and W is
the weight of sample in grams (Simpson and Haard,
1985). Results are presented as the mean values of triplicate measurements.

2.4 Astaxanthin Extraction Using Subcritical Fluid

Astaxanthin extraction was carried out in the laboratory-scale apparatus (Fig.1) using subcritical R134a. In
each experiment, approximately 1.00 g of ground Euphausia pacific was loaded to the center of the extraction
cell. The bottom and top of the extraction thimble were
filled with glass wool to prevent the entrainment of material and the blocking of the cap vessel holes. To avoid
fluid flowing through the vessel without adequately penetrating the sample matrix, the sample was packed moderately tight in the vessel. It should be noted that sample
overpacking can cause vessel blockage, restricting the
fluid flow rate and reducing the fluid permeability.
The extraction cell was placed in a heating bath, which
was used to control the operating temperature within 1 K
of the set point temperature for each run. To test its effects on the extraction rate, R134a was delivered by a
metering pump with a maximum pressure of 350 bar. The
system pressure was controlled by a back-pressure regulator, and the R134a flow rate regulated by the metering
pump. To improve the extraction efficiency, a static period of 30 min was included to allow the contact between
sample and subcritical R134a. The stream of subcritical
R134a fluid containing astaxanthin was then depressurised through a pressure restrictor, and the extracts were
collected with petroleum ether. The R134a flow rate was
measured by channelling it into a mass gas flow meter.
The extract was diluted to 50 mL, and its absorbance was
measured at 468 nm using a Spectronic 21 spectrophotometer. The extraction rate of astaxanthin was calculated
using the following formula:
Extraction rate (%) =

M1
100 ,
M0

chindra et al., 2006, 2007). The yield of carotenoids was

calculated as the amount of astaxanthin using the following formula:
A468 nm Vextract Dilution factor
,
0.2 Wsample

(1)

size and solvent flow rate, are shown in Figs.27.

3.1 Effects of Operating Temperature and Pressure

The effects of pressure on astaxanthin extraction under
constant conditions of temperature (333 K), time (30 min),
moisture content (5.5%) and R134a solvent flow rate
(10.0 g min1) are shown in Fig.2. Results showed that the
extraction pressure significantly affected the extraction
rate, with the latter increasing with the former when other
process parameters remained constant. The highest and
lowest astaxanthin extraction rates were obtained under a
high pressure of 120 bar and the lowest pressure of 30 bar,
respectively. These observations could be explained by
the principles of the polarisability parameter (*) established by Kamlet (1977) and Laurence (1994), who demonstrated the relationship between the solvent properties
and *. Abbott and Eardly (1998) measured the parameter
* for R134a as a function of temperature and pressure to
cover the liquid and supercritical states. The * value in
the liquid region increases roughly linearly with pressure
in the liquid state and decreases dramatically at temperatures above 473 K and pressures below 60 bar (Abbott and
Eardly, 1998). This indicates that the increases in pressure
elevate the * value of the R134a solvent, improving its
solvating power. As the density of the solvent increases,
the intermolecular interactions of solutes become more
intense. Consequently, astaxanthin and solvent dissolution can be promoted, thereby increasing the extraction
rate.

(2)

where M1 is the mass of astaxanthin extracted by subcritical R134a, and M0 is the mass of total astaxanthin
extracted by organic solvent. Results are presented as the
mean values of triplicate experiments.

3 Results and Discussion

The total amount of astaxanthin extracted from Euphausia pacific by the Soxhlet method was 189.37 g g1.
The effects of process parameters, such as the extraction
pressure, temperature, time, moisture content, particle

Fig.2 The extraction rate under various pressures and constant conditions of temperature (333 K), time (30 min),
flow rate (10 g min1) and moisture content (5.5 Wt%).

The increase in astaxanthin-solvent dissolution with

pressure during the solubility-dependent stage was much
slower compared to the increase in the diffusivity-dependent stage. The influence of pressure above 100 bar was
not significant on astaxanthin extraction, consistent with
previous findings in -carotene extraction (Sabio et al.,
2003). When the pressure was higher than 120 bar, the
extraction rate slightly decreased. Further increase in
pressure had a small effect on the solubility of astaxan-

HAN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (4): 562-568

thin. This was likely due to the fluid distant from a subcritical state under a high extraction pressure. Such a
change would influence the extraction ability of subcritical R134a. In addition, the viscosity of subcritical R134a
increased with pressure, thus would prevent the spreading
of solute to the fluid, decreasing the extraction rate.
The influence of temperature on astaxanthin extraction
rate is shown in Fig.3. The extraction was performed under constant conditions of time (30 min), moisture content
(5.5%), flow rate (10.0 g min1), and an optimised pressure of 100 bar. The astaxanthin extraction rate was found
increasing with temperature under a constant pressure,
consistent with the theory proposed by Machmudah et al.
(2006a) and in agreement with previous findings regarding astaxanthin extraction using scCO2 (Lpez et al.,
2004; Nobre et al., 2006; Qu et al., 2004).

Fig.3 The extraction rate under constant conditions of

pressure (100 bar), time (30 min), flow rate (10 g min1)
and moisture content (5.5 Wt%) at various temperatures.

The solubility of the analyte depends on a complex

balance between the subcritical fluid density and the solute vapour pressure, both of which are controlled by fluid
temperature and pressure. For volatile solutes, there is
competition between their solubility and volatility in
R134a, which respectively decreases and increases with
temperature. Fig.3 shows that the extraction rate increased with temperature. As the increase in temperature
reduced the fluid density while elevating the solute vapour pressure, we suggest that the solubility of astaxanthin largely depends on the solute vapour pressure (Roy et
al., 1996). The astaxanthin extraction rate was dependent
on temperature from the beginning of the extraction. An
apparent increase was observed in the extraction rate
when temperature varied from 303 to 323 K, and associated astaxanthin extraction rates were 52.30% and 73.42%,
respectively. However, when the temperature continuously increased to 333 K, the extraction rate only slightly
increased by 6%. The maximum extraction rate obtained
at a higher temperature can be attributed to the improvement in astaxanthin dissolution due to the increased mass
transfer rates caused by temperature increases, as suggested by Brunner (1994). Despite the low pressure tension of astaxanthin, the relative change in temperature
and the corresponding change in solubility can be large.
Therefore, temperature increase elevated the mass transfer rate and the vapour pressure of solutes, increasing the
astaxanthin extraction rate.
Of note is that the extraction rate decreased with temperature increases at above 333 K. This could be attributed to the main factor of subcritical fluid density, which

565

influenced the solubility of the solute at high temperatures. The increase in temperature reduced the density of
the fluid, thus decreasing the solubility of the R134a solvent. In addition, high temperatures could accelerate the
oxidation of astaxanthin, further decreasing the astaxanthin ex- traction rate. Using shrimp waste as the raw material for astaxanthin extraction, Zhang et al. (2008)
found that below 333 K, the temperature effect on astaxanthin oxidation was not significant. However, the oxidation of astaxanthin was accelerated at a temperature
above 333 K, and the astaxanthin was completely destroyed when temperature was raised to 353 K.

3.2 Effect of Time

It is important to maximise the contact of subcritical
R134a with the sample for improving the extraction efficiency. Variables that influence the contact of solvent
with samples include extraction time, flow rate, and extraction mode (static with no follow-through or dynamic
with follow-through) (Pourmortazavi and Hajimirsadeghi,
2007). Stahl et al. (1988) reported that a 10 to 20-min
static extraction prior to dynamic extraction improved the
extract recovery in SFE extraction. In the present study,
we performed a 30-min static extraction prior to the dynamic extraction. The extraction time varied between 10 to
50 min under a constant condition of pressure (100 bar), temperature (333 K), moisture content (5.5%) and flow rate (6
g min1). As shown in Fig.4, the extraction rate slightly
increased with extraction time and remained at the same
level with extended extraction time. The best extraction
recovery was obtained at 30 min, indicating that half an
hour was sufficient for the diffusion of astaxanthin to the
solvent stream. The increase in the extraction time improved the recovery of astaxanthin as it promoted the
contact of R134a molecules with astaxanthin, thus accelerating the intermolecular interaction between R134a and
the solute, further improving the solute dissolution. In
addition, extended extraction time increased the amount
of R134a for a more complete extraction. Therefore, the
yield of astaxanthin extract was improved.

Fig.4 The extraction rate at various extraction times under

constant conditions of pressure (100 bar), temperature (333
K), flow rate (10 g min1) and moisture content (5.5 Wt%).

3.3 Effect of R134a Flow Rate

As shown in Fig.5, the extraction rate increased with
the R134a flow rate within the range of 2.0 to 6.0 g min1,
with the extraction rate at a flow rate of 6 g min1. This

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increase could be attributed to the increase in solvent

penetrating the particle cell structure. In addition, the increasing flow rate could decrease the mass transfer resistance and cause the solvent to leave the extractor under
saturated conditions, eventually achieving the maximum
extraction rate (Saldana et al., 2002). However, the extraction rate only slightly increased with the R134a flow
rate at a higher consumption level (Fig.5).

high moisture content, water would separate from R134a

and form a water film on the material surface, impeding
the diffusion of solute to subcritical R134a. Consequently,
a two-phase system is created (Lagalante et al., 1998;
Jackson and Fulton, 1996). Therefore, increasing the
moisture content would influence the state of subcritical
R134a. In addition, the sample tended to clump and agglomerate at high moisture content levels, preventing
R134a from penetrating into the sample.

Fig.5 The extraction rate at various R134a flow rates under constant conditions of pressure (100 bar), temperature
(333 K), time (30 min), and moisture content (5.5 Wt%).

Fig.6 The extraction rate at various moisture content levels under constant conditions of pressure (100 bar), temperature (333 K), time (30 min), and flow rate (6 g min1).

The increased R134a flow rate not only shortened the

resistance time, but also promoted the contact between
R134a molecules and astaxanthin. This could accelerate
the intermolecular interactions between R134a and the
solutes, further improving the solute dissolution. In addition, the elevation of R134a flow rate increased the number of solvent molecules per unit volume in the extractor.
Under this condition, mass transfer was influenced by the
increase in R134a flow rate, and the intra-particle diffusion resistance became dominant (Machmudah et al.,
2006b).
The effect of flow rate on astaxanthin extraction was
two-fold. An increase in the flow rate promoted the mass
transfer while reducing the contact time. Consequently,
the extraction efficiency was dependent on the compensation between the two consequences. Fig.5 demonstrates a
lack of increase in the extraction efficiency by an increase
in flow rate to 10 g min1. In this case, increasing the flow
rate reduced the residence time for the solvent to reach
saturation, preventing the equilibrium at a higher mass
transfer rate (Mira et al., 1999). In addition, the excessive
solvent bypassed the extractable solutes instead of penetrating the solid matrix cellular structure and dissolving
the solute (Pourmortazavi and Hajimirsadeghi, 2007).

As shown in Fig.6, the extraction rate was 59.62%

when the moisture content was 32.79%. A higher extraction rate of 69.86% was obtained with lower moisture
content of 24.77%. Within these moisture content levels,
the sample was easily dispersed. Therefore, subcritical
R134a could easily penetrate into the sample and increase
the extraction rate. When the moisture content reached
63.61%, the extraction rate was substantially low (6.7%).
In addition, the sample could easily agglomerate at this
moisture content level and influence the mass transfer.
Many researchers have studied astaxanthin extraction
using scCO2 (Lpez et al., 2004; Thana et al., 2008) and
their samples were often dried tissues due to the low extraction efficiencies resulted from the high moisture content. With subcritical R134a, high extraction efficiency
was obtained at a moderate moisture level, indicating the
higher efficiency of subcritical R134a than that of scCO2
in extraction of astaxanthin from biological materials. By
stirring subcritical R134a and the sample during extraction, the contact between R134a and solute could be enhanced, further destroying the water film. Therefore, we
assumed that the extraction efficiency increased with
moisture content.

3.5 Effect of Particle Size

3.4 Effect of Moisture Content
As shown in Fig.6, the astaxanthin extraction rate decreased with increasing moisture content. With relatively
high moisture content, only a very small amount of
astaxanthin was extracted. In addition, astaxanthin was
extracted from fresh E. pacific with very high moisture
content (79.21%), but the extraction efficiency was extremely low (2.27%). The low extraction efficiency could
be attributed to the weak interaction between R134a and
water molecules (Selvam et al., 2006; Li et al., 2000).
R134a has a limited capacity of dissolving water. With

As shown in Fig.7, higher extraction efficiencies were

obtained by applying smaller particle sizes, which controlled the mass transfer kinetics and the access of R134a
to astaxanthin (Nagy and Simndi, 2008).
Smaller particle sizes increase the mass transfer surface
and the amount of soluble fraction on the surface. With
larger sizes, the contact surface contributed by all particles decreases and the diffusion distance increases, resulting in a decreased mass transfer rate. A smaller particle size can expand the mass transfer surface and the
quantity of the soluble fraction on the sample surface.

HAN et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (4): 562-568

However, if the material particle size is too small, the

particles will easily stack together, decreasing the mass
transfer rate. In addition, excessive grinding may hinder
the extraction due to re-adsorption of analytes onto matrix
surfaces and pressure drops inside the extraction chamber
(Pourmortazavi and Hajimirsadeghi, 2007), resulting in
the lower extraction rate at 0.109 mm than at 0.120 mm.

Fig.7 The extraction rate at different particle size levels

under constant conditions of pressure (100 bar), temperature (333 K), time (30 min), flow rate (6 g min1) and moisture content (5.5 Wt%).

4 Conclusions
This study investigated the extraction of astaxanthin
from E. pacific using subcritical R134a. The highest
astaxanthin extraction rate (87.74%) was obtained under
optimized conditions of pressure (100 bar), temperature
(333 K), time (30 min), flow rate (6 g min1) and moisture
content (5.5%). Without using large amounts of organic
solvent, the subcritical fluid extraction produced better
results compared to Soxhlet extraction. Higher extraction
efficiency was obtained at a moderate moisture level,
indicating that subcritical R134a is applicable for materials with relatively high moisture contents. Due to the
modest pressures, low operating temperatures and high
permanent dipole moment (2.05 D), R134a is ideal for
extracting many other valuable substances, broadening its
range of applications.

Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.31071541).

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(Edited by Wei Liuzhi)