Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: https://www.tandfonline.com/loi/lanl20

A Green Deep Eutectic Solvent-Based Ultrasound-

Assisted Method to Extract Astaxanthin from
Shrimp Byproducts

Heng Zhang , Baokun Tang & Kyung Ho Row

To cite this article: Heng Zhang , Baokun Tang & Kyung Ho Row (2014) A Green Deep Eutectic
Solvent-Based Ultrasound-Assisted Method to Extract Astaxanthin from Shrimp Byproducts,
Analytical Letters, 47:5, 742-749, DOI: 10.1080/00032719.2013.855783

To link to this article: https://doi.org/10.1080/00032719.2013.855783

Accepted author version posted online: 01

Feb 2014.
Published online: 10 Mar 2014.

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Analytical Letters, 47: 742–749, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 0003-2719 print/1532-236X online
DOI: 10.1080/00032719.2013.855783

Preconcentration Techniques

A GREEN DEEP EUTECTIC SOLVENT-BASED

ULTRASOUND-ASSISTED METHOD TO EXTRACT
ASTAXANTHIN FROM SHRIMP BYPRODUCTS

Heng Zhang, Baokun Tang, and Kyung Ho Row

Department of Chemistry and Chemical Engineering, Inha University,
Incheon, Korea

The extraction of valuable compounds from waste products using green and inexpensive
solvents is a significant strategy for sample preparation. Accordingly, in the present
study, the use of deep eutectic solvents, an emerging green approach, was used to
extract astaxanthin, a well-known and widely-used antioxidant, from shrimp byproducts.
After evaluating different combinations of extraction conditions and deep eutectic
solvents, an ultrasonication method was established and optimized by a systematic
investigation of the influencing factors. A comparison of the amount of astaxanthin
(102
g g−1  extracted using a traditional organic solvent, ethanol, showed that more
astaxanthin (146
g g−1  was obtained using the reported eco-friendly method. The
excellent properties of deep eutectic solvents highlight their advantages as promising
inexpensive green solvents for the extraction and determination of a range of bioactive
compounds from natural products.

Keywords: Astaxanthin; Deep eutectic solvents; Extraction; Shrimp waste

INTRODUCTION
Astaxanthin, as a remarkable scavenger of singlet oxygen, is the strongest
antioxidant among the carotenoids. These antioxidant properties, associated with
the ability to cross the blood-brain barrier, highlight its potential for protecting
against several diseases, such as immune-system disorders, cardiovascular issues, and
cancer (Holanda and Netto 2006).
Almost all the astaxanthin on the market is produced synthetically using
complex steps, making it quite expensive. On the other hand, synthetic astaxanthin
contains a mixture of stereoisomers and is easier to oxidize than the natural
product (Boussiba, Cohen, and Richmond 2000). Astaxanthin is found in large
quantities in natural sources, mainly in crustaceans, salmonids, yeast, alga, and

Received 20 August 2013; accepted 5 October 2013.

Address correspondence to Kyung Ho Row, Department of Chemistry and Chemical
Engineering, Inha University, Incheon 402-751, Korea. E-mail: rowkho@inha.ac.kr
Color versions of one or more of the figures in the article can be found online at www.
tandfonline.com/lanl.

742
 DEEP EUTECTIC SOLVENT EXTRACTION FOR ASTAXANTHIN 743

microalga (López, Arce, and Garrido 2004), and hence there is interest in extracting
it from raw materials. A number of methods have been developed to extract
astaxanthin from natural sources, such as fermentation (Ni, Chen, and Ruan 2007),
organic solvent extraction (Sanches, Ribeiro, and Albuquerque 2013), supercritical
fluid extraction (Fujii 2012), and ionic liquid based solid-phase extraction using
a molecularly imprinted polymer sorbent (Bi et al. 2010). All these methods were
designed to be more convenient and efficient. From a green chemistry point of
view, supercritical fluid and ionic liquid-assisted extraction have been used to extract
astaxanthin efficiently. Nevertheless, the high cost of these methods has hindered
their application and development.
A new type of ionic solvent, called a deep eutectic solvent, has addressed
the disadvantages of ionic liquids that include high cost and toxicity. A deep
eutectic solvent is formed by mixing two low toxicity components. In general, one
of the components used in deep eutectic solvent is an inexpensive, biodegradable,
and nontoxic quaternary ammonium salt called choline chloride. Choline chloride
forms a deep eutectic solvent with another component that includes hydrogen
bond containing functional groups (carboxylic acids, urea, or polyols) (Bi, Tian,
and Row 2013). In addition to making contributions to electrochemistry, catalysis,
material chemistry, and organic synthesis, deep eutectic solvents also have potential
for extraction (Zhang De Oliveira Vigier, and Royer 2012; Oliveira, Pereiro, and
Luís 2013). Considering the chemical structure and properties of astaxanthin, deep
eutectic solvents with a lower polarity are preferred. Therefore, deep eutectic
solvents of composed of choline chloride and polyols were selected as the extraction
solvent.
A simple, effective, and green deep eutectic solvent-based ultrasound-assisted
method was proposed for the extraction of astaxanthin from shrimp byproducts.
Significant variables, such as the identity of the solvent, ratio of water to deep
eutectic solvent, ratio of sample to solvent, ultrasonic power, and time were
optimized systematically. Conditions were also optimized for the determination of
the analyte by high-performance liquid chromatography (HPLC).

EXPERIMENTAL
Chemicals
Astaxanthin (98%) was obtained from Sigma (St. Louis, MO, USA).
Choline chloride (>98.0%), ethylene glycol (>99.5%), glycerol (>99.0%), 1,2-
butanediol (>98.0%), 2,3-butanediol (>97.0%) 1,3-butanediol (>99.0%), 1,4-
butanediol (>99.0%) and 1,6-hexanediol (>97.0%) were purchased from Tokyo
Chemical Industry Co. Ltd. (Tokyo, Japan). Methanol, ethanol, acetonitrile, and
dichloromethane were supplied by Duksan Pure Chemical Co. Ltd. (Ansan, Korea).
All solvents used in the experiment were of HPLC or analytical grade. Distilled
water was vacuum filtered (HA-0.45, Division of Millipore, Waters, USA) and
(Division of Millipore, Waters, USA). All the samples were passed through a filter
(MFS-25, 0.2
m TF, Whatman, USA) before HPLC analysis.
Deep eutectic solvents were obtained by heating choline chloride and hydrogen
bond donors to 80
C with constant stirring until a homogeneous liquid was formed.
Table 1lists the abbreviations of the deep eutectic solvents produced.
744 H. ZHANG ET AL.

Table 1. Abbreviation of the studied deep eutectic solvents

Mole ratio of salt to

Abbreviation Salt Hydrogen bond donor hydrogen bond donor

Deep eutectic solvent-1 Choline chloride Ethyl glycol 1/2

Deep eutectic solvent-2 Glycerol
Deep eutectic solvent-3 1,2-Butanediol
Deep eutectic solvent-4 1,3-Butanediol
Deep eutectic solvent-5 1,4-Butanediol
Deep eutectic solvent-6 2,3-Butanediol
Deep eutectic solvent-7 1,6-Hexanediol

Extraction Procedure
Shrimp byproducts were collected from a local market, and washed, frozen,
dried, ground, and sieved. The shrimp powder was stored in a refrigerator. Shrimp
shell powder (0.1 g) was mixed with 1.0 mL of different deep eutectic solvents over
a range of solid/liquid ratios in sealed vials. The suspensions were processed by
ultrasonic extraction at a frequency of 20 kHz and an output power of 200 W max
(Mirae Ultrasonic Tech. Co., Bucheon, Korea). An amount of 200
L of the extract
were mixed with an equal volume of the mobile phase. After centrifugation, the
extracts were filtered (0.2 m) before being analyzed by HPLC. Each sample was
injected 3 times to evaluate the precision.

HPLC Instrumentation
The HPLC system consisted of an M930 solvent delivery pump (Young
Lin Co., Anyang, Korea), an ultraviolet detector (M 720 Absorbance Detector,
Young-In Scientific Co., Anyang, Korea), and an integrated data system
(Autochrowin version 1.42, Young Lin Co.). Injection valves with 20.0
L
sample loops were used. HPLC was performed using a commercial C18 column
(4.6 × 150 mm, 5
m) purchased from RStech Co. (Daejeon, Korea). The mobile
phase, dichloromethane/methanol/acetonitrile/water (5/85/5.5/4.5, v/v, was used
for isocratic elution at room temperature. The flow-rate, ultraviolet wavelength, and
injection volume were set to 0.5 mL/min, 476 nm, and 5.0
L, respectively.

RESULTS AND DISCUSSION

Selection of Deep Eutectic Solvent
The dissolution of the analyte in deep eutectic solvents is affected by
the types and ratios of the hydrogen bond donors. A comparison of three
extraction methods revealed ultrasonic extraction to be the most efficient.
The amount of astaxanthin obtained by ultrasonic extraction was 75
g g−1
(solid/liquid ratio = 1/10 g mL−1 , ultrasonic power = 85 W, time = 30 min), which
was higher than the amounts obtained by room temperature stirring (34
g g−1
with a solid/liquid ratio = 1/10 g mL−1 , temperature = 20
C, and time = 30 min)
and by extraction with heating (62
g g−1 , solid/liquid ratio = 1/10 g mL−1 ,
 DEEP EUTECTIC SOLVENT EXTRACTION FOR ASTAXANTHIN 745

Figure 1. Schematic diagram of extract preparation.

temperature = 60
C, time = 15 min). Moreover, after addition of the mobile phase
(dichloromethane/methanol/acetonitrile/water) to the extract, proteins were
precipitated to decrease the deep eutectic solvent viscosity prior to HPLC analysis
(Fig. 1).
Based on the results in Fig. 2A, the amount of astaxanthin extracted using
deep eutectic solvent system 3 was optimum compared to the other solvent systems.
The physicochemical properties, such as viscosity, surface tension, and polarity have
a significant effect on the extraction efficiency of deep eutectic solvents. The viscosity
and surface tension of deep eutectic solvent-3 were lower than the others except
for deep eutectic solvent-1 (Bi et al. 2013). The high viscosity of deep eutectic
solvents results in a lower mobility of free species, which can affect the extraction

Figure 2. Optimization of solvent conditions. (A) Choline chloride/ hydrogen bond donor ratio: the
choline chloride/ hydrogen bond donor ratio was 1/2, the sample/deep eutectic solvent ratio was
1/10, ultrasound time was 30 min, and ultrasonic power: 85 W. (B) Optimization of choline chloride/
hydrogen bond donor ratios: the deep eutectic solvent type was deep eutectic solvent-3, sample/deep
eutectic solvent ratio was 1/10, the ultrasound time was 30 min, and the ultrasonic power was 85 W.
(C) Optimization of water percentage: sample/deep eutectic solvent: 1/10, ultrasonic time: 30 min,
ultrasonic power: 85 W.
746 H. ZHANG ET AL.

efficiency because of the extensive hydrogen bonding between each component of

deep eutectic solvent (Pang, Hou, and Wu 2012). The polarity of deep eutectic
solvent-3 appears to be most favorable for astaxanthin.
In addition, different choline chloride/ hydrogen bond donor ratios (1:2, 1:3,
1:4, 1:5, 1:6) were also evaluated. As shown in Fig. 2B, the amount of astaxanthin
increased with decreasing ratio from 1:2 to 1:5 (mol mol−1 . Overall, a choline
chloride/ hydrogen bond donor ratio of 1:5 (mol mol−1  of deep eutectic solvent-3
was deemed to be optimal.

Effect of the Water Percentage in the Deep Eutectic Solvent

The water content in the deep eutectic solvent may contribute to a decrease
in viscosity, and hence the effect of the water percentage (0–20%) on the extraction
efficiency was investigated. Figure 2C shows that the extraction efficiency was better
at a concentration of 10% water. On the other hand, a higher water percentage
of the deep eutectic solvent increased the polarity and decreased the interaction
between the solvent and target. Therefore, the water/deep eutectic solvent ratio was
set to 10% for the further extraction procedures.

Effect of the Ultrasonication Time and Frequency

The ultrasonication time was optimized for the extraction. The amount of
astaxanthin extracted increased with ultrasonication time to 40 min (Fig. 3). On the
other hand, the increase in efficiency from 30 min to 40 min was negligible compared
to the energy cost, and hence 30 min was employed for economic consideration.
It was well known that the ultrasonic power is a driving force for developing
contact between the deep eutectic solvent and sample, which can affect the

Figure 3. The effect of ultrasound time on extracted amount of astaxanthin: choline chloride/hydrogen
bond donor ratio: 1:5, water percentage: 10%, sample/deep eutectic solvent: 1:10, ultrasonic power:
85 W.
 DEEP EUTECTIC SOLVENT EXTRACTION FOR ASTAXANTHIN 747

Figure 4. Effect of ultrasonic power on extracted amount of astaxanthin: choline chloride/hydrogen

bond donor ratio: 1:5, water percentage: 10%, sample/deep eutectic solvent: 1:10, ultrasonic time:
30 min.

performance of the solvent significantly. The sample size will be diminished by

solid disruption which leads to increasing the total solid surface in contact with the
solvent by the ultrasonication process. The importance of the ultrasonic frequency
has been shown in recent applications (Zhu et al. 2013; Tian et al. 2013). The
ultrasonication frequency was varied from 50 to 100 W to optimize the extraction
power (Fig. 4). The efficiency increased up to 70 W, after which it reached a constant
value. Therefore, an ultrasonic power of 70 W was selected as optimal.

Effect of Sample/Liquid Ratio

A range of sample/liquid ratios (1:5, 1:10, 1:15, 1:20, 1:25 g mL−1  were
employed to obtain an optimum value. Generally, larger solvent volumes adversely
affect the efficiency, decrease the economic feasibility, and create unnecessary
byproducts. As shown in Fig. 5, the extracted amount of astaxanthin increased
with solvent volume. However, a further increase in the sample/liquid ratio above
1/15 g mL−1 did not significantly affect the amount extracted. The highest extraction
efficiency was obtained when the sample/solvent ratio was set to 1/15 g mL−1 .
Under these conditions, the amount of analyte obtained was 146
g g−1 .

Method Analytical Figures of Merit

The linearity, precision, detection limits, and other characteristics for the
method were obtained under the optimized conditions. The linear dynamic range
was 10–50
g mL−1 , with a correlation coefficient of 0.994. The precision was
determined using five extractions of astaxanthin using deep eutectic solvent, which
results in a relative standard deviation (RSD) was 1.4%. Based on signal-to-
noise ratios of 3 and 10, the limit of detection (LOD) and limit of quantification
748 H. ZHANG ET AL.

Figure 5. Effect of ratio of sample and deep eutectic solvent on extracted amount of astaxanthin:
choline chloride/hydrogen bond donor ratio: 1:5, water percentage: 10%, ultrasonic time: 30 min,
ultrasonic power: 70 W.

(LOQ) for astaxanthin were 124 ng mL−1 and 410 ng mL−1 , respectively. The average
recoveries of astaxanthin were from 76% to 102% at three concentration levels.
This highlights the stability of the proposed method and its potential application
for natural products.

CONCLUSIONS
The application of deep eutectic solvents in the ultrasonic-assisted method was
developed to extract astaxanthin from shrimp byproducts. In this study, a choline
chloride/ hydrogen bond donor ratio of 1/5 (mol mol−1  with deep eutectic solvent-
3 was used with a sample/solvent ratio of 1/15 (g mL−1  in 30 min. The water
percentage in deep eutectic solvent-3 was set to 10%. The amount of astaxanthin
extracted in shrimp shells was 146
g g−1 . The amount of astaxanthin in the shrimp
heads was also examined; the amount reached a maximum value of 218
g g−1 .
Masses of 102
g g−1 and 158
g g−1 astaxanthin were extracted by ethanol from
shrimp shells and shrimp head, respectively. In contrast, the amount extracted by the
deep eutectic solvent increased to 146
g g−1 and 218
g g−1 , respectively. This new
method demonstrates the application of a deep eutectic solvent in an environmental-
friendly extraction process.

FUNDING
This study was a part of the project titled “Gyeonggi Sea Grant Program,”
funded by the Ministry of Oceans and Fisheries, Korean.
 DEEP EUTECTIC SOLVENT EXTRACTION FOR ASTAXANTHIN 749

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