Analytical Letters, 45: 26752686, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032719.2012.702179

Separations
ZINC ION DOPED SOLID-PHASE EXTRACTION OF
EICOSAPENTAENOIC ACID AND DOCOSAHEXAENOIC
ACID FROM ANTARCTIC KRILL
Baokun Tang, Minglei Tian, and Kyung Ho Row
Department of Chemical Engineering, Inha University, Incheon, Korea
Antarctic krill crude extracts contain high levels of eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA). Accordingly, the solid phase extraction of EPA and DHA
from Antarctic krill crude extracts has attracted signicant research interest. This study
compared the extraction of EPA and DHA from Antarctic krill crude extracts using an
aminopropyl, zinc ion-doped silica, and C18 and zinc ion-doped C18 solid-phase column.
The best extraction effect was obtained using the zinc ion-doped C18 SPE with water containing methanol as the eluant. The efciency increased gradually with increasing methanol
concentration from 12.5 to 25% in the washing stage, and when pure methanol (5.0 mL) or
acetonitrile (3.0 mL) was used as the eluant. To detect EPA and DHA, the acids were rst
converted to their methyl esters and detected by gas chromatography with ame ionization
detection (GCFID). In the zinc ion-doped C18 elution fractions, EPA and DHA were
isolated from the crude extracts in high yield (8591% (r2 4.86.3%)).
Keywords: Antarctic Krill; Aminopropyl; Docosahexaenoic acid; Eicosapentaenoic acid; Zinc ion doped
C18; Zinc ion doped silica

INTRODUCTION
Antarctic krill is a species of krill found in the Antarctic waters of the Southern
Ocean. Beginning with the discovery of Antarctic Krill, many researchers have paid
increasing attention to this potential source of food (Hamner et al. 1983; Ross and
Quetin 1986; Hempel and Hempel 1986). Estimates suggest that although the stock
of Antarctic krill in the Southern Ocean is difcult to determine accurately, the abundance of biomass is likely to be the highest of any multi-cellular animal species on
Earth (Nicol, James, and Pitcher 1987; Priddle et al. 1998). Reports in the 1980s suggested that this abundant source may allow yearly catches in the range of 50100 million tons, which was in the same order as the total catch of sh in the world at that
Received 27 April 2012; accepted 4 June 2012.
This study was a part of the project titled Korea Sea Grant Program (Gyeong-gi Sea Grant)
funded by the Ministry of Land, Transport, and Maritime Affairs, Korea.
Address correspondence to Kyung Ho Row, Department of Chemical Engineering, Inha University, Incheon 402-751, Korea. E-mail: rowkho@inha.ac.kr
2675

2676

B. TANG ET AL.

time (Ellingsen and Mohr 1979). In recent years, the annual sustainable capture from
Antarctic krill has increased to 70200 million tons (Suzuki and Shibata 1990). By
comparison, the total annual capture from all sheries has been approximately
130 million tons since 2000 (Gigliotti et al. 2011). This suggests that Antarctic krill
may be comparable to the biomass of all other aquatic species currently harvested.
Eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) are two
omega-3 polyunsaturated fatty acids (PUFA) that are essential to normal growth
and health. An adequate dietary intake of EPA and DHA is considered benecial
for alleviating a range of chronic diseases, such as cardiovascular, hypertensive,
inammatory and autoimmune disorders, depression and certain disrupted neurological functions (Simopoulos 1991, 1997; Dyerberg 1986; Mehta et al. 1988; Kinsella 1986; Shahidi and Wanasundara 1998; Mansour 2005). Fricke et al. (1984)
reported that free fatty acids (FFA) (8-16%) in the main lipid cases of Antarctic krill,
and EPA and DHA were identied as the major components of FFA (Fricke et al.
1984; Kolakowska, Kolakowski, and Szczygielski 1994).
The general chemical and biochemical composition of Antarctic krill has been
the subject of many studies (Ellingsen and Mohr 1979; Fricke et al. 1984; Kolakowska et al. 1994; Kolakowski 1986). A small number of comprehensive studies
examined PUFA but most used crude preparations and detection methods (Wilson
et al. 1993; Belarbi, Molina, and Chisti 2000; Fountoulaki et al. 2003; Giogios et al.
2009). Few studies of EPA and DHA in Antarctic krill have been reported. This may
be because Antarctic krill contains large quantities of saturated and unsaturated fatty
acids as well as other compounds, such as lipids, sterols, amino acids, alcohols, and
so forth (Fricke et al. 1984). Isolating EPA and DHA from these complicated compositions in a sample is quite difcult. Moreover, it is difcult to detect EPA and
DHA correctly and accurately among the many compounds in the samples. Some
researchers attempted a range of techniques to isolate and prepare PUFA from shellsh, sh oil and edible oils etc. samples. These include solid phase extraction,
reversed-phase high-performance liquid chromatography, silver-ion high-performance liquid chromatography, argentated silica gel chromatography, aminopropyl
solid phase extraction, and so forth (Mansour 2005; Wilson et al. 1993; Lacaze
et al. 2007; Adlof and List 2004; Guil-Guerrero, Campra-Madrid, and
Navarro-Juarez 2003). On the other hand, these processes involved the use of too
many processing operations that reduce the target recovery and magnication time.
Furthermore, HPLC may be better used to separate and detect the targets of PUFA
(Mehta et al. 1988; Adlof and List 2004) but it is an expensive technique that is not
easily scalable to obtain large quantities of puried targets. Therefore, there is no
simple and agreed method for further purifying PUFA from different samples.
More recently, solid-phase extraction (SPE) was applied to the isolation of lipid
classes by step-wise sequential elution. SPE with commercially available and autonomous sorbent cartridges was applied recently and offers a viable alternative to conventional sample preparation methods (Gelencser et al. 1995). Speed, reliability, low
cost, and the use of minimal amounts of solvent are the main advantages of SPE over
thin layer chromatography (TLC) and column chromatography (Giacometti, Milosevic, and Milin 2002). Different types of SPE packing, such as silica, C18, and bonded
aminopropyl-silica have been used to separate targets into classes (Giacometti et al.
2002; Poerschmann et al. 2006; Kaluzny et al. 1985). The SPE isolation of the PUFA

SOLID-PHASE EXTRACTION FROM ANTARCTIC KRILL

2677

fraction from lipid samples can be achieved using small volumes of solvent and with a
high degree of purity and high target recovery (Lacaze et al. 2007).
Normally, the PUFA in samples is rst transformed to methyl esters, and then
detected by capillary column gas chromatography with ame ionization detection
(GCFID) (Lacaze et al. 2007; Brondz 2002). The esterication of PUFA is essential
for accurate detection. The reaction conditions were optimized in another study
(Tang et al. 2012).
This report describes the use of different packing materials for isolating EPA and
DHA from Antarctic krill by solid phase extraction, as well as an efcient SPE method
to characterize the EPA and DHA composition in different Antarctic krill samples.
EXPERIMENTAL
Chemicals
Methanol, dichloromethane, hexane, acetonitrile (HPLC grade), and 95% sulfuric acid (extra pure) were supplied by Duksan Pure Chemical Co., Ltd. (Ansan, Korea).
Double distilled water was ltered through a vacuum pump (Waters, Milford, MA,
USA) and a lter (HA-0.45, Waters, Milford, MA, USA) prior to use in SPE washing
or elution. Methanol and acetonitrile were used in the SPE washing or elution stage,
and dichloromethane was utilized as the extractant in Soxhlet extraction. Methanol
and sulfuric acid were employed as the esterifying agent and catalyst, respectively,
for EPA and DHA esterication. There is a key sulfuric acid to methanol ratio (v=v)
in esterication (as described in a following section). Hexane was used to extract the
EPA and DHA methyl esters in the mixture solution after esterication. The C18
(15 mm, E. Merck, Germany), aminopropylsilyl (50 mm, Alltech, US), silica (25 mm,
YMC, Japan), and zinc sulfate (extra pure, Duksan pure chemical CO. LTD, Korea)
were used as the SPE solid phase in the packing cartridge.
The EPA (C20:5, analytical standard), DHA (C22:6, purity
98%) EPA methyl
ester (analytical standard), DHA methyl ester (analytical standard), and 3,5-di-tertbutyl-4-hydroxytoluene (BHT) (analytical standard, as anti-oxidant) were acquired
from SigmaAldrich, Co. and used to optimize the esterication process and quantitative analysis. The BHT (0.01%, based on PUFA or PUFA esters solution) was added to
all solutions, which included EPA, DHA, EPA methyl esters, and DHA methyl esters,
to prevent oxidation. Both EPA and DHA methyl ester standards were initially diluted
in acetonitrile to give a calibration stock mixture solution (both 5.0 mg=mL) depending
on the composition of the EPA and DHA methyl esters. The stock mixture solutions
and acetonitrile were combined to produce seven calibration solutions (ranging from
0.0001 to 1.0 mg=mL depending on the EPA and DHA methyl esters composition)
for quantitative analysis of the EPA and DHA methyl esters in each sample solution.
Standard EPA and DHA mixture solutions (both 1.0 mg=mL) based on acetonitrile
were used to optimize the esterication process.
Antarctic Krill Preparation
Whole fresh and dried Antarctic krill samples were supplied by Insung Food
and Biological Products Co., Ltd. (Seoul, Korea). The Antarctic krill was collected

2678

B. TANG ET AL.

from the Southern Ocean in July 2011 and processed by Insung Food and Biological
Products Co., Ltd. The fresh Antarctic krill measured 45 cm from head to tail and
the dried product measured 23 cm. The fresh and dried krill blocks were transported to the laboratory in heavily insulated industrial strength boxes lled with
dry ice. Upon arrival, the samples were removed from the boxes and stored immediately in a refrigerator until needed.
PUFA Extraction
The structure of one whole Antarctic krill includes mainly head and pleon
parts. In this study, the two parts of fresh or dried Antarctic krill were separated
using stainless tweezers and examined. The fresh or dried head and pleon samples
were extracted with a dichloromethane solvent using a Soxhlet extraction method
(AOAC 1998). After extraction, 0.01% BHT (v=v, based on the extraction solution)
for anti-oxidation of EPA and DHA were added to the solution. All the sample solutions were stored in a refrigerator.
Solid-Phase Extraction
The SPE was used to separate PUFA from a complicated mixture of compounds. The separation of EPA and DHA from the Antarctic krill crude extracts
was achieved using different stationary phases, such as C18, aminopropyl-silica, zinc
sulfate-silica, and zinc sulfate-C18. Each type of SPE cartridge was prepared using a
similar process as follows: 200 mg of adsorbent (20 mg zinc sulfate if needed) for column chromatography was added to methanol (20 mL). The mixture was agitated for
20 min and the slurry mixture was added to the cartridge (9 cm  0.9 cm i.d.). Methanol was pumped using a vacuum pump. The packed SPE cartridges were activated
in a dryer heated to 50 C, at which point they were removed from the dryer and
cooled to room temperature.
The SPE cartridges were conditioned with water (1.0 mL) and methanol
(1.0 mL), and each Antarctic krill crude extract (1.0 mL) was loaded onto a cartridge.
During SPE, all the crude extracts were the same as the Soxhlet extracts of fresh Antarctic krill pleon. Each SPE process is reported in the following section. The interferents were washed with watermethanol, and the PUFAs were eluted with
methanol or acetonitrile into a 5 mL glass tube. The contents of the tubes were esteried and detected as follows.
GC-FID Analysis
The optimization conditions for the esterication of EPA and DHA are
reported elsewhere. Therefore, the EPA and DHA from the SPE fractions were esteried directly under the optimal conditions. The EPA and DHA methyl esters standards as well as the fractions of the EPA and DHA esters obtained from the SPE
fractions were analyzed by GC=FID on a Yong Lin Instrument (Korea) GC-6100
with a DB-1701 capillary column (30 m  0.320 mm  1.00 mm) (Agilent Technologies) and detected using a FID detector. Ultra-high purity hydrogen (purity
99.999%) was used as the carrier gas with a ow rate of 1.80 mL=min. The oven

SOLID-PHASE EXTRACTION FROM ANTARCTIC KRILL

2679

temperature was programmed as follows: initial temperature of 140 C increasing to

220 C at 5 C=min, followed by a further increase to 280 C at 10 C =min, and held at
that temperature for 4 min. The total time for a single GC run was 26 min. The FID
and injector temperature was 300 C and 280 C, respectively. The injection was performed in split mode at a rate of 42:1.
RESULTS AND DISCUSSION
SPE of EPA and DHA From Antarctic Krill Extracts
SPE is adsorption chromatography using columns of different materials and is
useful for isolating target molecules from complicated mixtures, making them easy to
detect. The methods used to obtain fractions rich in PUFA are commonly based on
the differences in polarity and spatial conguration of the PUFA present in the crude
extracts. These differences are associated mainly with the number of double bonds in
the carbon chain. Therefore, PUFA can be extracted and separated according to
their degree of unsaturation. The results of the extraction of PUFA using different
solid phase columns were compared.
The crude extracts and fractions obtained by SPE were analyzed by gas chromatography to establish the recovery. The initial amount of EPA or DHA in the Antarctic
krill crude extract loading on SPE was detected and recorded. After the SPE process,
the amount of PUFA in the different fractions was also detected and recorded. The
recovery of EPA or DHA in SPE was calculated using the following equation:
Recovery %

Amount of target in SPE fraction

 100%
Amount of target in loading crude extract

In this paper, the capacity factor (k) of the target on each SPE fraction was calculated
using the equation described by Gelencser et al. (1995):
Capacity factor k

Amount of target adsorbed on the stationary phase

Amount of target in SPE fraction

In the aforementioned equation, the corresponding capacity factor of a target

was calculated when each of the SPE fractions was nished. Capacity factor means
changes in the target between the solid and mobile phases.

Aminopropyl Bonded Silica Column

The SPE isolation of the fatty acids fraction from the other lipid classes was
achieved using aminopropyl-silica cartridges (Wilson et al. 1993; Lacaze et al.
2007; Giacometti et al. 2002; Kaluzny et al. 1985). Therefore, the separation of
EPA and DHA from krill crude extracts was attempted to achieve bonded phase
aminopropyl-silica cartridges.
The crude extracts were applied to silica-aminopropyl columns, as listed in
Table 1. The recovery and capacity factor of EPA and of DHA in each SPE step
is also shown. In the loading step fraction, the amounts of EPA and DHA were

2680

B. TANG ET AL.

Table 1. Amounts, recovery, and capacity factor of EPA and DHA from the Antarctic krill extracts using
silica-aminopropyl SPE
Solvent

EPA

Methanol
Volume Amount Recovery
content (%, v:v) (mL)
(mg)
(%)
Loading
step
Washing
step
Elution
step
Total

DHA
Capacity
factor (k)

Amount Recovery
(mg)
(%)

Capacity
factor (k)

1.0

121.0

17.5

4.8

110.3

16.1

5.2

2.5
5
100

1.0
1.0
2.0

37.1
73.3
462

5.3
10.4
66.2

14.6
6.4

25.4
34.1
514

3.7
5.1
74.8

21.7
14.7

693

99.4

683

99.7

Note: means that the value is not in the range of determination.

121.0 and 110.3 mg, respectively, corresponding to 17.5% and 16.1% recovery in the
crude sample. The two targets in the crude extracts were not absorbed completely by
the silica-aminopropyl solid phase, and the amount of EPA absorbed was less than that
of DHA. After loading, 5.3% and 3.7% recovery of EPA and DHA, respectively, were
also washed with 1.0 mL of a methanol to water mixture (2.5%, v:v) was rst used to
wash the referents in the crude extracts. Although some referents were washed in this
fraction, a certain amount of the targets was also washed away. Therefore, the referents
and targets could not be separated well. Increasing the methanol content in the washing
solution to 5.0% resulted in more EPA and DHA being washed with the referents, as
listed in Table 1. One reason may be the bonding force between the column azyl and
low concentration of carboxyl groups. Accordingly, the targets combined with the
interferents were washed easily in the same washing fraction. In addition, the capacity
factors in the washing steps were small, meaning the targets interacted weakly with the
solid phase. Despite more referents being washed in this fraction, the targets were also
lost from the solid phase. Consequently, in the next elution fraction, only 66.2% and
74.8% of EPA and DHA, respectively, were obtained in the elution fraction. On the
other hand, the total recovery of EPA and DHA were both approximately 100%. This
compared favorably with the SPE method detailed by Kaluzny et al. (1985), where the
recovery of one C18:1 unsaturated fatty acid contained in a lipid calibration standard
was 101%. This was better than a similar SPE method reported by Lacaze et al. (2007))
where the recovery of EPA and DHA from shellsh extracts was 8490%. Although the
total recovery of EPA or DHA was high, the recovery in the elution fraction was not
ideal and the targets were not separated completely from the referents. Possibly, the disadvantage of the silica-aminopropyl SPE of EPA and DHA can be overcome by selecting an appropriate washing and elution solution system. The solution system might
require further research. This study attempted to change the solid phase to achieve
the research objective.
Zinc Ion Doped Silica Column
The use of Ag doped silica solid phase column extraction of PUFA had been
described (Belarbi et al. 2000; Guil-Guerrero et al. 2003; Ryu et al. 1997). The Ag

SOLID-PHASE EXTRACTION FROM ANTARCTIC KRILL

2681

Table 2. Amounts, recovery, and capacity factor of EPA and DHA from Antarctic krill extracts using
silica-zinc ion SPE
Solvent

EPA

Methanol
Volume Amount Recovery
content (%, v:v) (mL)
(mg)
(%)
Loading
step
Washing
step
Elution
step
Total

DHA
Capacity
factor (k)

Amount Recovery
(mg)
(%)

Capacity
factor (k)

1.0

131.4

19.5

4.1

112.3

16.6

5.0

2.5
5
100

1.0
1.0
2.0

83.2
170.6
292.4

11.8
24.7
41.9

5.8
1.8

71.3
153.7
292.6

10.5
23.6
42.9

6.9
2.1

677.6

97.9

629.9

93.6

Note: means that the value is not in the range of determination.

doped silica gel fractionation of PUFA is based on the bonding force between the
Ag blank outermost orbit and the olenic p electrons of PUFA. Considering that
Zn2 has a similar electron conguration to Ag, the Zn2 doped silica solid phase
was selected to isolate EPA and DHA from the krill crude extracts. The retention of
EPA and DHA by zinc ion chromatography, which was attributed primarily to the
interaction of Zn2 ions and the olenic p electrons of the sample molecule(s), can be
correlated to some extent with these double bonds in the targets. The highly unsaturated PUFA might be retained more strongly at the Zn2 doped silica column.
The Zn2 doped silica solid phase was applied to the extraction of EPA and
DHA from krill crude extracts. Table 2 lists these SPE fractions and target recovery,
capacity factor. The recovery of EPA and DHA in the loading fraction was 19.5 and
16.6%, respectively, which suggests that the absorbing force of Zn2 doped silica was
insufcient to retain the two targets in 1.0 mL of the krill crude extracts. A washing
fraction containing 2.5% methanol resulted in 11.8% and 10.5% recovery of EPA and
DHA, respectively. Increasing the methanol fraction to 5% resulted in EPA and
DHA recovery of 24.7 and 23.6%, respectively, based on the crude extract. The
capacity of the two targets in the loading and washing fractions were small, so most
of the EPA and DHA had been obtained in the two fractions. The interferents obviously decreased and there was greater EPA and DHA purity (41.9 and 42.9% recovery, respectively) in the elution fraction. The total recovery of EPA or DHA (97.9
and 93.6% recovery, respectively) was similar to that reported by Guil-Guerrero
et al. (2003) in that several unsaturated fatty esters were obtained by Ag doped silica extraction. The objective of isolating the targets from the crude extracts was
effective using this method but it was not enough to completely separate the targets
from the crude mixtures. Therefore, a search was made to identify a perfect solid
phase to achieve this research aim.

C18 Column
After previously employing aminopropyl-silica and ion-doped silica to purify
PUFA, a solid phase of C18 was assessed for purifying PUFA with a range of chain

2682

B. TANG ET AL.

Table 3. Amounts, recovery, and capacity factor of EPA and DHA from Antarctic krill extract using C18
SPE
Solvent

EPA

Methanol
Volume Amount Recovery
content (%, v:v) (mL)
(mg)
(%)
Loading
step
Washing
step

Elution
step
Total

DHA
Capacity
factor (k)

Amount Recovery
(mg)
(%)

Capacity
factor (k)

1.0

5
10
12.5
15
20
100

5.0
3.0
2.0
2.0
1.0
2.0

8.5
20.3
26.6
53.9
530.6

1.2
5.9
3.8
7.7
75.8

82.3
15.7
23.4
10.6

8.1
17.5
37.8
546.1

1.2
2.6
5.6
80.9

82.3
37.0
16.2

667.8

91.4

609.5

90.3

Note: means that the value is not in the range of determination.

lengths and unsaturation (Lacaze et al. 2007; Giacometti et al. 2002; Kaluzny et al.
1985; Ryu et al. 1997; Ulberth and Achs 1990; Wilson and Sargent 1992). Although
one paper (Mansour 2005) reported that RP-HPLC (C18 packed) had been optimized for the isolation and detection of PUFA, there is no published method using
C18 as a solid phase in the cartridge to extract and purify PUFA. Therefore, C18 as a
solid phase in the cartridge was developed as follows.
Table 3 lists the sequence of operations in the isolation of EPA and DHA
from an Antarctic krill extract by C18 SPE. The table also shows the EPA and
DHA recovery at various steps, such as loading, washing, and elution. Neither
EPA nor DHA were detected in the loading fraction or 5% methanol washing fraction. There might be a relationship between the length of the compounds and C18,
which shows that longer chain compounds are eluted later on a C18 solid phase
than shorter chain analogs (Mansour 2005). Therefore, when a polar solvent is
used for washing, the longer chains of EPA and DHA are retained on the column,
and the shorter chain interferents are washed away. Hence, the capacity factors of
the two targets in the washing step were obviously higher than the previous two
methods. When the methanol content in the washing solution was increased, some
less polar interferents were washed and only a small amount of the targets washed
away (recovery <8%). If the methanol content in the washing solution increased
sharply, the target with similar polarity interferents would be washed in the same
fraction. Consequently, the methanol content was increased slowly, and the interferents could be washed away. The two targets were not isolated from some of the
longer chain interferents, and the total recovery of the target was approximately
90%91%, which is lower than that of front SPE. The adsorption force between
the target and solid phase might have been so strong that more targets were lost
on the column and were not detected. On the other hand, 75.8 and 80.9% of
EPA and DHA, respectively, were obtained based on the crude extract in the elution fraction. Higher recovery targets were obtained in the elution fraction of C18
SPE than the front SPEs.

SOLID-PHASE EXTRACTION FROM ANTARCTIC KRILL

2683

Table 4. Amounts, recovery, and capacity factor of EPA and DHA from Antarctic krill extracts using zinc
ion-doped C18 SPE
Solvent

EPA

Methanol
Volume Amount Recovery
content (%, v:v) (mL)
(mg)
(%)
Loading
step
Washing
step

Elution
step
Total

DHA
Capacity
factor (k)

Amount Recovery
(mg)
(%)

Capacity
factor (k)

1.0

12.5
15
20
25
100

5.0
3.0
3.0
3.0
2.0

12.5
22.3
45.1
604.5

1.8
3.1
6.4
85.5

54.6
30.7
13.9

15.9
29.1
584.4

2.4
4.4
88.4

40.7
21.2

673.2

96.8

629.3

95.2

Note: means that the value is not in the range of determination.

C18 Column and Zinc Sulfate

To better isolate EPA and DHA completely from the crude extracts, the
method of C18 SPE was improved by doping Zn2 into C18 as a solid phase. The reason for combining C18 and zinc ions was that while high recovery of the targets in the
elution fraction was maintained, the targets were isolated completely from the crude
extracts. In C18 SPE, although a high elution recovery was achieved, interferents
with a similar length chain to the targets were also combined with the targets in
the washing and elution fraction. This method employed C18 with a long chain target
and Zn2 with double bonds of the target to isolate it from the interferents. This
combined the advantages of the ion force and C18 alkyl chain force. The recovery
of EPA and DHA in the elution fraction was 96.8 and 95.2%, respectively, as shown
in Table 4. Although the capacity factor of the target on Zn2-C18 phase was smaller
than the C18 phase in the washing and elution stage, the targets were apparently
separated from the interferents on the Zn2-C18 phase, as shown in Figure 1. The
unsaturated targets were adsorbed strongly on the Zn2-C18 stationary phase due
to the formation of very stable coordination complexes, and require pure methanol
or acetonitrile to elute them.
Based on the aforementioned discussion, apparently different effects on different SPE were observed. The C18 and zinc-doped C18 SPE were better methods for
enriching the EPA and DHA targets. Moreover, the latter was the best method
for enriching the targets. Highly unsaturated EPA and DHA are strongly adsorbed
on the zinc ion-doped C18 stationary phase due to the formation of stable forces.
Therefore, the targets can remain in the column during the washing stage and can
be eluted with high recovery in the elution fractions.

Applications
Table 5 lists the results of the zinc ion-doped C18 SPE to isolate EPA and DHA
from 12 Antarctic krill samples. The targets were then transformed to methyl esters

2684

B. TANG ET AL.

Figure 1. GC chromatograms of different fractions of zinc ion-doped C18 SPE of Antarctic krill extract.

Table 5. Amounts of EPA and DHA from different Antarctic krill crude extracts, and recovery of EPA
and DHA from the different Antarctic krill extracts using zinc ion doped C18 SPE
Sample
Fresh
Head
Pleon
Dried
Head
Pleon

Target

Amount mg g1

Recovery (%)

EPA
DHA
EPA
DHA

4.87  0.23
4.26  0.22
2.79  0.13
2.62  0.14

86.7  5.3
89.4  6.1
85.5  5.6
88.4  5.9

EPA
DHA
EPA
DHA

27.80  1.08
24.68  1.01
19.89  0.78
18.46  0.68

87.2  4.8
90.1  5.1
91.1  6.3
90.4  5.7

Note: Amount is expressed as the amount of EPA or DHA in sample and represents means  standard
deviation of 3 separate experiments.

and detected by GC=FID. Higher recovery of EPA and DHA was noted in each
sample. The application of the developed zinc ion-doped C18 SPE method to the
12 samples showed similar EPA and DHA recovery with some differences between
species; the recovery of EPA and of DHA ranged from 85 to 91%. In fresh samples,
80% water was detected in the fresh sample. Overall, the amount of target in the
fresh samples was lower than in the dried sample.
CONCLUSIONS
A zinc ion doped C18 SPE method was developed to isolate EPA and DHA
from complex Antarctic krill crude extract mixtures. This method was more effective

SOLID-PHASE EXTRACTION FROM ANTARCTIC KRILL

2685

in separating the targets than the other SPE techniques. A gradient increase in the
methanol to water ratio in the extraction procedure was used to extract the two targets from different types of samples. This method, which showed good selectivity
and high recovery, was applied successfully to extract the EPA and DHA present
in different species. After separation, the targets were esteried and analyzed by
GC=FID. This method was applied to the extraction and detection of PUFA from
12 Antarctic krill samples. The amounts of EPA and DHA in the fresh Antarctic krill
detected were respectively, 4.87 and 4.26 mg=g in the head, and 2.79 and 2.62 mg=g in
the pleon. In the dried Antarctic krill, the amount of EPA and DHA was, respectively, 27.80 and 24.68 mg=g in the head, and 19.89 and 18.46 mg=g in the pleon.

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