Journal of Chromatography A, 1351 (2014) 1220

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Covalent immobilization of graphene onto stainless steel wire for

jacket-free stir bar sorptive extraction
Wenpeng Zhang, Zixin Zhang, Juan Zhang, Jiawei Meng, Tao Bao, Zilin Chen
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of
Pharmaceutical Sciences, Wuhan 430071, China

a r t i c l e

i n f o

Article history:
Received 19 March 2014
Received in revised form 15 May 2014
Accepted 15 May 2014
Available online 23 May 2014
Keywords:
Covalent immobilization
Stainless steel wire
Polydopamine
Graphene
Stir bar sorptive extraction

a b s t r a c t
Stainless steel wire is a good substrate for jacket-free stir bar sorptive extraction (SBSE). In this work,
we present the rst example of chemical modication of graphene on stainless steel wire (SSW) for
SBSE. Bio-inspired polydopamine was rstly modied on the SSW covalently; then graphene oxide was
introduced and reacted with amino groups of polydopamine layer. The modication of polydopamine and
graphene can be repeated by a layer-by-layer strategy, resulting in control of the thickness of graphene
layer and increase of extraction capability. The prepared covalently immobilized graphene-stainless steel
wire (G-SSW) exhibited good stability under stirring, ultrasonication and treatment with commonly used
organic solvents, basic and acidic solutions. Application as stir bar for SBSE, G-SSW was found to possess
good extraction efciency towards pollutant polyaromatic hydrocarbons (PAHs), with enrichment factors from 10086 to 16896. After optimization of conditions, G-SSW based SBSE was coupled HPLC for
determination of PAHs in environmental and food samples. The quantitative method has low limits of
detection of 0.250 pg/mL, which is better than some reported SBSE-HPLC methods. The method shows
wide linear range (2005000, 101000 and 11000 pg/mL), good linearity (r 0.9950) and good reproducibility (RSD 4.97%). The method has been applied to soil and food samples, with good selectivity and
good recoveries ranging from 88.5113.6%.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Determination of organic or inorganic compounds in complex matrices such as environmental, food and biological samples
often requires sample pretreatment before separation or detection
[1]. Green sample pretreatment techniques such as stir bar sorptive extraction (SBSE) and solid-phase microextraction (SPME) are
desirable for they consume little organic solvent [2].
Stir bar sorptive extraction was rst developed in 1999 [3].
In comparison with SPME, SBSE usually exhibits better extraction
capability due to its larger volume and surface area. However, at
present only a few kinds of stir bars such as polydimethylsiloxane
(PDMS) and ethyleneglycol silicone-coated stir bars are commercially available, therefore, to develop stir bar with different kinds
of sorbents is useful for the purpose of extending the application
of SBSE [1,4]. Generally, stir bars consist of three essential parts:

Corresponding author. Luojia Chair Professor, Vice Dean and Institute Director,
School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China.
Tel.: +86 27 68759893; fax: +86 27 68759850.
E-mail address: chenzl@whu.edu.cn (Z. Chen).
http://dx.doi.org/10.1016/j.chroma.2014.05.038
0021-9673/ 2014 Elsevier B.V. All rights reserved.

a magnetic stirring rod, which rotates the stir bar, as well as the
liquid sample; a thin glass jacket that covers the magnetic stirring
rod and a layer of sorbent into which the analytes are extracted.
The glass jacket is useful to immobilized sorbents onto the stir
bar as its chemical activity. For example, for polymer sorbents, the
glass jacket can be rst silanized with 3-(trimethoxysilyl) propyl
methacrylate (-MAPS), which can co-polymerize with monomers
and resulting in immobilization of polymer sorbents [5]; and PDMS
can be immobilized by solgel process [6]. However, the glass is
fragile and easy to be broken during stirring and ultrasonication,
which would lead to bad stability and reproducibility.
Stainless steel wire (SSW) can stir spontaneously under the
magnetic force given by a commonly used magnetic stirring apparatus. Besides, they are easy to obtain and of high mechanical
strength, making them good substrates for SBSE. However, as the
stainless steel is chemically resistant, it is difcult to immobilize
sorbents onto the surface of the stainless steel. Few works were
reported to coat sorbents onto the stainless steel for SBSE. Conductive polymer was electrochemically deposited onto the surface of
a stainless steel rod for SBSE [7]. However, as the sorbents were
just coated physically, the immobilization was not rmly enough,
and just a thin lm of conductive polymer could be fabricated on

W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

the surface of the stir bar, it is difcult to increase the thickness

of the sorbent for the purpose of increasing the extraction efciency. Moreover, sorbents can be immobilized are also limited. The
presence of chemical bonding between sorbents and the substrate
can lead to high stability and lifetime [8]. However, to our knowledge, no work has been reported for covalent bond of sorbents onto
stainless steel for SBSE.
Graphene is a typical two-dimension material that has been
attracting considerable interest since its rst discovery in 2004
[911]. Graphene possesses large delocalized -electro system
which can form a strong -stacking interaction with aromatic
compounds and their derivatives [12], along with its large surface
area (theoretically about 2700 m2 /g) [13] and ease of preparation
[14,15], graphene has been used in extraction techniques in recent
years, such as for solid-phase extraction (SPE), SPME and matrix
solid-phase dispersion [8,1619]. However, graphene as sorbent
for SBSE has not been reported. The reason may be that conditions
in SBSE such as stirring and ultrasonication are more damageable
than that in SPE or SPME, so that methods for immobilization of
graphene in SPE or SPME are not t for SBSE.
Inspired by mussels unique binding behavior onto rocks,
dopamine was revealed to be essential to the binding [20] and
a polydopamine method has been developed as a versatile surface modication protocol [21]. On the basis of this discovery, we
have developed a method for immobilization of sorbents or stationary phase inside micro tubes in our previous works [16,22,23].
Polydopamine can grow on the surface of plastic tubes; the polydopamine layer is chemically active and can be used for binding of
polymer sorbents or graphene. The functionalized polydopamine
layer was found to be highly stable and we also testied the
rm binding between the plastic surface and sorbents. However,
the interaction between polydopamine and plastic surface is noncovalent, it is still not adequate to use for SBSE. Fortunately, the
catechol group was found to covalently bond with metals [24]; the
presence of catechol groups in polydopamine layer make it promising to covalent modify the stainless steel by the polydopamine
method.
In this work, we proposed a simple chemical-bonding procedure to immobilize sorbents on SSW for SBSE. Bio-inspired
polydopamine method was used to covalently functionalize SSW
rstly. Graphene oxide (GO) was then introduced to react with
polydopamine to form a layer of graphene-based sorbent. The
process can be repeated by a layer-by-layer strategy to control
the thickness of the sorbents of SBSE. In comparison with that
for polyetheretherketone (PEEK) or polytetrauoroethylene (PTFE)
surfaces, polydopamine can covalently react with SSW, resulting
in good stability of the proposed stir bar. The formation of multilayer graphene was characterized by Fourier transform infrared
spectra (FTIR) and scanning electron microscopy (SEM). Extraction
efciency of the multilayer graphene-modied SSW stir bar was
investigated and an SBSE-HPLC method has also been developed
for determination of polyaromatic hydrocarbons (PAHs) in environmental and food samples. To the best of our knowledge, this
is the rst time that graphene is chemically modied onto metal
surfaces for SBSE.

13

purchased from Alfa Aesar (Tianjing, China). Acetonitrile (Tedia,

OH, USA) was HPLC grade and water used was puried by a Milli-Q
system (MA, USA). Stainless steel wires were purchased from a
local store.
2.2. Instrumentation
A Shimadzu 20A HPLC system (Tokyo, Japan) was used, which
equipped with two 20A pumps, a six-port valve and a 10A uorescent detector. A column (C-18, 150 mm 4.6 mm i.d.) packed
with 5 m particle size (GL Science, Tokyo, Japan) was used for
chromatographic separation. Data collection and handling was
performed on a Shimadzu LC Solution software. The detection
wavelength of uorescent detector was set at 300 nm (exiting
wavelength) and 440 nm (emission wavelength). The temperature
for HPLC separation was set at 30 C, controlled by a thermostat
controlled column compartment.
FTIR spectra was determined a by Thermo Nexus 470 FT-IR
system (MA, UAS). The morphology of GO was investigated by
a JEM-2100 (HR) transmission electron microscope (TEM) (JEOL,
Tokyo, Japan), and the surface morphology of the graphene modied wires was characterized by a Quanta 200 scanning electron
microscope (SEM, FEI, Holand). X-ray photoelectron spectroscopy
(XPS) experiments were performed by a KRATOS XSAM800 XPS
instrument (Manchester, UK).
2.3. Preparation of GO
Graphene oxide was prepared by a modied Hummers method
[14,15]. The graphite powder was rstly preoxidized by H2 SO4 ,
K2 S2 O8 and P2 O5 . It was then subjected to oxidation by Hummers
method. The GO product was suspended in distilled water to give
a viscous, brown dispersion, which was subjected to dialysis to
remove metal ions and residual acids. Exfoliation was achieved by
dilution of the GO dispersion with deionized water (1/24, v/v), followed by sonication. GO sheets were obtained by centrifugation
(10,000 rmp) and further dried in an oven (60 C) for 12 h.
2.4. Fabrication of graphene-polydopamine modied SSW
SSW (3 cm) was washed sufciently with ethanol to clean the
surface, and then dried in an oven (60 C) for further use. For polydopamine modication, dopamine solution (2 mg/mL, pH 8.5) was
agitated with a vortex mixer for peroxidation. When the solution
became slightly brown, the SSW was immersed and put under room
temperature for 12 h. After drying in an oven (60 C) for 3 h, polydopamine modied SSW was obtained. For graphene modication,
GO aqueous solution (1 mg/mL) was prepared in a screw-cap vial,
polydopamine modied SSW was then put into the solution. The
vial was sealed and put into a water bath for reaction (65 C, 10 h).
After washing with ethanol and drying in the oven (60 C, 3 h),
graphene-polydopamine modied SSW was obtained. The above
procedures were repeated for three times to obtain multilayer
graphene modied SSW (G-SSW). The schematic is shown in Fig. 1.

2. Experimental

2.5. SBSE procedures

2.1. Chemicals and materials

SBSE was performed in a 100 mL beaker with a cover of paralm.

Sample solution of 30 mL (containing 1% acetonitrile, v/v) was
added into the beaker with the G-SSW in the solution. G-SSW can
stir by controlling with a magnetic stirring apparatus, at a speed of
300 r/min. After sample loading for 2 h, the wire was taken out of
the sample solution and put into a small tube led with 200 L
acetonitrile, which was then sonicated for 5 min to desorb the

PAHs standards (uoranthene (FLU), pyrene (PYR),

benz[a]anthracene (B[a]AN), benzo[a]uorathene (B[a]FL),
benzo[a]pyrene (B[a]PY) and dibenz[a,h]anthracene (D[a,h]AN))
and dopamine hydrochloride were obtained from SigmaAldrich
(MO, USA). Graphite powder (natural, 215 m, 99.9995%) was

14

W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

Fig. 1. Schematic of modication of stainless steel wire (A) and reaction process (B).

analytes. Eluent of 20 L was then injected into the HPLC system

for analysis.

and loaded for SBSE. For comparison, 20 L of the extract of the

grilled eggplant by acetonitrile was also injected directly into the
HPLC system for analysis.

2.6. Sample preparation

3. Results and discussion
PAHs stock solution with a concentration of 10 g/mL was
prepared with acetonitrile and diluted step-by-step to certain concentrations with acetonitrile. For SBSE, 20 L of standard solution
was added into water to make aqueous sample solutions.
Soil and food samples were used to demonstrate the application
of the method in real environmental samples. Soil sample was collected from lakeshore of the East Lake. After collection, it was dried
in an oven (60 C) for 10 h, grounded and sieved by a 48-m sieve.
Then 5 g soil particles were added into a tube containing 20 mL
acetonitrile, the solution was ultrasonicated for 1 h for extraction.
The supernatant was collected after centrifugation (10,000 rmp,
10 min), dried by rotary evaporation and redissolved with 1 mL acetonitrile. Sample of 50 L and 250 L acetonitrile were diluted to
30 mL with water and loaded for SBSE. For comparison, 20 L of
the extract by acetonitrile was also injected directly into the HPLC
system for analysis. Barbecue samples were from a local barbecue restaurant, for pretreatment, 1 g grilled was collected, cut into
small pieces and put into a tube containing 20 mL acetonitrile; the
solution was ultrasonicated for 1 h for extraction. The supernatant
was collected after centrifugation (10,000 rmp, 10 min), dried by
rotary evaporation and redissolved with 1 mL acetonitrile. Sample
of 50 L and 550 L acetonitrile were diluted to 30 mL with water

3.1. Covalent immobilization of multilayer graphene on SSW

Stainless steel is of high strength and convenient to get, although
it has been used as substrate for SPME, as its chemically resistant
property, it is still not easy to rmly immobilize sorbents into the
surface of stainless steel [25]. Some methods such as magnetronsputtering [26] and coating with noble metals [27] have been
employed to enhance the stability of the immobilization of sorbents
on stainless steel for SPME. However, procedures of these methods
were not easy to operate, and the applications were also limited.
In this work, a simple and versatile bio-inspired polydopamine
method was developed to covalently functionalize stainless steel,
which could be used to immobilize different kinds of sorbents for
SBSE. The schematic is shown in Fig. 1. Dopamine basic solution was
prepared, after oxidization, rearrangement and inter-molecular
cross-linking, polydopamine layers would grow on the surface
of SSW. Unlike non-covalent bonding in the PEEK or PTFE surfaces, catechol groups of polydopamine can react covalently with
SSW, as shown in Fig. 1. Amino groups in the polydopamine layer
were active to covalently react with carboxyl and epoxy groups of
GO [28], realizing chemical immobilization of graphene on SSW.

W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

15

Fig. 2. Transmission electron micrograph of synthesized graphene oxide.

Fig. 3. X-ray photoelectron spectrum of synthesized graphene oxide.

When introducing dopamine solution again, dopamine could polymerize inter-molecularly and formed another polydopamine layer
on immobilized graphene layer. The procedures could repeat three
times to obtain multilayer graphene modied SSW, which would
possess better extraction capability.
In general, the thickness of the sorbents on the stir bar is usually difcult to control when immobilization of sorbents for SBSE,
which would cause problems such as bad reproducibility and stability. As for the bio-inspired polydopamine method, the sorbent was
synthesized by a novel non-covalent and covalent combined layerby-layer strategy, where polydopamine was served as binders and
interlayers. Therefore, the thickness of the sorbent can be increased
and controlled by times of coatings.

3.2. Characterization
The morphology of GO was investigated by TEM, as shown in
Fig. 2. Disordered paper-like sheet was observed, which could testify nanosheet structure of GO. The chemical composition of the
GO was investigated by XPS. C 1s and O 1s is found in GO (Fig. 3A),
peak tting of the C 1s bands of GO (Fig. 3B) yields related functional groups, including non-oxygenated ring C (CC), CO bonds,
carbonyl (C O) and carboxylate C (HOC O), which indicated the
oxidation of graphene sheet. Fig. 4 shows SEM images of modied
SSW. After polydopamine modication (Fig. 4A and B), continuous small particles are observed on the surface of SSW, which are

Fig. 4. Scanning electron micrograph of polydopamine modied SSW (A and B), and G-SSW (C and D).

16

W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

3.3. Stability of the multilayer graphene modied SSW

Fig. 5. Scanning electron micrograph of G-SSW (cross section).

formed by polymerization of dopamine. Some large particles were

also found (Fig. 4A), which from the polymerization in the solution where the SSW put for polydopamine modication. Comparing
with that on plastic surfaces, polydopamine shows more continuous on stainless steel, even some layer structures were observed
(Fig. 4B), probably due to covalent bonding with SSW and the
good electrical conductivity of SSW, which benets to the electron
transfer during the inter-molecule polymerization of dopamine.
After modication with GO (Fig. 4C and D), compact lm structure was observed, indicating the immobilization of graphene on
polydopamine. As the graphene lm was covalent reacted with
polydopamine and rmly covered on the particles, the surface of
the SSW has become smooth compared with polydopamine-SSW.
The cross section of G-SSW is shown in Fig. 5, and the thickness of
the graphene layer about 15 m was observed.
The immobilized graphene based layers were also studied by
FTIR. As shown in Fig. 6, the characteristic peaks for GO of stretching around at 3420 cm1 (OH), 1726 cm1 (C O), vibration at
1393 cm1 (OH of COH) and stretching at 1104 cm1 (CO of
epoxy) were observed. After reaction with polydopamine, CO
stretching of epoxy at 1104 cm1 and OH vibration of C-OH at
1393 cm1 decreased signicantly. Moreover, a red shift of C O is
observed, from 1725.98 to 1718.96 cm1 , probably due to structure
change from C( O) OH to C( O)NH. The results of FTIR could
testify the covalent bonding between GO and polydopamine.

In the process of SBSE, the G-SSW was immersed in the sample

solution and stirred under magnetic force at a speed of 400 r/min.
And for desorption, the G-SSW was suffered with ultrasonication.
The coating of stir bar is easy to be damaged during both these
two process, therefore, the stability of the coating is important for
SBSE. To investigate its stability, the G-SSW was treated with stirring, ultrasonication and solvents, respectively. G-SSW was put into
an aqueous solution and stirred for continuous 48 h; no obvious
stripping of the layers was found, indicating its good tolerance to
stirring. The stir bar was also found to be stable to ultrasonication
for 20 min, which is useful in the process of desorption of the analytes. To study its stability in different kinds of solvents, G-SSW
was put into 2 mL of commonly used organic solvents including
methanol, ethanol, isopropanol, dodecanol, acetonitrile, acetone,
toluene, dimethylsulfoxide and chloroform, and 1 M HCl and 1 M
NaOH solutions, respectively; after ultrasonication for 5 min, no
obvious stripping of the graphene layers was observed for all of
the solvents and solutions. Interestingly, polydopamine layer was
tolerant to strong basic conditions, which was intolerant to 1 M
NaOH on PEEK or PTFE surfaces. The results showed that covalent bonding of polydopamine on metal surfaces was more stable
than non-covalent bonding on other surfaces, and G-SSW is stable
enough to be used in SBSE. The prepared G-SSW was also treated
with high temperature of 300 C in a GC oven for 2 h, no obvious
stripping of the layer of graphenepolydopamine was observed,
indicating the good thermal stability of the G-SSW. The stability of
the stir bar has also been investigated in the aspect of consecutive
use. A G-SSW can be used about 50 times for consecutive extraction
without signicant decrease of extraction efciency.
3.4. Application of the multilayer graphene-modied SSW for
SBSE
PAHs compounds were selected to demonstrate the extraction
efciency of G-SSW for their strong interaction with graphene
and demands of their low-content quantication [29,30]. Some
parameters possible to affect SBSE were systematically studied and
optimized.
3.4.1. Salt content
Salt in the aqueous sample solution may contribute to extraction due to the decrease of solubility of some analytes, especially
in headspace sampling mode [8]. In this work, NaCl was added to
sample solutions to study the effect of salt content on G-SSW based
SBSE. The content of NaCl from 0 to 5% (w/v) was investigated; the
sample volumes were 20 mL, with 1% acetonitrile, and extraction
for 30 min at a stirring rate of 200 r/min. As shown in Fig. 7A, extraction efciency of PAHs decreased along with the increase of NaCl
content. It was probably resulted from the decrease distribution
coefcient (K) of PAHs along with the increase of NaCl content.
Similar with that in SPME, the amount (n) of PAHs extracted by
immobilized graphene layer can be calculated by [31]:
n=

KVg Vs
c0
Vs + KVg

(1)

Where c0 is the concentration of PAHs in the sample solution

before extraction, Vs and Vg are the volume of the sample solution
and the graphene layer, respectively. As the volume of the graphene
layer on the surface of SSW is very small (for example, for a wire of
3 cm long, with diameter of 300 m, if the thickness of the coating
was 15 m, the volume of the graphene layer was calculated to be
less than 1 L), therefore, Vs > >KVg and Eq. (1) can be simplied to
Fig. 6. FTIR spectra of polydopamine (A), GO (B) and GO-polydopamine (C).

n = KVg c0

(2)

W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

17

Fig. 7. Effect of the salt content (A), acetonitrile content (B), stirring rate (C), stirring time (D) and sample volume (E) on the extraction efciency of SBSE.

As the content of NaCl increased, K decreased, PAHs that could

be extracted by graphene would decrease as a result. In order to
obtain best extraction efciency, salts were not added in the sample
solution for further studies.
3.4.2. Acetonitrile content
PAHs are highly hydrophobic compounds; therefore, acetonitrile was added into aqueous sample solution to help the dissolution
of PAHs. However, too much acetonitrile in the aqueous sample
solution may be not benecial to the extraction of PAHs. Acetonitrile content was optimized in the range of 0.510% (v/v); the
sample volumes were 20 mL, and extraction for 30 min at the stir
rate of 200 r/min. The curves of peak area-acetonitrile content are
shown in Fig. 7B. Peak areas of PAHs were found to increase within
certain acetonitrile contents, and then decrease along with further
increase of acetonitrile contents. Partition coefcient of PAH (K)
would decrease along with the increase of acetonitrile content, but
when acetonitrile content was low, c0 was also inuenced which
increased along with the increase of acetonitrile content (Eq. (2)).
Inversely proportional relationship between K and c0 at low acetonitrile concentration results in the high points in Fig. 7B, for the
consideration of extraction efciency, acetonitrile condition of 1%
(v/v) was selected.
3.4.3. Stirring rate for extraction
Stirring could contribute to the fast equilibrium of samples on
the surface of stir bar. Stirring rate was studied from 0 to 400 r/min;
the sample volumes were 20 mL, and extraction for 30 min. As
shown in Fig. 7C, the extraction efciency for six PAHs was relatively low when the G-SSW was not stirred during the SBSE process.
The extraction efciency increased dramatically with stirring and

reached equilibrium at the rate of 300 r/min. Therefore, stirring rate

of 300 r/min was maintained in further studies.
3.4.4. Time for extraction
SBSE using G-SSW as stir bar was performed in an aqueous
solution under the control by a magnetic stirring apparatus, the
extraction is a process of adsorption equilibrium. Time for extraction was investigated in the range of 10 min to 4 h; the sample
volumes were 20 mL, extraction at the stir rate of 300 r/min. Peak
areas of all of six PAHs increased signicantly when the extraction
time was 10 min to 1 h (Fig. 7D), equilibrium reached at about 2 h,
and the extraction efciency remained almost unchanged after 2 h.
Therefore, extraction was performed for 2 h in further studies.
3.4.5. Volume of sample solution
Extraction efciency can be enhanced by increase the volume
of sample solution to some extent. PAHs sample solutions from
5 to 40 mL were loaded for extraction, respectively. As shown in
Fig. 7E, extraction efciency of PAHs increased steadily along with
sampling from 5 to 30 mL, and increased little when larger sample solution was loaded. Therefore, sample solution of 30 mL was
selected in further studies.
Enrichment factors of G-SSW towards PAHs were determined
under optimized conditions. Enrichment factor (FE ) was calculated
by:
FE =

cg
c0

(3)

Where c0 is the PAHs concentration of sample solution and

cg is the PAHs concentration in the immobilized graphene layers.
Aqueous samples containing 0.1 ng/mL PAHs were loaded onto the

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W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

Table 1
Analytical performance of SBSE-HPLC method for determination of PAHs.
Linearity

FLU
PYR
B[a]AN
B[a]FL
B[a]PY
D[a,h]AN

LOD (pg/mL)

Range (pg/mL)

Regression equation

2005000
2005000
101000
11000
11000
101000

y = 176x + 132
y = 275x + 172
y = 8320x + 823
y = 18952x + 1136
y = 36062x + 1578
y = 14148x + 417

0.9980
0.9986
0.9958
0.9954
0.9982
0.9950

50
50
1.5
0.3
0.2
2

LOQ (pg/mL)

200
200
5
1
1
5

RSD% (n = 5)

FE

3.53
4.58
2.28
2.24
4.97
1.44

5.12
6.11
5.61
4.53
6.45
3.13

11746
16544
16730
16898
14016
10086

a: between runs; b: between G-SSWs.

prepared G-SSW, after extraction and desorption, PAHs extracted

by G-SSW were calculated by comparison with that of a standard
sample. Counting the volume of graphene layers on SSW as 1 L,
FE for six PAHs were calculated to be 11746 for FLU, 16544 for PYR,
16730 for B[a]AN, 16898 for B[a]FL, 14016 for B[a]PY and 10086 for
D[a,h]AN (Table 1). The high FE showed good enrichment efciency
of the G-SSW. Enrichment folds by comparing the injected samples
were also evaluated. Sample solutions with and without extraction
(20 L) were injected into HPLC, and the increase of the peak area
was calculated. The folds of the peak area increased with SBSE were
found to be 49 for FLU, 64 for PYR, 77 for B[a]AN, 64 for B[a]FL, 58
for B[a]PY and 35 for D[a,h]AN.
Extraction efciency of SSW, polydopamine-SSW, G-SSW, and
SSW coated by polydopamine (P-SSW) and graphene oxide for one
time (G1-SSW) were compared. The concentration of PAHs was
100 pg/mL and the extraction was performed under optimized conditions. Taking B[a]PY as an example, the enrichment efciency is
shown in Fig. 8. SSW exhibited some adsorptive capability towards
PAH, after coated with polydopamine, the extraction efciency
increased a little; when the graphene layer were coated, the enrichment efciency increased signicantly, larger enrichment factor
was also observed when the coating procedures were repeated
for three times. The results indicated that SSW and polydopamine
can help extraction of PAHs, but graphene is the essential for the
excellent extraction efciency. Moreover, layer-by-layer growing
of graphene layer can increase extraction efciency by increase of
the thickness of the sorbent.
3.5. SBSE-HPLC method for analysis of PAHs
Graphene was found to interact strongly with aromatic compounds, and the adsorption mechanism of are mainly based
on stacking and hydrophobic effect. In this work, the
graphene modied SSW-based SBSE was coupled with HPLC for
the determination of PAHs in environmental and food samples.

Fig. 8. Comparison of extraction efciency of stir bar modied with different layers.

Analytical performances of the proposed SBSE-HPLC method was

investigated, parameters including the calibration equation, linearity, correlation coefcients, low limits of detection (LOD),
limit of qualication (LOQ) and the repeatability were performed
under the optimized conditions and the results are listed in
Table 1. All of six PAHs possess wide linearity in the range of
2005000, 101000 and 11000 pg/mL, respectively, with correlation coefcients (r) 0.9950. LODs (S/N = 3) of the graphene based
SBSE-HPLC method can reach 50 pg/mL for FLU and PYR, 1.5 pg/mL
for B[a]AN, 0.3 pg/mL for B[a]FL, 0.2 mg/mL for B[a]PY and 2 pg/mL
for D[a,h]AN. And LOQs (S/N = 10) of six PAHs is 1200 pg/mL,
which is useful to determine trace amount of PAHs in real samples. The repeatability was determined by loading PAHs solution of
100 pg/mL for 5 times and relative standard deviations (RSDs) for
each peak areas were calculated; RSDs between 5 runs less than
4.97%, and RSDs between 5 G-SSWs less than 6.45% were obtained,
indicating the good reproducibility of the SBSE-HPLC method.
The proposed graphene based SBSE-HPLC method was compared with that with PDMS as the sorbent for PAHs enrichment and
determination. As listed in Table 2, G-SSW based method showed
better or similar sensitivity for some PAHs such as B[a]FL, B[a]PY,
and D[a,h]AN. The relatively higher LODs of some PAHs such as FLU
and PYR were probably due to the single wavelength for uorescent detection in this work while multiple wavelengths were set in
the literature [32,33].

3.6. Application in environmental and food samples

PAHs are pollutants of concern which are considered to be carcinogenic, mutagenic, and teratogenic to human beings. PAHs are
widely distributed in environment such as water, soil and air, they
are also found in cooked foods. Control of the amount of PAHs in
environment and food is important to protect human health. In
this work, the proposed graphene-based SBSE-HPLC method was
applied to extract and determine PAHs in environmental and barbecue samples. Soil from the lakeshore of the East Lake was taken
as an example for environmental samples. As shown in Fig. 9A, all
six PAHs were well detected after SBSE, the contents are calculated to be 64.8 ng/g for FLU, 22.0 ng/g for PYR, 1.4 ng/g for B[a]AN,
11.0 ng/g for B[a]FL, 4.3 ng/g for B[a]PY and 11.2 ng/g for D[a,h]AN.
The contents of PAHs were consistent with that obtained by online
SPME-HPLC [16]. Interestingly, comparing with the chromatogram
obtained from online SPME-HPLC, SBSE seems to exhibit better
clean-up efciency since most disrupting peaks from matrices has
been removed in the chromatogram of SBSE-HPLC. The method has
also been applied in barbecue samples. Chromatogram of grilled
eggplant sample is shown in Fig. 9C. All of six PAHs were found in
the grilled eggplant sample and the contents were calculated to be
32.6 ng/g for FLU, 6.8 ng/g for PYR, 0.6 ng/g for B[a]AN, 1.4 ng/g for
B[a]FL, 2.6 ng/g for B[a]PY and 2.7 ng/g for D[a,h]AN. Besides, the
signicant enhancement of the signal of PAHs by comparing the
chromatograms before and after SBSE (Fig. 9AD) also shows great
enrichment efciency of G-SSW.

W. Zhang et al. / J. Chromatogr. A 1351 (2014) 1220

19

Table 2
Comparison the proposed method with reported PDMS-SBSE-HPLC methods.
Sorbents

Graphene
PDMS [32]
PDMS[33]

LOD (pg/mL)
FLU

PYR

B[a]AN

B[a]FL

B[a]PY

D[a,h]AN

50
0.1
1.5

50
0.5
0.7

1.5
0.9
0.5

0.3
2.4
0.4

0.2
1.2
0.2

2
3.5
0.4

FL wavelength

Samples

Single
Multiple
Multiple

Foods/soil
Mate tea
Water

Table 3
Results and recoveries for determination of PAHs in soil and barbecue samples.
Compounds

FLU
PYR
B[a]AN
B[a]FL
B[a]PY
D[a,h]AN

Barbecue

Soil
Content (ng/g)

Added (ng/g)

Recovery (100%)

Content (ng/g)

Added (ng/g)

Recovery (100%)

64.8
22.0
1.4
11.0
4.3
11.2

60.0
60.0
60.0
60.0
60.0
60.0

98.5
112.0
94.5
111.5
89.0
91.5

32.6
6.8
0.6
1.4
2.6
2.7

25.7
25.7
25.7
25.7
25.7
25.7

105.1
91.6
113.6
112.4
113.0
88.5

in solutions. The G-SSW stir bar showed good extraction efciency

towards PAHs, and an SBSE-HPLC method was developed to developed to enrich and determine pollutant PAHs in environmental. The
method showed high sensitivity, wide linear range, good linearity
and good reproducibility. The bio-inspired polydopamine method
is a versatile for covalent immobilization of graphene on stainless
steel, it can also be used for immobilization of other sorbents for
SBSE.
Acknowledgement
This work was supported by the National Scientic Foundation
of China (Grant nos. 21375101 and 30973672), Wuhan Science
and Technology Bureau (No: 20140601010057), Innovation Seed
Fund and Translational Medical Research Fund of Wuhan University School of Medicine, and the Fundamental Research Funds for
the Central Universities (No. 2012306020203).
Fig. 9. Chromatograms of the soil sample with and without SBSE (A and B), and the
grilled eggplant sample with and without SBSE (C and D).

Recoveries in environmental and barbecue samples were tested

by for the lakeshore soil of the East Lake and grilled eggplant
examples. Six PAHs standards were spiked at the content of 60.0
and 25.7 ng/g, respectively. By calculating the mean value of three
duplicates, recoveries in the soil sample were in the range of
89.0112.0%, and recoveries in the grilled eggplant sample were in
the range of 88.5113.6% (Table 3). The results of recovery showed
good accuracy of the proposed SBSE-HPLC method.
4. Conclusions
In this work, we proposed the rst example of covalent bonding of sorbents on SSW for SBSE. Multilayer graphene immobilized
by layer-by-layer method was also applied for sorbents of SBSE for
the rst time. Bio-inspired polydopamine method was used to functionalize the surface of SSW, GO was then introduced to react with
polydopamine, resulting in the formation of graphene layer on the
SSW; the procedures could also be repeated to control the thickness
of the sorbent. The reaction was performed under mild conditions
and it was environmental friendly as free of organic solvents or
toxic reagents. The immobilized graphene layer was stable in commonly used solvents, as well as under ultrasonication, and stirring

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