Open-Tubular Capillary Electrochromatography Using A Capillary Coatedwith Octadecylaminecapped Gold Nanoparticles

901
Electrophoresis 2008, 29, 901909
Qishu Qu1, 2
Xinxin Zhang1
Ming Shen1*
Yin Liu1
Xiaoya Hu1
Gongjun Yang1
Chengyin Wang1
Yukui Zhang2
Chao Yan2, 3
1
Jiangsu Key Laboratory of

Environmental Materials and
Environmental Engineering,
College of Chemistry
and Chemical Engineering,
Yangzhou University,
Yangzhou, P. R. China
2
National Chromatography
R&A Center, Dalian Institute
of Chemical Physics,
Chinese Academy of Sciences,
Dalian, P. R. China
3
School of Pharmacy,
Shanghai Jiao Tong University,
Shanghai, P. R. China
Received June 6, 2007

Revised September 21, 2007
Accepted September 22, 2007
Research Article
Open-tubular capillary electrochromatography

using a capillary coated with octadecylaminecapped gold nanoparticles
Octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and characterized by using UVVis adsorption spectrum, transmission electron chromatography (TEM),
SEM, and FT-IR. A simple but robust hydrophobic coating was easily developed by flushing
a capillary with a solution of ODA-Au-NPs, because the positive charges were carried by the
nanoparticles which strongly adsorb to the negatively charged inner surface of a fused-silica
capillary via electrostatic and hydrophobic interactions. The chromatographic characteristics of the coated capillary was investigated by varying the experimental parameters such
as buffer pH, buffer concentration, and percentage of organic modifier in the mobile phase.
The results show that (i) resolution between thiourea and naphthalene is almost the same
when comparing the electrochromatograms obtained using pH 7 buffer as mobile phase
after and before the capillary column was operated using pH 11 and 3 mobile phase; (ii) no
significant changes in retention time and deterioration in peak efficiency were found after
60 runs of test aromatic mixtures; and (iii) column efficiency up to 189 000 theoretical
plates/meter for testosterone was obtained. All of the results indicated that the coating
could act as a stable stationary phase for open tubular CEC as well as for bioanalysis.
Keywords:
Gold nanoparticle
chromatography
Introduction
CEC is a hybrid of CE and micro HPLC (mHPLC). The most

common approach for performing CEC involves the use of a
fused-silica capillary column packed with a stationary phase,
or a monolithic column, or an open tubular capillary column
coated with stationary phase on the surface of inner wall.
Open tubular capillaries have advantages over packed capillary columns in several aspects including ease of preparation,
no bubble formation, and simple instrumental handling. The
main methods used to prepare the stationary phase for the
open tubular CEC (OTCEC) include chemical bonding [14],
solgel derived phases [5, 6], molecular imprinting [7], porous
Correspondence: Dr. Qishu Qu, Jiangsu Key Laboratory of Environmental Materials and Environmental Engineering, College of
Chemistry and Chemical Engineering, Yangzhou University,
Yangzhou 225002, P. R. China
E-mail: quqishu@gmail.com
Fax: 186-514-7975244
Abbreviations: ATR-FTIR, attenuated total reflectance FTIR;
MPTMS, 3-mercaptopropyltrimethoxysilane; ODA-Au-NPs, octadecylamine-capped gold nanoparticles; OTCEC, open tubular
CEC; TEM, transmission electron microscopy
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Octadecylamine
Open-tubular capillary electroDOI 10.1002/elps.200700409
layers [8, 9], physical coating [1012], and nanoparticle phases

[1318]. Among these techniques, coating of capillaries with
nanoparticles provided high separation efficiencies for various analytes [19]. However, due to the tedious coating processes, only a few types of nanoparticles such as titanium
dioxide and gold nanoparticles were used as coating materials
for OTCEC even though significant advances were found. To
date, nanoparticles were used mostly as pseudostationary
phases to enhance separation performance of CEC [2035].
It has been suggested that gold nanoparticles served as
large surface area platforms for adsorption of organic molecules that alter the interactions of nanoparticles with the capillary wall, the analytes, or both. However, gold nanoparticles
acting as coating materials have been employed in OTCEC to
enhance the separation efficiency by only a few groups [13
16]. Glennon and co-workers [13] reported the immobilized
dodecanethiol gold nanoparticles on prederivatized 3-aminopropyltrimethoxysilane (APT-MS) or 3-mercaptopropyltrimethoxysilane (MPTMS) fused-silica capillaries, which
confirmed the use of gold nanoparticles as a novel stationary
phase for OTCEC. Two steps were involved in the preparation
of these capillary columns: (i) modification of the inner capil* Additional corresponding author: Dr. Ming Shen, E-mail: shen
ming@yzu.edu.cn
www.electrophoresis-journal.com
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Q. Qu et al.
lary wall with APT-MS or MPTMS; (ii) immobilization of the

gold particles to the prederivatized inner surface of the capillary column. Thereafter, an etching method was developed by
the same group to further increase the inner surface area of
the capillary before the chemical modification procedure [14].
Better separation results were obtained by using an etched
capillary column, but the HF etching process is time-consuming and complicated. Liu et al. [16] achieved good results
by using alkanethiol self-assembly and dithiol layer-by-layer
self-assembly processes to prepare a multilayer gold nanoparticle-coated OTCEC. However, six major steps were
involved in the fabrication procedures.
To date, only dodecanethiol-capped gold nanoparticles were
ever used as coating materials for CEC separation. The drawback is that the materials could not be directly adsorbed onto the
inner surface of the fused-silica capillary column [13]. A possible
explanation is that negatively charged dodecanethiol was repelled by the negatively charged inner surface of a fused-silica capillary column. Therefore, to obtain a stable coating using dodecanethiol-capped gold nanoparticles, a pretreatment of the capillary column with APTMS or MPTMS was necessary, which
made the fabrication process much complicated.
Amine-capped gold nanoparticles can be adsorbed
strongly onto the surface of glassy carbon electrode surface
[36]. It is estimated that amine-capped gold nanoparticles can
also be adsorbed onto the inner surface of a fused-silica capillary column as a coating material. Since amines bear positive charges and the inner surface of a fused-silica capillary
bear negative charges, amine-capped gold nanoparticles can
be strongly adsorbed onto the inner surface of a silica capillary through electrostatic interaction and hydrophobic interaction. Therefore, the coating layer formed by using aminecapped gold nanoparticles would be more stable than that
formed by using thiol-capped gold nanoparticles.
Amine-capped gold nanoparticles were first prepared by
Leff et al. [37] and it was found that amine-capped gold nanoparticles were nearly as stable as their thiol-capped counterparts. Thereafter, Sastry and co-workers [3840] prepared the
fatty amine-capped gold nanoparticles with the simplified
Brust method and studied the interaction between surfacebound alkylamines and gold nanoparticles. We reported the
use of microwave irradiation to prepare amine-capped gold
nanoparticles using a safe organic solvent mixture n-heptane/
ethanol [41]. The average diameters of the obtained gold
nanoparticles were about 4.53 nm. Since gold nanoparitcles
have unique physical and chemical properties and octadecylamine-capped gold nanoparticles (ODA-Au-NPs) can be selfassembled onto a glass surface to form a thick film, we
describe here the application of these ODA-Au-NPs as coating
material for OTCEC. The synthesis of amine-capped gold
nanoparticles and the subsequent coating of the nanoparticles
onto the fused-silica capillary column are described in detail.
The coatings were characterized by UVVis, scanning electron microscopy (SEM), transmission electron microscopy
(TEM), and attenuated total reflectance FTIR spectroscopy
(ATR-FTIR). The effects of various separation parameters
including the percentage of the organic modifier, pH value,

and concentration of the buffer on the separation performance of the OTCEC column were evaluated.
Materials and methods
2.1 Chemicals and materials

Octadecylamine (C18NH2, .98%) was purchased from
Merck (Darmstadt, Germany). Hydrogen tetrachloloroaurate
tetrahydrate (HAuCl4 ?4H2O, A.R.), anhydrous ethanol
(A.R.), 95% ethanol (A.R.), sodium hydroxide (NaOH, A.R.),
sodium dihydrogen phosphate, disodium hydrogen phosphoric acid, chloroform, thiourea, naphthalene, biphenyl,
and HPLC-grade methanol were purchased from Shanghai
Chemical Reagent (Shanghai, China). Testosterone, progesterone, and testosterone propionate were obtained from
SigmaAldrich (St. Louis, MO, USA). Water used in all of the
experiments was doubly distilled and purified by a Milli-Q
system (Millipore, Milford, MA, USA).
2.2 Equipment
A Beckman MDQ P/ACE system (Fullerton, CA, USA) with
an on-column DAD detector was used for all experiments.
Data collection and instrument control were done with the
Beckman 32 Karat software version 4.01 (19992000 Beckman Coulter). Bare fused-silica capillaries (50 mm id, 365 mm
od) were obtained from Yongnian Optic Fiber (Hebei,
China). The total length of the capillary was 60 cm (10 cm
after the detection window). The temperature of the capillary
was maintained at 257C. A Philips (The Netherlands) XL-30
ESEM SEM and a Philips TECNAI-12 TEM were employed
to characterize the surface features of the inner surface of
capillary and the sizes of gold nanoparticles, respectively. An
ATR-FTIR spectroscopy (IFS66/S, BRUKER, Germany) was
used to verify the formation of the layer of ODA-Au-NPs on
the inner surface of capillary column. UV adsorption spectra
were measured on a TU-1800spc UVVis spectro-photometer (Beijing Purkinje General Instrument, China).
2.3 Mobile phase and sample preparation
Stock solution (1 mg/mL) of each sample was prepared in
methanol. Standard working solution was prepared by diluting
the standard stock solution with methanolwater (70/30, v/v) to
reach a final concentration of 0.05 mg/mL for thiourea,
0.03 mg/mL for biphenyl, 0.05 mg/mL for naphthalene, and
0.1 mg/mL for testosterone, progesterone, and testosterone
propionate, respectively. Prior to carry out the chromatographic
separation, all solutions were filtered through a 0.2 mm membrane filter (Schleicher & Schuell, Dassel, Germany). A stock
buffer electrolyte was prepared by dissolving an exact amount of
phosphate in water. The pH value of the phosphate solution was
adjusted to the range of 3.0211.0 by the addition of 0.1 mol/L
CE and CEC
NaOH or 0.1 mol/L HCl solutions. The mobile phase was

obtained by mixing the phosphate solution with the appropriate
amount of water and MeOH. The final mobile phase was filtered through a 0.2 mm membrane filter and degassed in an
ultrasonic bath for 15 min before use.
903
0.5 psi (1 psi = 6894.76 Pa) for 3 s. Before each run, the capillary was rinsed with running buffer for 1 min. In a long
series of runs, the buffer solution was changed after every
four runs to ensure the quality.
2.4 Synthesis of ODA-Au-NPs
3
The method for the synthesis of the ODA-Au-NPs was similar
to that previously reported [41]. Aqueous solution of 9.7 mM
HAuCl4 ?4H2O (0.50 mL) was evaporated to dry in a 100 mL
beaker, followed by the successive addition of 16.00 mL of nheptane, 3.50 mL of ethanol, 0.0523 g of octadecylamine
(molar ratio of octadecylamine to HAuCl4 is 40:1), and
0.50 mL of solution of NaOH in ethanol (0.20 M). The mixture was sonicated to a transparent solution at room temperature. Then the beaker was placed at the center of a domestic
2450 MHz microwave oven (Samsung N8A78, Suzhou Samsung Electronics, China) with a stirring apparatus. After
about 45 s of microwave irradiation at the maximum power
output of 700 W, the orange solution changed to purplish red
and the octadecylamine-capped gold colloid was obtained
after stirring for another 20 min. The volume of the colloid
was reduced to about 2 mL by rotary evaporation and the gold
particles were precipitated from 80 mL anhydrous ethanol at
187C after 20 h. The purplish red solid was filtered out
through a 0.45 mm PTFE filter film, washed several times with
anhydrous ethanol and 95% ethanol, successively, and redispersed into an appropriate amount of chloroform. The drying
C18NH2-capped gold particles were readily soluble in some
nonpolar organic solvents such as chloroform and toluene,
and insoluble in polar solvents such as water and acetone.
2.5 Preparation of OTCEC capillary column
In order to expose the maximum number of silanol groups
on the silica surface, the fused-silica capillaries were pretreated with 1 M NaOH solution for 1 h, followed by rinsing
with deionized water for another hour and then by drying
at ,1807C under nitrogen flow for 1 h. Three methods were
utilized to coat such capillary columns: (i) a solution of ODAAu-NPs in chloroform was pumped through the capillary
with a hand-held plastic syringe and allowed to stay in the
column for 5 min. Then the excess amount of gold solution
was removed by pumping water and the column was stored
in water until use (type I); (ii) the injected solution of ODAAu-NPs was allowed to stand for 30 min and other procedures are the same as type I (type II); (iii) the injection of the
solution of ODA-Au-NPs were repeated for three times
before excess amount of gold solution was removed by water
and other procedures are the same as type I (type III).
Results and discussion
3.1 Characterization of ODA-Au-NPs coating capillary

UVVis spectrophotometry was already reported as an effective method to monitor the evolution of metal species in the
synthesis of colloidal metal clusters [42, 43], because it could
quantitatively measure the concentration and particle diameter of species in the solution based on their corresponding
adsorption spectrum. The ODA-Au-NPs displayed a typical
signal for gold colloids 2 a single plasmon resonance in the
visible region with lmax< 500550 nm [44]. Figure 1 shows
the adsorption spectrum of the colloid formed by ODA-AuNPs dispersion in chloroform. The wavelength of the
adsorption peak maximum of Au nanoparticles is 517 nm.
The TEM micrograph of Au nanoparticles in Fig. 2 shows
that the particle shape of Au nanoparticle is nearly spherical
and the size of them is mostly distributed to the 57 nm
range. The average diameter and the SD of the prepared gold
nanoparticles determined from the histogram (Fig. 2b) are
6.06 nm 6 1.08 nm. The particle size distribution histogram
is obtained on the basis of the measurement of about 700
particles. The result of electron diffraction pattern of gold
nanoparticles, as shown in the insert of Fig. 2a, indicates that
they are face-centered cubic (fcc) polycrystalline gold. Figure
3 shows an SEM of a bare fused-silica capillary column and
an ODA-Au-NPs modified capillary column. Although the
particles can not be observed due to the small size around
6.06 nm, the SEM indicates a dense layer formed by the
adsorption of the ODA-Au-NPs onto the inner surface of the
2.6 Electrochromatographic conditions

The capillary electrochromatographic separation conditions
were as follows: applied voltage 25 kV; sample injection at
Figure 1. UVVis adsorption spectrum of octadecylamine-capped gold colloid in chloroform.
904
Q. Qu et al.
capillary column. ART-FTIR (Fig. 4) shows characteristics of

NH stretching vibration (3302 cm21), CH stretching
vibration (2848, 2917 cm21), and CH bending vibration
(1660 cm21). For comparison, we also used ART-FTIR to
examine the wall of a bare fused-silica capillary column. As
anticipated, no signals of these bonds were found on the
wall. It proves again that ODA-Au-NPs were adsorbed onto
the inner surface of capillary column.
3.2 Effect of ODA-Au-NPs modification on separation
Figure 2. TEM micrograph, electron diffraction pattern (the inset

of a) and histogram of size distribution (b) of the octadecylaminecapped Au nanoparticles. The bar is 50 nm in (a)
Because octadecylamine is a protonated and highly hydrophobic molecule, it can be adsorbed onto the inner surface of
a fused-silica capillary column by electrostatic force and
hydrophobic interaction. The electrostatic force is generated
between negatively charged inner wall of capillary column
and positively charged octadecylamine. The hydrophobic
interaction is generated between hydrophobic SiOSi bond
and octadecylamines long hydrocarbon chains. The adsorption of ODA-Au-NPs onto the fused-silica capillary column is
like the adsorption of CTAB. Both of them have long hydrocarbon bonds and have an amine group. It was found that a
CTAB-modified capillary column could only last one run
[45]. However, our ODA-Au-NPs-modified capillary columns
could last at least 60 runs. One possible reason is the different solubility of CTAB and octadecylamine in polar solution,
while CTAB can dissolve easily in methanolwater solution
and octadecylamine is almost insoluble in polar solution.
Furthermore, the adsorption sites of ODA-Au-NPs and inner
surface of the capillary are covered by ODA hydrocarbon
chains. The polar solution or charged solvent cannot affect
the structure between the inner surface of the capillary column and ODA-Au-NPs easily.
The ODA-Au-NPs-modified capillary column (type I) was
evaluated in the EOF rate measurement using 45 mM phosphate buffer as background solution (Table 1). Thiourea was
chosen as the EOF marker because of no interaction of this
sample with the stationary phase [13]. The suppressed EOF
Figure 3. Scanning electron micrographs of (A) bare fused-silica capillary column and (B) ODA-Au-NPs modified capillary column (type I).
Figure 4. ATR-FTIR spectrograms of an untreated silica capillary

and ODA-Au-NPs-modified capillary (type I).
indicates that the inner surface of the capillary column was

covered partially by ODA-Au-NPs. The residual silanol group
is partially ionized at pH 3 and deprotonated completely at
pH.8 [46]. Therefore, gradually deprotonation of silanol
group with pH value leads to an increase of the EOF mobility.
CE and CEC
905
Octadecylamine with a pKa value around 10.6 is not expected

to influence the EOF behavior in the pH range from 5.0 to
9.0. However, when the pH value is higher than 11, both EOF
behavior and stability of the coating will be changed with pH
value. At much higher pH value, octadecylamine possesses
very few positive charges, which results in the decrease of the
electrostatic force between octadecylamine and ionized silanol group. Furthermore, the electrostatic force between
octadecylamine and negatively charged gold nanoparticles
(surface bound AuCl42 /AuCl22 ions) also decreases, leading
to the desorption of gold nanoparticles from the inner surface. However, there is no obvious evidence to prove the desorption of the ODA-Au-NPs at this high pH value. Compare
the electrochromatograms obtained using pH 7 buffer as
mobile phase after and before the capillary column was
operated using pH 11 and 3 mobile phase, resolution between thiourea and naphthalene was almost the same
(Fig. 5). It demonstrates that most of the ODA-Au-NPs coating is still adsorbed on the inner surface. It is possibly due to
the hydrophobic chain of octadecylamine which effectively
resists pH value change. At low pH value such as 3, the silanol groups are slightly ionized and the electrostatic interaction between inner surface of the capillary wall and ODA-AuNPs should be weak which may lead to the loss of the ODAAu-NPs coating. But there is still no evidence for the desorp-
Figure 5. Comparison of the

electrochromatograms obtained
before and after the ODA-AuNPs-modified capillary column
(type I) was operated with pH 11
and 3 mobile phases. OTCEC
conditions: 60 cm (effective
length 50 cm)650 mm id, mobile
phase, 45 mM phosphate buffer
at pH 7.0 containing 70% v/v
MeOH, injection, 0.5 psi, 3 s,
applied voltage, 25 kV, detection, 200 nm. Analyte peaks in
order of elution: thiourea, naphthalene, and biphenyl.
906
Q. Qu et al.
Table 1. Electroosmotic mobilities in the prepared capillaries and

effect of pH on the resolution and number of theoretical
plates height
Bare fused-silica
capillarya)
meo (61024 cm2 ?V21 ?s21)
Nc) (Rs)
3.32
n/ad)
After treatment with ODA-Au-NPsb)

pH 9.0
pH 8.0
pH 7.0
pH 6.0
pH 5.0
1.50
1.44
1.27
0.90
0.62
26.8 (0.9)
24.1 (1.1)
22.6 (1.4)
16.9 (1.3)
10.3 (1.0)
a) EOF
marker,
thiourea.
Bare
fused-silica
capillary
(60 cm650 mm id); the effective length of the column is 50 cm;
running buffer, 45 mM phosphate buffer at pH 7.0; injection,
0.5 psi for 3 s; UV detection wavelength at 200 nm; applied
voltage, 25 kV. n = 4.
b) Column with an ODA-Au-NPs coating (type I). Mobile phase:
45 mM phosphate buffer containing 70% v/v MeOH. Other
conditions as in (a).
c) N, average theoretical plate in 10 000/m for naphthalene; Rs,
resolution between thiourea and naphthalene.
d) n/a, not applicable.
tion of the ODA-Au-NPs coating (Fig. 5). This test was done
in 3 days and three injections were performed for each pH
value every day. Totally nine injections were carried out at
each pH value. However, no significant deviation of retention
times and deterioration of peak shapes were found for the
three compounds tested (1.2 and 1.6% RSD of retention time
for naphthalene and biphenyl, respectively). These results
confirmed the function of the highly hydrophobic chain of
ODA-Au-NPs. Thus, the ODA-Au-NPs-modified capillary
column is stable at pH values from 3 to 11.
In order to study the effect of buffer pH value on the
separation efficiency, OTCEC was performed in the mobile
phase containing 70% v/v methanol and 45 mM phosphate
buffer in a pH range of 5.0 to 9.0. The effects of mobile phase
pH value on the EOF, theoretical plate number of naphthalene, and resolution between thiourea and naphthalene were
listed in Table 1. In accordance with electrophoretic theory, the
EOF increased with the pH value of the mobile phase due to a
higher charge density at the silica surface [47]. The theoretical
plate number also decreased with decreasing pH value because
the constant B in Golay equation increased with the decreasing
EOF. However, resolution did not decrease as expected with
pH value. In the pH range of 9.0 to 7.0, the resolution
increased with decreasing pH value. It was because the decrease of pH value resulted in a decrease of EOF which
increased the interaction time between the analytes and the
stationary phase. However, the theoretical plate number of the
analytes only decreased slightly with decreasing pH value from
9.0 to 7.0. That means the peak width only broaden a little.
Therefore, the resolution increased with decreasing pH value
in the pH range from 9.0 to 7.0. However, it was noticed that

the retention time became longer with a running electrolyte at
low pH value, which resulted in some loss of column efficiency. This could explain the decrease of resolution when the
pH value changed from 7.0 to 5.0. Relatively high column
efficiency and optimum resolution were achieved at pH 7.0.
3.3 Effect of ionic strength on chromatographic
retention
Another factor that influences both separation efficiency and
resolution on this ODA-Au-NPs-modified capillary column in
OTCEC mode is the concentration of the aqueous buffer. In
order to examine the effect of ionic strength, the experiments
were performed in phosphate buffer containing 70% methanol
at pH 7.0 by using type I capillary column. It is found that EOF
decreased gradually, while peak efficiency and resolution
increased at higher ionic strengths and decreased at very high
ionic strength (higher than 55 mM) (Table 2). The increase of
peak efficiency and resolution with increase of ionic concentration from 35 to 55 mM is easy to explain because the electroosmotic mobility decreased at higher ionic strength resulting
from the decrease of double layer thickness and the increase of
viscosity [48]. The peak efficiency and resolution decreased at an
ionic concentration higher than 55 mM. These effects have also
been observed by other authors [4851]. It was reported that in
OTCEC, term B in plate height equation only changed a little bit
while term C dramatically decreased with the increase of buffer
concentration [51]. Their experimental results suggest that the
mass transfer resistance decreased with increasing buffer concentration because of the decrease of EOF, consequently the
peak efficiency increased. However, too high ionic strength of
mobile phase would cause Joule heating and lead to bubble formation and current breakdown. Thus, a relatively optimal
phosphate concentration of 55 mM was selected.
Table 2. Effect of ionic strength on separation efficiency and resolution on the ODA-Au-NPs-modified capillary
Concentration
(mM)
Thiourea and
naphthalene
35
N
a)
45
Rs
20.9 1.1
55
Rs
22.6 1.4
65
Rs
28.3 1.8
Rs
24.1 1.3
Experimental conditions are the same as in Fig. 5.

a) n = 4; N, average theoretical plate in 10 000/m for naphthalene.
3.4 Effect of MeOH on electrochromatographic

separation
Under the optimum ionic strength and pH of the mobile
phase (55 mM phosphate buffer, pH 7.0), the effect of MeOH
in the mobile phase on the EOF mobility, resolution, and
CE and CEC
number of theoretical plates was studied (Fig. 6, in this figure,

the y-axis on the right side represents both resolution and
column efficiency (100 000) and the left side represents EOF).
Obviously, MeOH in the mobile phase has an essential effect
on the retention factor. The results showed that EOF velocity
decreased slightly when MeOH concentration increased from
40 to 50%, but further increase of MeOH concentration
caused a sharp increase of the EOFmobility. From Fig. 6, it can
be found that the number of theoretical plates of naphthalene
always increased with increasing MeOH concentration. This
is because the more organic modifier in the mobile phases the
more solutes partition into the mobile phase. As a result, the
interaction between the solutes and the stationary phase was
reduced. Therefore, the retention times of the solutes
decreased and consequently the B term decreased when a
high percentage of MeOH was used. Thus, the number of
theoretical plates of the solutes increased with the increase of
MeOH percentage. The resolution decreased dramatically
with MeOH concentration in the range from 40 to 60% v/v.
When MeOH concentration was increased from 60 to 70% v/
v, the resolution only decreased a little bit. It is probably due to
the stationary phase that was wetted better in the higher
organic range, leading to high column efficiency, which, in
turn, would offset the decrease in resolution. MeOH (70%)
was selected as the optimum organic modifier content.
Figure 6. Effect of MeOH in polar organic mobile phase on EOF

mobility, resolution of thiourea and naphthalene, and peak efficiency of naphthalene. Separation conditions: mobile phase,
55 mM phosphate at pH 7.0. The y-axis on the right side represents both resolution and column efficiency (100 000) and the left
side represent EOF. All other conditions are held constant and are
the same as in Fig. 5.
RSDs were 2.2, 3.1, and 4.3%, respectively, using the same
optimum experimental conditions as for type I. These experimental results indicated that ODA-Au-NPs can be adsorbed
onto the inner surface of the capillary column very quickly.
Therefore, the procedure for type I column preparation was
chosen as the best method since it needs the shortest time.
3.6 Stability of ODA-Au-NPs-modified capillary
columns
The repeatability of the ODA-Au-NPs-coated capillaries was
investigated by conducting 15 consecutive runs in the same
capillary within 10 h; the electrochromatograms obtained after
1st and 15th injection on ODA-Au-NPs modified capillary column are shown in Fig. 7. The run-to-run repeatability of retention time for biphenyl was good with an RSD value of 1.6%
(Table 3). The retention time from the 60th injection for naph-
Figure 7. Comparison of electrochromatograms obtained from a

ODA-Au-NPs-modified capillary column after 1st and 15th injection. Experimental conditions: mobile phase, 55 mM phosphate
buffer at pH 7.0 containing 70% v/v MeOH. Other conditions are
the same as in Fig. 5.
Table 3. Retention time repeatabilities of ODA-Au-NPs-modified

capillary columns (RSD%)
3.5 Character of different types of ODA-Au-NPsmodified capillary columns

Two other types of ODA-Au-NPs-modified capillary columns
were tested under the optimum conditions developed for the
type I. The column-to-column reproducibility of retention time
for thiourea, naphthalene, and biphenyl was investigated. The
907
Run-to-run (n = 15)
Day-to-day (n = 15 within
first 5 days)
Column-to-column (n = 6)
Thiourea
Naphthalene
Biphenyl
1.1
2.3
1.7
3.8
1.6
4.2
4.5
5.1
6.3
908
Q. Qu et al.
thalene only slightly decreased 17.23 min (n = 1) to

17.06 min (n = 15). These capillaries were stable for up to
20 days and no observable changes in the separation efficiency were found after at least 60 injections. The retention
time from the 60th injection for naphthalene only slightly
decreased from 17.23 min (n = 1) to 17.03 (n = 60). However,
when dodecanethiol gold nanoparticles were used directly as
coating material for CEC separation, the capillary was not
stable even only after nine runs [13]. Although the ODA-AuNPs coating is more stable than dodecanethiol gold nanoparticles coating, there are obvious tailings in the peaks at
the 15th and 60th injections. It indicated that small amount
of ODA-Au-NPs coatings were desorbed from the capillary
column during the separation. So more silanols appeared
and tailings occurred. However, the tailing did not become
worse even after 60 times injection. That means the amount
of coating desorbed from the surface of the capillary column
become less and less during the separation. Thus, ODA-AuNPs-modified column can last a long time. The low RSD
values of day-to-day and column to column reproducibility of
retention time for the test compounds (Table 3) proved again
this standpoint.
increase the phase ratio, such as using etching method to

increase the inner surface area of the wall or using layer-bylayer technology to increase the thickness of the coating, is a
logic step for the next project. Notwithstanding its limitation,
this study suggests that ODA-Au-NPs can be utilized easily
for separation of many types of compounds.
Concluding remarks
This study shows that ODA-Au-NPs can be easily adsorbed

onto the inner surface of a fused-silica capillary column and
act as a novel stationary phase for CEC to be used for
separation of neutral aromatic compounds and pharmaceutical steroid drugs. This stationary phase is stable at varying
pH and percentage of methanol in the mobile phase. In
addition, the experimental results demonstrate that this system exhibits reproducible retention times and high separation efficiencies. Since there is a strong interaction between
gold nanoparticles and biomolecules, the ODA-Au-NPsmodified capillary column is expected to be more useful in
bioanalysis.
3.7 Separation of steroid hormones

The modified capillary column was used to separate three
neutral steroid hormones: testosterone, progesterone, and
testosterone propionate (Fig. 8). Baseline separation of these
three compounds was achieved with theoretical plate numbers per meter of 189 000, 155 000, and 87 800 for
testosterone, progesterone, and testosterone propionate,
respectively. Nevertheless, as can be seen from Figs. 7 and 8,
the signals are low and the baseline noise is relative big.
Apparently, this is the drawback of the ODA-Au-NPs-coated
capillary caused from the low loading capacity of this type of
column which is also the drawback of the separation mode of
open tubular column. Thus, developing some methods to
Valuable discussions of the manuscript with Dr. Yanhua

Zhang, Department of Chemistry, University of Chicago, during
its preparation are highly appreciated. This work was supported
by the Natural Science Foundation of Yangzhou University
(HK0513103), Foundation of Jiangsu Provincial Key Program
of Physical Chemistry in Yangzhou University, and the National
Natural Science Foundation of China (20675071, 20773105).
The authors have declared no conflict of interest.
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Open-Tubular Capillary Electrochromatography Using A Capillary Coatedwith Octadecylaminecapped Gold Nanoparticles

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Electrophoresis 2008, 29, 901909

Jiangsu Key Laboratory of

Received June 6, 2007