Journal of Chromatography A, 1218 (2011) 7663–7669

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

On-line concentration sample stacking coupled with water-in-oil microemulsion

electrokinetic chromatography
Hsi-Ya Huang ∗ , Wan-Ling Liu, Brenda Singco, Shih-Huan Hsieh, Yung-Han Shih
Department of Chemistry and Center for Nanotechnology at CYCU, Chung Yuan Christian University, 200 Chung Pei Road, Chung-Li, 320, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: This study describes for the first time, the ability of a normal stacking mode (NSM) on-line concentration
Available online 1 June 2011 step coupled with water-in-oil (W/O) microemulsion electrokinetic chromatography (MEEKC), using six
common penicillin antibiotics (oxacillin, penicillin V, penicillin G, nafcillin, ampicillin, and amoxicillin) as
Keywords: test analytes. Optimization of penicillin separation in the conventional W/O MEEKC system demonstrated
Water-in-oil microemulsion that change in the type and concentration of the oil phase (1-butanol) and column temperature had a
Microemulsion electrokinetic
pronounced effect on the separation. With the subsequent development of the NSM coupled with W/O
chromatography
MEEKC, improved separation and detection sensitivities were observed when an organic solvent plug
On-line concentration
Food sample
(1-propanol; 1.04 cm) was placed between the W/O microemulsion and the sample solutions. This could
be attributed to the solution viscosity difference between the aqueous sample zone and the organic
solvent plug causing the penicillin to be stacked in this 1-propanol plug. The optimal NSM W/O MEEKC
provided about 12-fold increase in detection sensitivity compared with conventional sample injection
(50 mbar, 3 s). Finally, this proposed method was successfully applied in the analyses of several food
samples (porcine organs) spiked with penicillin.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction of W/O MEEKC as separation strategy for a range of acids, bases

Microemulsions are composed of water-immiscible organic sol-
vent (oil), water, surfactant and/or cosurfactant (medium chain
alkyl alcohol). When the surface tension between an immiscible
organic solvent (oil) and water is reduced with the aid of surfac-
tants and/or co-surfactants, stable, dispersed, and surfactant coated
nanometer-sized droplets are created in microemulsion solutions
[1–3]. These nanometer-sized spherical droplets, which can either
consist of water droplets in an oil continuous phase (W/O) or oil
droplets in a water continuous phase (O/W), have been demon-
strated as promising separation media to influence the separation
behavior of analytes in microemulsion electrokinetic chromatogra-
phy (MEEKC) [4–19]. Most of the MEEKC reports are focused on the
evaluation of O/W microemulsions for the separation of lipophilic
and hydrophilic compounds [7–16]; however, there have been
very few reports on the use of W/O microemulsions as separation
media [17–19]. Altria et al. used water-in-oil (W/O) microemul-
sions as running solution in MEEKC for the first time, and a uniquely
different separation from O/W MEEKC was obtained [17]. More-
over, the same research group also demonstrated the usefulness
∗ Corresponding author. Tel.: +886 3 2653319; fax: +886 3 2653399.
E-mail address: hyhuang@cycu.edu.tw (H.-Y. Huang).
and neutrals; with emphasis on the method’s suitability for very
water-insoluble drug compounds [18]. Recently, Li et al. compared
the separation ability of both O/W and W/O MEEKC with thirteen
phenolic acids and diterpenoid compounds. They found out that
oil and organic modifier concentrations were the main influenc-
ing factors in both MEEKC systems, whereas oil type and surfactant
concentration had only little effect on W/O MEEKC separation [19].
Regardless of the high separation efficiency of MEEKC for a
wide range of analytes [4–19], inadequate detection sensitivity was
still observed when it was used for the trace compound analyses.
Presently, there have been many reports on the use of an on-line
sample concentration technique such as field-amplified sample
injection (FASI), anion-selective exhaustive injection (ASEI) and
reversed electrode polarity stacking mode (REPSM) in combination
with O/W MEEKC separation in order to enhance their detection
ability [20–28]. To our knowledge, no studies employing an on-
line concentration step coupled to W/O MEEKC analyses have been
reported to date.
There have been more than 30 chromatographic reports every
year on penicillin analyses for the last decade. This implies that
the development of novel chromatographic methods for peni-
cillin determination still receives considerable attention. Penicillin
residues analyses in environmental and food samples by O/W
MEEKC coupled with an on-line sample concentration step have

0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2011.05.091
7664 H.-Y. Huang et al. / J. Chromatogr. A 1218 (2011) 7663–7669
been previously reported [21,25], but separation by W/O MEEKC
method has never been documented.
The purpose of this study was to develop a novel W/O MEEKC
method for penicillin antibiotics; concomitantly, the feasibility of
an on-line concentration procedure combined with W/O MEEKC
was evaluated for the first time. Several on-line concentration
strategies coupled with O/W MEEKC mentioned above can provide
high sensitivity with a large-amount sample introduction. Intro-
duction of a large sample zone into the capillary, however, would
likely cause phase separation if applied in W/O microemulsion.
In addition, an acidic running solution (pH 2) was often used in
FASI- or ASEI-O/W MEEKC reports to suppress EOF. In this study a
basic solution (pH 8) W/O microemulsion is needed to convert test
molecules into anions to maintain an adequate migration velocity
because of a very low EOF produced in W/O MEEKC. Consequently,
a normal stacking mode (NSM) on line concentration W/O MEEKC
using a basic running buffer and a medium amount of sample
requirement would be used. The NSM strategy reported for the
first time by Liu et al. [29] has been successfully used to concen-
trate cationic or neutral analytes in a single-run sweeping step
[30–33]. Since a specific combination of surfactant, oil and water
concentration is required to form the W/O microemulsion phase,
this NSM strategy used for penicillin analyses needs to be modified
so that a stable microemulsion phase can be maintained when cou-
pled to W/O MEEKC separation. Finally, after a simple solid phase
extraction (SPE), as low as 50 ␮g/L level of penicillin antibiotics was
determined in real food samples (porcine organs) by the optimal
NSM-W/O MEEKC method, in which this spiked concentration was
referred to the maximum residue limits (MRLs) of penicillins in ani-
mal tissues (25–300 ␮g/kg) in the European Union and in Taiwan
[34].
2. Experimental
2.1. Chemicals and reagents
Oxacillin (O) (pKa1 = 2.61) was purchased from Fluka (Buchs,
Switzerland). Penicillin V (PV) (pKa1 = 2.62) was purchased from
ICN (OH, USA). Penicillin G (PG) (pKa1 = 2.62), ampicillin (Amp)
(pKa1 = 2.62, pKa2 = 7.4), amoxicillin (Amo) (pKa1 = 2.62, pKa2 = 7.2,
pKa3 = 9.6) and methyl paraben were purchased from Sigma (St.
Louis, MO, USA). Nafcillin (N) (pKa1 = 2.61) was purchased from MP
(Eschwege, Germany). The above-mentioned test analytes were
individually dissolved in deionized water at a stock concentra-
tion of 1 mg/mL. Internal standard (methyl paraben, 100 ␮g/mL)
was dissolved in deionized water. Stock solutions of standards and
internal standard were prepared weekly. All other chemicals were
reagent-grade.
Fig. 1. Illustrations of a modified NSM step coupled to W/O MEEKC model. (Step 1) A capillary was conditioned with a W/O microemulsion solution of pH 8.0, and then
an organic solvent plug (1-propanol; 50 mbar for 10 s) was introduced into the capillary. The penicillin sample prepared in deionized water was pressure-injected into the
capillary (50 mbar for 60 s). (Step 2) W/O microemulsion solution was placed at the inlet end of the capillary followed by the application of a 29 kV.
2.2. Sample pretreatment
Test samples (porcine liver and kidney) were obtained from
supermarkets in Taiwan. The porcine samples (100 g) with or with-
out spiking penicillin standards (50 ␮g/L) and IS (25 ␮g/L) were
vortex-mixed twice with 50 mL of acetonitrile for 10 min. The mix-
ture was centrifuged for 10 min at 6000 rpm, and the resulted clear
liquid was diluted with 50 mL of n-hexane followed by vortex-
mixing for 30 min. After the n-hexane organic layer was discarded,
the acetonitrile–aqueous liquid was collected and then was oven-
dried at 120 ◦ C. This extraction procedure followed the previous
method [35], with slight modification. The dry residues were dis-
solved in deionized water (10 mL) ready for SPE treatment as
described below.
C18 column (LC-18; Supelco, Bellefonte, PA, USA) used in SPE
was conditioned prior to use by washing with methanol (5 mL)
and deionized water (5 mL). After the addition of the clear sample,
the extraction column was washed with deionized water (2 mL)
and then air-flushed to dry. The absorbed penicillins and I.S. were
eluted from the column with 5 mL ACN at an approximate rate of
0.5 mL min−1 , and the eluted solution was then dried in an oven at
120 ◦ C. Finally, a 1 mL deionized water was added to the final dried
residues followed with a 0.45 ␮m membrane filtration, and then
the filtrate was ready to be analyzed by NSM-W/O MEEKC.
2.3. Preparation of microemulsion for NSM-W/O MEEKC
All microemulsions were prepared on a w/v basis (g/mL) in
70 mM sodium acetate solution (pH 8). A sodium acetate solu-
tion of pH 8 at a concentration of 70 mM was prepared by adding
0.574 g sodium acetate into 100 mL of deionized water. The opti-
mal W/O microemulsion solution was composed of 5% surfactant
(sodium dodecyl sulfate, SDS), 80% oil (1-butanol), and 15% sodium
acetate solution at pH 8. After the addition of SDS to the oil
phase, the solution was mixed with sodium acetate solution fol-
lowed by sonication for 30 min until an optically transmittance W/O
microemulsion was formed. This sequence was to ensure that the
W/O system will not pass through an emulsion phase, which might
be difficult to break [17,18]. The microemulsion solution was not
filtered prior to use.
2.4. Apparatus and operating conditions for CE
All experiments were performed with a Hewlett-Packard 3D
capillary electrophoresis system equipped with a 3D UV–vis detec-
tor (Waldbronn, Germany). Agilent Chemstation software was used
for instrumental control and data analysis. Separations were per-
formed in a 48.5 cm total length (40 cm to detector) of 50 ␮m ID
 H.-Y. Huang et al. / J. Chromatogr. A 1218 (2011) 7663–7669
uncoated fused-silica capillary (Polymicro Technologies, Phoenix,
AZ, USA). The capillaries were conditioned prior to separation by
washing with 0.1 M sodium hydroxide (1 min), deionized water
(2 min), then with running solution (5 min). Sample and standard
solutions were pressure-injected into the capillary column for W/O
MEEKC (0.12 cm) (50 mbar, 3 s) and NSM-W/O MEEKC (2.4 cm)
(50 mbar, 60 s). Separations were carried out using an electrical
voltage of −29 kV. The temperature of the capillary was maintained
at 37.5 ◦ C, while the detection wavelength was fixed at 200 nm.
2.5. NSM-W/O MEEKC model
The optimal NSM W/O MEEKC method used in this study is
shown in Fig. 1. First, the capillary was filled with a pH 8 microemul-
sion solution, and then an organic solvent plug (1-propanol,
50 mbar for 10 s, 1.04 cm) was introduced into the capillary. The
penicillin sample prepared in deionized water was introduced
hydrodynamically into the capillary (50 mbar for 60 s) (step 1).
After the sample was injected, a separation voltage of −29 kV was
applied with the microemulsion solution in the inlet vial (step 2).
This step allowed the analytes to be stacked in the organic solvent
zone because of the difference in solution viscosity; meanwhile, the
negatively charged water droplets also entered the stacked sample
zone to sweep the analytes, then the separation proceeded by W/O
MEEKC.
3. Results and discussion
3.1. Optimization W/O MEEKC method for penicillin separation
First, a W/O MEEKC method was developed for the penicillin
analyses. In order to achieve a baseline separation, microemulsion
composition (concentration or type of surfactant and oil) and col-
umn condition (temperature and applied voltage) were examined.
SDS, which is a commonly used surfactant either in O/W or W/O
MEEKC system, was used in the study, and the influence of SDS con-
centration on penicillin separation was then examined. Unlike in
most O/W MEEKC wherein the SDS concentration was around 3%, a
SDS level higher than 10% was often used in W/O MEEKC to increase
solution conductivity and lower surface tension [17–19]. However,
deposition of SDS on the CE electrodes occurred and a frequent
electrode cleanup had to proceed between several runs. To avoid
surfactant deposition, the SDS level used in this W/O MEEKC study
was kept in the range of 3–6%. The results indicated that variation in
the SDS concentration influenced the resolutions significantly, but
changed the migration velocities slightly for all analytes. Since a 5%
SDS level produced more efficient separation, thus it was chosen as
the optimal surfactant level in the study.
Next, the effect of oil type on the penicillin separation was
examined. Two short-chain alcohols, 1-butanol and 1-pentanol,
which are more miscible in water than other long-chain alkanes
or alcohols, are commonly used as oil phases in W/O microemul-
sion. Due to the high viscosity of 1-butanol and 1-pentanol,
the W/O microemulsion with high levels of alcohol (e.g., 70%
or 80%) only produced a little electroosmotic flow (EOF) dur-
ing electrophoresis; as a result, penicillin migration was only
dependent on its electrophoretic mobility or its affinity with
negatively charged water droplets. To increase the EOF mobil-
ity, a lower viscosity of alcohol (1-propanol) was used as the oil
phase in the study (e.g., the EOF mobility is 4.1 × 10−9 , 1.5 × 10−8
and 4.5 × 10−8 m2 V−1 s−1 for 1-propanol, ethanol and methanol,
respectively [36]). Penicillin separations with different oil phases
(1-propanol, 1-butanol or 1-pentanol) in W/O MEEKC were com-
pared (data not shown), and better resolutions and rapid migrations
were still obtained with 1-butanol as the oil phase. Subsequently,
7665
a series of W/O microemulsion solutions with different concen-
trations of 1-butanol (75%, 77.5% and 80%) were evaluated for
penicillin separations, in which the total amount of 1-butanol and
aqueous buffer was kept constant (i.e. 95% in microemulsion solu-
tion). The result indicated that with an increase in 1-butanol level
(i.e. decreased water level) the separation velocity and the peak
resolution for all tested analytes improved (data not shown). This
phenomenon could be attributed to fewer water droplets formation
at higher 1-butanol level resulting to a more negatively charged SDS
carried by each water droplet. Therefore, these hydrophilic analytes
that can strongly partition with the water droplets would migrate
rapidly toward the detector (i.e. positive polarity electrode). Thus,
the 80% 1-butanol that provided the highest resolution and best
peak symmetry was chosen as the optimal oil level in the study.
Similar to previous W/O MEEKC studies wherein an additional
oil phase (e.g., 2% heptanes or octane) improved microemulsion
stability but no influence in the separation [17,18], the incorpora-
tion of long-chain alkane to this three-component microemulsion
system of SDS–1-butanol–sodium acetate buffer had no improve-
ment in the penicillin separation, therefore, no additional oil phase
was used.
Several column variables such as applied voltage and column
temperature were also evaluated for enhancing the penicillin
separations. An increase in the applied voltage (or column tem-
perature) sped up penicillin separation, thus, 37.5 ◦ C and −29 kV
were chosen as the optimal column temperature and applied volt-
age, respectively; where these analytes had resolutions of 1 and
the whole separation was completed within 9 min (Fig. S1, supple-
mentary data). In contrast to previous W/O MEEKC reports wherein
10–18% SDS and 2–8% octane or 2-propanol was often used to
improve separation [17], lower surfactant amount (5% SDS) and
no extra oil phase were necessary in this case.
3.2. On-line concentration NSM step coupled to W/O MEEKC
The above results indicated that a W/O microemulsion solution
composed of 5% (w/v) SDS, 80% (w/v) 1-butanol (oil phase), and
15% (v/v) sodium acetate solution (pH 8, 70 mM) provided good
separation within 9 min but with low sensitivities (Fig. S1, supple-
mentary data), with LODs in the range of 0.6–1.0 ␮g/mL). Various
on-line concentration strategies including FASI, ASEI and REPSM
have been successfully combined with O/W MEEKC in the past,
however, failed separations often happen when these modes were
used to concentrate analytes in the W/O MEEKC. This is possibly due
to the low-conductivity solution or sample zone introduced into the
capillary, by which the “extra” solution zone created would likely
cause the phase separation of W/O microemulsion. Furthermore,
in contrast to previous FASI- or ASEI-O/W MEEKC reports wherein
an acidic running solution (pH 2) was used to suppress EOF and to
speed up analyte separation, in this study a W/O microemulsion
composed of basic sodium acetate solution (pH 8) is necessary in
order to convert penicillin molecules into anion forms (pKa s (or
pKa1 ) are around 2.6) and, in turn, maintain an adequate migra-
tion velocity. Consequently, a NSM procedure was used to on-line
concentrate penicillin solutes in this W/O MEEKC.
3.2.1. Effect of acidic solution plug
A conventional NSM mode was first used to concentrate peni-
cillin solutes, in which the penicillin sample prepared in deionized
water was pressure-injected into the capillary (10 ␮g/mL; 50 mbar
for 30 s). The results indicated that all analytes’ signals were obvi-
ously enhanced by the conventional NSM mode; however, the
resolution of penicillin G and nafcillin became worse (R = 0.96) as
well as broaden analyte signals were observed (Fig. 2a). Subse-
quently, this NSM procedure was further modified as shown in
Fig. 1: an acidic solution or organic solvent plug was placed between
7666 H.-Y. Huang et al. / J. Chromatogr. A 1218 (2011) 7663–7669

60 60
(a) No acidic or organic solvent plug
40 Cyclohexanol
40 1 2 3,4
20 6
5
mAU

5
20 123 6 0
4
60
0
40 1-Butanol
12 5
34 6 I.S.
60 20
(b) acidic plug (0.3 cm; 50 mbar, 10 s) 0
40 60

mAU
1-Propanol
mAU

40
20 2 5
1 3 4 6 I.S.
1 23, 4, 56 20

0 0
60 I.S.
60 Ethanol
40
(c) organic solvent plug (1.04 cm; 50 mbar,10 s) 12 5
34 6
40 20
23 5 0
mAU

1 6
20 4 60

40
Methanol
0 12 5
20 34 6 I.S.

7 8 9 10
Time (min)
Fig. 2. Electropherograms of penicillin compounds on NSM-W/O MEEKC with or
without acidic/organic solvent plug. W/O microemulsions composed of 5% SDS, 80%
1-butanol, and 15% sodium acetate solution (pH 8) was used as the running solution.
No acidic or organic solvent plug was used in (a); while an acidic plug (b) or organic
solvent plug (c) was pressure-injected into the capillary for 10 s (50 mbar) before
sample injection. The concentration of analyte standard was maintained at 10 ␮g/mL
for each analyte, and deionized water was used as sample matrix. Samples were
pressure-injected into the capillary (50 mbar, 30 s). Oxacillin (1), penicillin V (2),
penicillin G (3), nafcillin (4), ampicillin (5), and amoxicillin (6).
the W/O microemulsion and the sample zone. All penicillins that
have pKa s (or pKa1 ) around 2.6 are present as cation or neutral
forms in an acidic solution. Once the penicillin anions entered an
acidic zone (pH 2), they would be converted to neutral or cationic
solutes, thus a better stacking effect was possibly obtained. How-
ever, the detection sensitivity of all the analytes did not improve
and even a poor separation was observed when a short acidic plug
(50 mM, phosphate solution of pH 2) was used (Fig. 2b). A most
probable reason for this could be that the presence of a phosphate
solution (0.3 cm length) in the capillary inlet end would cause a
phase change in the subsequent W/O microemulsion leading to an
inadequate separation and stacking effects.
3.2.2. Effect of organic solvent plug
Next, an organic solvent zone (1-propanol) in lieu of an acidic
plug was further employed in this modified NSM (Fig. 1). All peni-
cillins had better sensitivity and resolution (Fig. 2c) compared
with conventional NSM mode (i.e. without organic solvent plug)
(Fig. 2a) (N and R were in the range of 320,000–634,000 plates and
1.38–2.38 for Fig. 2c, and were in the range of 200,000–552,000
plates and 0.96–2.10 for Fig. 2a). 1-Propanol has a higher viscosity
than the aqueous sample zone (i.e. deionized water and penicillin
anions), thus its presence in the study should play a similar role
with poly(ethylene oxide) (PEO) in protein stacking [37–39] (e.g.,
0.89 cP and 1.9 cP at 25 ◦ C for water and 1-propanol, respectively).
When the penicillin anions in the sample zone migrated against
0
7 8 9 10 11
Time (min)
Fig. 3. Electropherograms of penicillin compounds on NSM-W/O MEEKC with
different alcohols used as organic solvent plugs. Several alcohols including cyclo-
hexanol, 1-butanol, 1-propanol, ethanol and methanol were used as organic solvent
plug, and the injection length for each organic solvent was maintained at 1.04 cm.
All other conditions were the same as in Fig. 2.
the reversed EOF (produced in the sample zone) and entered the
organic solvent zone (1-propanol), their electrophoretic mobili-
ties were likely retarded via this high-viscosity 1-propanol zone
(1.04 cm in length) placed in front of the sample zone. Therefore,
the decrease in migration velocity caused the stacking of peni-
cillin anions in the interior of the organic solvent zone, which
resulted in a higher detection sensitivity and resolution. To clarify
this assumption, several alcohols with differing viscosities includ-
ing cyclohexanol, 1-butanol, 1-propanol, ethanol, and methanol
(with viscosities of 41.7, 3.0, 1.9, 1.0 and 0.5 cP at 25 ◦ C, respec-
tively) were used as the organic solvent plugs, and their effect
on the penicillin stacking were compared (Fig. 3). Compared to 1-
propanol, these two alcohols, 1-butanol and cyclohexanol, created
a larger viscosity difference with the sample zone, but their insol-
uble nature with aqueous sample reduced the penicillin stacking
in the modified NSM step. Consequently, 1-propanol improved the
sensitivities and resolution of the test analytes separation, thus it
was employed as the organic solvent zone in the modified NSM
procedure.
In addition, the length of the organic solvent plug (1-propanol),
which varied from 0.52 to 1.56 cm (50 mbar; 5–15 s) was examined
to optimize penicillin stacking, and results indicated that the migra-
tion time of penicillin analytes was increased with the 1-propanol
length (e.g., amoxicillin that has the slowest migration among six
penicillins was detected at 9.1 min and 9.7 min at 0.52 cm and
1.56 cm 1-propanol, respectively), but there was no apparent influ-
ence on the stacking abilities (supplemental information, Fig. S2).
This observation confirmed that the migration of penicillin anions
toward the detector was really restricted by the higher-viscosity 1-
 H.-Y. Huang et al. / J. Chromatogr. A 1218 (2011) 7663–7669 7667

60 respectively [36]). Finally, a 1.04 cm 1-propanol (50 mbar, 10 s) that

(a) W/O MEEKC provided the best penicillin resolution was used as the optimal
(50 mbar, 3 s) organic solvent plug in the study.
40
23
5 6
1 4 3.2.3. Effect of sample injection time on penicillin stacking
20
The analyte amount that was pressure-injected into the capil-
lary possibly influenced the detection sensitivity. Thus, the sample
0 injection time variation from 30 s to 90 s (50 mbar) was evalu-
ated, and their effects on penicillin stacking were also compared
60 (supplemental information, Fig. S3). Results obtained from dif-
(b) W/O MEEKC ferent sample injection time showed that when this was raised
(50 mbar, 60 s) from 30 s to 60 s, a 2-fold enhancement in penicillin sensitivity
40
was observed with no reduction in resolution and performance.
mAU

A further increase in the sample injection time to 90 s, worsen

12 5
20 34 the resolution and peak intensity of the three analytes (penicillin
6
G, nafcillin, and ampicillin). In addition, the penicillin separation
time became longer, which could be attributed to the stronger EOF
0
formed in this longer sample zone that limited the penicillin anions
migration toward the organic solvent zone. Consequently, 60 s sam-
60 ple injection (50 mbar) that provided a baseline separation and
(c) NSM-W/O MEEKC
(50 mbar, 60 s) better sensitivity enhancement was employed in this NSM-W/O
40 MEEKC method.
2
3
5 In addition, to investigate if the variation in the peak intensities
1 4 6 I.S. observed in Fig. 3 is due to a sharpening phenomenon caused by a
20
small solvent plug or some variations in the sample loading, the test
samples (5 ppm per analyte) were introduced with the same sam-
0 ple injection time (60 s, 50 mbar) by normal (i.e. without 1-propanol
7 8 9 10 11 plug) and NSM modes (i.e. with 1-propanol plug, 1.04 cm), respec-
Time (min) tively. Results indicated that the presence of 1-propanol improved
the peak intensities, efficiencies as well as resolutions (Fig. 4) and
Fig. 4. Electropherograms of penicillin standards determined by W/O MEEKC (a, the observed peak intensity variations were due to the sharpening
b) and NSM-W/O MEEKC (c). Penicillin standards were pressure-injected into the effect produced by the short organic plug.
capillary at 50 mbar for 3 s (100 ␮g/mL) (a) or 60 s (5 ␮g/mL) (b) for W/O MEEKC,
and 50 mbar for 60 s (5 ␮g/mL) (c) for NSM-W/O MEEKC. All other conditions were
the same as in Fig. 2. 3.3. Performance, qualitative precision, and recovery of
NSM-W/O MEEKC

propanol zone. The reversed EOF produced in the 1-propanol plug
possibly caused the decrease in the analyte’s migration, however,
compared with that of the aqueous sample zone, this reversed EOF
seemed too weak to slow down these analytes (EOF mobilities (eo )
are 7.6 × 10−8 and 4.1 × 10−9 m2 V−1 s−1 for water and 1-propanol,
Comparison of the electropherograms indicated that the reso-
lutions of most penicillins became better with NSM W/O MEEKC
separation method than with conventional mode (Fig. 4) (R was
in the range of 1.9–2.1 and 1.2–2.5 for the NSM and conven-
tional modes, respectively; N was in the range of 194,000–402,000

Table 1
Performance of on-line concentration W/O MEEKC method for penicillin compounds.

Analytes SERheight a LOD (␮g/mL)b Migration Calibration curvesd Theoretical

time (%)c plate numbers
(N, plates)e

W/O MEEKC NSM-W/O Coefficient of Standard Standard

MEEKC determination deviations of deviations of
(r2 ) the slopes the intercepts

Oxacillin 21.7 1.20 0.08 9.05 (0.19) 0.9989 0.005 0.025 194,000

Penicillin V 16.9 0.74 0.06 9.17 (0.20) 0.9965 0.008 0.043 402,000

Penicillin G 15.8 0.82 0.08 9.30 (0.21) 0.9993 0.003 0.015 328,000

Nafcillin 17.4 1.28 0.11 9.37 (0.10) 0.9991 0.002 0.012 305,000

Ampicillin 21.4 0.95 0.07 9.46 (0.23) 1 0.001 0.004 386,000

Amoxicillin 16.7 1.00 0.09 9.59 (0.21) 0.9992 0.004 0.021 289,000

a
SERheight , sensitivity enhancement factor in terms of peak height = dilution factor × (peak height obtained with NSM-W/O MEEKC/peak height obtained with W/O MEEKC).
b
S/N = 3.
c
n = 3, The values in parentheses are the relative standard deviations (RSD) of migration time.
d
The calibration curves were constructed from three replicate measurements by NSM-W/O MEEKC at each concentration in the range of 0.5–10 ␮g/mL (0.5, 1, 2.5, 5 and
10 ␮g/mL).
e
N = 5.54 × (tR /W1/2 )2 .
7668 H.-Y. Huang et al. / J. Chromatogr. A 1218 (2011) 7663–7669

Table 2
Quantitative precisions and accuracies of on-line concentration MEEKC method.a

Analytes Repeatability of Reproducibility Recovery 1 (%) Recovery 2 (%)

peak areab (%) of peak areac (spiked (spiked
(%) 2.5 ␮g/mL) 5 ␮g/mL)

Oxacillin 0.34 3.45 105 (4.77) 98.3 (0.44)

Penicillin V 0.97 4.58 102 (1.28) 90.9 (0.95)
Penicillin G 3.62 2.54 107 (4.20) 99.8 (3.80)
Nafcillin 1.93 4.53 101 (2.93) 104 (1.97)
Ampicillin 1.91 1.55 101 (2.60) 100 (2.02)
Amoxicillin 1.37 3.90 101 (3.05) 96.3 (1.36)
a
Values were obtained at 5 ␮g/mL of penicillin standard with 2.5 ␮g/mL methyl paraben (internal standard). Recovery = (concentration of penicillin standards obtained
by NSM-W/O MEEKC/spiked concentration of penicillin standards) × 100%.
b
The RSD of each peak area for three intra-day replicated injections represented repeatability of quantification.
c
The RSD of each peak area for three inter-day replicated injections represented reproducibility of quantification.

plates/m and 223,000–763,000 plates/m for the NSM and con-
ventional modes, respectively). Additionally, the large background
signal between 9.5 and 10 min observed in the conventional MEEKC
method (Fig. 4a), became smaller in the NSM mode (Fig. 4b).
The LODs of the six analytes were reduced from the range of
0.7–1.2 ␮g/mL to 0.06–0.11 ␮g/mL by the NSM step, and thus an
almost 12-fold enhancement in the detection limit was achieved
when compared to normal injection (50 mbar, 3 s). In order to eval-
uate the feasibility of the proposed on-line concentration method
for the penicillin analyses, the qualitative and quantitative perfor-
mances were examined (Table 1). The quantitative precisions of
the sample stacking method are shown in Table 2. Without internal
standard (IS), the relative standard deviation (RSD) of the peak area
for three intra-day replicate injections ranged from 2.0 to 5.9%, and
three inter-day replicate injections ranged from 3.9 to 11%. After
the IS addition (methyl paraben, 2.5 ␮g/mL), the RSD values of peak
area were in the range of 0.3–3.6% and 1.6–4.6% for three intra-
day and inter-day replicate injections, respectively. These results
indicated that the addition of IS was useful for the improvement
of quantitative precisions in this proposed method. Furthermore,
the quantitative accuracies of six penicillins by the MEEKC method
were in the range of 90.9–107% (Table 2). The results indicated that
this on-line concentration method provided good qualitative and
quantitative performances for the penicillin compounds analyses
after internal standard calibration.
3.4. Comparison of NSM-W/O MEEKC with other methods
The performances of several penicillin separations in both W/O
and O/W MEEKC systems were first compared. Comparing NSM
W/O MEEKC with previous on-line concentration O/W MEEKC
method (FASI [25] and REPSM [21]), different selectivities were
observed (i.e. the order of elution in O/W is different from in W/O),
that showed different separation mechanisms exist between them.
Similarities in retention times were observed between the 2 modes
(e.g., 9 min [25] or 12 min [21] for O/W and 10 min for this W/O,
respectively), but a narrower separation window was found in this
W/O mode or in O/W with Brij 35 as surfactant [21]. The efficiency
in FASI O/W MEEKC was higher than in NSM W/O MEEKC (N was
112,000–2,066,000 plates for O/W and 194,000–402,000 plates for
W/O, respectively). The detection limits were similar in both sys-
tems with conventional sample injection (50 mbar, 3 or 5 s) (LODs
were in the range of 0.7–5.2 ␮g/mL [25] or 0.5–1.0 ␮g/mL [21] for
O/W and were 0.74–1.28 ␮g/mL for W/O), but with on-line concen-
tration step O/W MEEKC, better detection sensitivities were noted
(LODs were in the range of 0.5–2.9 ␮g/mL (FASI) or 10–25 ␮g/mL
(REPSM) for O/W and were 60–110 ␮g/mL for NSM W/O MEEKC),
due to a longer sample injection time allowed in O/W system (i.e.
900 s (10 kV) (FASI) or 270 s (50 mbar) (REPSM) for O/W and 60 s
(50 mbar) for this NSM W/O).
Next, the detection ability of this proposed W/O MEEKC with
other CE and LC methods was compared. Previous studies on the
analyses of penicillin compounds reported the detection limits of
around 0.7–5.2 ␮g/mL in CE/UV (normal sample injection) and was
improved to 0.2–10 ␮g/L when an on-line concentration step was
employed [25]; while LODs were around 0.44–1.99 ␮g/mL in LC/UV
[40] and around 0.1–20 ␮g/L in LC/MS [41]. This indicated that the
proposed NSM W/O MEEKC method has better detection abilities
than LC/UV and CE/UV, but poorer sensitivity compared with LC/MS
or other on-line concentration CE methods.

60 60

porcine liver spiked with

50 mg/L penicillins and 25 mg/L I.S. porcine kidney spiked with
40 40 50 mg/L penicillins and 25 mg/L I.S.
mAU

mAU

1 1
5 3 5
2 3 6 I.S. 2 6 I.S.
20 4 20 4

0 0

8 9 10 8 9 10
Time (min) Time (min)

Fig. 5. Electropherograms of porcine organ samples determined by NSM-W/O MEEKC. The porcine samples (porcine liver and kidney), which were spiked with or without
the pencillin standards (50 ␮g/L for each one) followed with a sample treatment procedure described in the experimental section, were analyzed by the NSM-W/O MEEKC.
Samples were pressure-injected into the capillary at 50 mbar for 60 s.
 H.-Y. Huang et al. / J. Chromatogr. A 1218 (2011) 7663–7669 7669

3.5. Real sample analyses by NSM-W/O MEEKC Supplementary data

Subsequently, the optimal NSM W/O MEEKC method was used Supplementary data associated with this article can be found, in
to analyze porcine liver and kidney samples (Section 2.2). The the online version, at doi:10.1016/j.chroma.2011.05.091.
results indicated that no penicillin residue was found in these sam-
ples. To examine the separation and detection ability of the on-line References
concentration W/O MEEKC method for all tested analytes, these
samples, which were spiked with penicillins (50 ␮g/L) (its MRLs [1] S.E. Friberg, C. Brancemicz, Langmuir 10 (1994) 2945.
[2] A.M. Bellocq, D. Roux, Microemulsions: Struct. Dyn. (1987) 33.
in animal tissues is 25–300 ␮g/kg in the European Union and in [3] Y. Yang, S. Donegan, R.C. Patel, A.J.I. Ward, Chemosphere 28 (1994) 1967.
Taiwan) were separated by the optimal condition. The trace-level [4] A. Cifuentes, Electrophoresis 27 (2006) 283.
penicillins in these samples were successfully determined without [5] V. García-Cañas, A. Cifuentes, Electrophoresis 29 (2008) 294.
[6] M. Herrero, V. García-Cañas, C. Simo, A. Cifuentes, Electrophoresis 31 (2010)
any interference (Fig. 5). The extraction recoveries of penicillins 205.
were in the range of 79.4–97.6% for porcine liver sample, and were [7] C.W. Klampfl, Electrophoresis 24 (2003) 1537.
in the range of 77.4–94.4% for porcine kidney sample (n = 3). These [8] K.D. Altria, P.E. Mahuzier, B.J. Clark, Electrophoresis 24 (2003) 315.
[9] X. Cahours, S. Cherkaoui, G. Rozing, J.L. Veuthey, Electrophoresis 22 (2002)
results demonstrated that the NSM W/O MEEKC method is feasible
2320.
for the analyses of penicillin residues in these samples after a SPE [10] C.G. Jensen, S.H. Hansen, S.P. Bjergaard, Electrophoresis 22 (2001) 1330.
sample pretreatment. [11] S. Gong, T. Bo, L. Huang, K.A. Li, H. Liu, Electrophoresis 25 (2004) 1058.
[12] H.Y. Huang, M. Wei, Y.R. Lin, P.H. Lu, J. Chromatogr. A 1216 (2009) 2560.
[13] H.Y. Huang, W.C. Lien, C.W. Chiu, J. Sep. Sci. 28 (2005) 973.
4. Conclusion [14] L. Suntornsuk, Anal. Bioanal. Chem. 398 (2010) 29.
[15] A. Jouyban, E. Kenndler, Electrophoresis 29 (2008) 3531.
In this paper, a NSM on-line sample concentration step cou- [16] M.C. Puyana, A.L. Crego, M.L. Marina, Electrophoresis 29 (2008) 274.
[17] K.D. Altria, M.F. Broderick, S. Donegan, J. Power, Electrophoresis 25 (2004) 645.
pled to W/O MEEKC method is reported for the first time. Since a [18] M. Broderick, S. Donegan, J. Power, K. Altria, J. Pharm. Biomed. Anal. 37 (2005)
specific combination of surfactant, oil and water concentration is 877.
required to form a stable W/O microemulsion, the strategy of using [19] J. Cao, J. Chen, L. Yi, P. Li, L.W. Qi, Electrophoresis 29 (2008) 2310.
[20] P. Puig, F. Borrull, M. Calull, C. Aguilar, Chromatographia 62 (2005) 603.
a large amount of low-conductivity solution or sample zone that [21] P. Puig, F. Borrull, C. Aguilar, M. Calull, J. Chromatogr. B 831 (2006) 196.
is often used in most on-line concentration O/W MEEKC reports [22] A. Macià, F. Borrull, C. Aguilar, M. Calull, Electrophoresis 24 (2003) 2779.
to enhance analyte sensitivity, was not applicable in this W/O [23] A. Macià, F. Borrull, M. Calull, C. Aguilar, Electrophoresis 26 (2005) 970.
[24] Y.T. Lin, Y.W. Liu, Y.J. Cheng, H.Y. Huang, Electrophoresis 31 (2010) 2260.
MEEKC system. Therefore, a modified NSM step was used to on- [25] H.Y. Huang, S.H. Hsieh, Electrophoresis 29 (2008) 3905.
line concentrate penicillin antibiotics in the study. In contrast to [26] R. Ryan, S. Donegan, J. Power, E. McEvoy, K. Altria, Electrophoresis 30 (2009)
previous NSM reports wherein only a little longer sample zone 65.
[27] R. Ryan, S. Donegan, J. Power, K. Altria, Electrophoresis 31 (2010) 755.
was pressure-injected into the capillary, a high-viscosity organic [28] R. Ryan, E. McEvoy, S. Donegan, J. Power, K. Altria, Electrophoresis 32 (2011)
solvent plug (1-propanol, 50 mbar for 10 s) was introduced into 184.
the capillary before the sample injection. In addition to the sen- [29] Z. Liu, P.S. Sam, S.R. McClure, J. Chromatogr. A 673 (1994) 125.
[30] J.P. Quirino, S. Terabe, J. Chromatogr. A 781 (1997) 119.
sitivity enhancement, the resolution of several test compounds
[31] J.H. Borges, M.A.R. Delgado, F.J.G. Montelongo, A. Cifuentes, J. Sep. Sci. 28 (2005)
was improved by combining the NSM step with W/O MEEKC sep- 948.
aration as compared to that of conventional W/O MEEKC. This [32] N.A. Santagati, S. Tropea, G. Ronsisvalle, J. Chromatogr. A 1081 (2005) 77.
study demonstrated that the proposed NSM W/O MEEKC method [33] J.H. Borges, A. Cifuentes, F.J.G. Montelongo, M.A.R. Delgado, Electrophoresis 26
(2005) 980.
was successfully used to detect trace levels of penicillin com- [34] Establishment of Maximum Residues Levels of Veterinary Medical Products in
pounds in porcine organs samples after a simple SPE sample Foodstuffs of Animal Origin, European community council regulation 2377/90,
treatment. Off. J. Eur. Commun. L224 (1990) 1.
[35] M. Ma, H.S. Zhang, L.Y. Xiao, L. Xiao, P. Wang, H.R. Cui, H. Wang, Electrophoresis
28 (2007) 4091.
Acknowledgements [36] E. István, H. Valkó, M.L. Sirén, J. Riekkola, J. Microcolumn Sep. 11 (1999) 199.
[37] Y.F. Huang, M.M. Hsieh, W.L. Tseng, H.T. Chang, J. Proteome Res. 5 (2006) 429.
[38] W.L. Tseng, H.T. Chang, Anal. Chem. 72 (2000) 4805.
This study was supported by both Grants NSC-98-2113- [39] T.C. Chiu, Y.W. Lin, C.C. Huang, A. Chrambach, H.T. Chang, Electrophoresis 24
M-033-004-MY3 from the National Science Council of (2003) 1730.
Taiwan, and the project of the specific research fields in [40] L. Kantiani, M. Farré, D. Barceló, Trends Anal. Chem. 28 (2009) 729.
[41] C. Chiaochan, U. Koesukwiwat, S. Yudthavorasit, N. Leepipatpiboon, Anal. Chim.
the Chung Yuan Christian University, Taiwan, under grant Acta 682 (2010) 117.
CYCU-98-CR-CH.