Journal of Chromatography A, 1291 (2013) 174–178

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

Differential pulsed amperometry coupled to microchip capillary

electrophoresis
Xinchun Li a,b , Zuanguang Chen a,∗ , Jianbin Pan a , Fan Yang a,c , Yinbao Li a , Meicun Yao a,∗
a
School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road of Higher Education Mega Centre, Guangzhou 510006, China
b
School of Pharmaceutical Sciences, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, China
c
Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 2019 Jialuo Road, Shanghai 201800, China

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

Article history: In this work, we describe a novel electrochemical detection method, differential pulsed amperometry
Received 24 November 2012 (DPA) on microchip capillary electrophoresis (MCE). In a pulse period, a sequential two-step sampling is
Received in revised form 22 March 2013 executed at two different potentials (E1 and E2 ). Differential current signal of the duplex sampling events
Accepted 25 March 2013
is recorded that functions as time domain. The performance of this detection scheme was evaluated
Available online 30 March 2013
by separating and detecting three model analytes including tyramine (Tym), tryptophan (Trp), and p-
aminobenzoic acid (PABA). Multiple parameters that would affect electrochemical response and peak
Keywords:
shape, such as sampling potential, sampling time, and electrode cleaning time, were investigated. This
Microfluidic
Capillary electrophoresis
pulse technique exhibits better sensitivity over constant potential amperometry (CPA), nearly equal to
Electrochemical detection triple pulsed amperometry (TPA). More importantly, DPA can generate more stable baseline than TPA,
Differential pulsed amperometry primarily due to the background subtraction through the two-step sampling, which is beneficial to further
improve analytical sensitivity. In the optimal condition, the limits of detection for Tym, Trp and PABA,
were down to 0.27 ␮M, 0.32 ␮M and 1.1 ␮M, respectively. DPA detection opens up a new avenue for
microchip electrochemistry, and can be virtually extended to other fluid analysis techniques.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction An effective method to improve the repeatability of amper-

Microchip capillary electrophoresis (MCE) has grown into a
competent analytical technique in the past decades, and contin-
ues to broaden the application fields [1–6]. Detector is a core
component that directly affects the analytical performance of
MCE. Compared to other readout strategy, amperometric detection
(AMD) is particularly attractive, as it has several remarkable merits
including high sensitivity, low cost, and easy miniaturization [7,8].
Therefore, AMD has thus far been a focus of research efforts in the
territory of microfluidic chemical analysis.
Generally, amperometric detection is performed at a given elec-
trolytic potential, namely constant potential amperometry (CPA).
Unfortunately, poor reproducibility is an obstinate problem of CPA
detection [9], which is especially serious for metal electrodes (such
as platinum, gold) at the detection of carbohydrates, amino acids,
bioamines and thiols [10,11]. This is principally ascribed to the
buildup of reaction products on electrode surface [12], which atten-
uates electrochemical response of target analytes, and leads to poor
repeatability on bulk samples assay.
∗ Corresponding authors. Tel.: +86 20 3994 3044; fax: +86 20 3994 3071.
E-mail addresses: chenzg@mail.sysu.edu.cn (Z. Chen), Lssymc@mail.sysu.edu.cn
(M. Yao).
ometry is pulsed electrochemical detection (PED). This detection
mode can trace back to the use on HPLC in 1980s, introduced by
Johnson [13]. To date, PED has found a variety of applications in
connection with HPLC, FIA, CE, and MCE [14]. Garcia [11] and co-
workers employed PDMS electrophoretic microchip to implement
pulse electrochemical detection of carbohydrates, amino acids and
antibiotics using a gold microwire electrode. Vickers and Henry [15]
reported another MCE-PED device using a palladium microwire as
decoupling electrode, with detection limits at nanomole level for
dopamine, glutathione and glucose. Practically, a widely used elec-
trochemical pulse technique is triple pulsed amperometry (TPA).
Fig. 1A schematically depicts the pulse waveform of TPA. In this
mode, the sampling (signal detection) follows the preceding elec-
trochemical treatments for working electrode, thus amperometric
detection virtually occurs at a refreshing electrode surface. The
response sensitivity and repeatability are consequently amelio-
rated.
However, baseline drift is usually observed at TPA, due to the
alteration and slow reconstruction of electrode surface caused by
the oxide on–off cycle in the applied pulse waveform. Obviously,
baseline noise is detrimental to detection sensitivity [16]. To
overcome this disadvantage, integrated pulsed electrochemical
detection (iPED) was developed. In this mode, signal detection
is acquired in a triangle potential waveform. Baseline drift is

0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2013.03.064
 X. Li et al. / J. Chromatogr. A 1291 (2013) 174–178
Fig. 1. Schematic drawings of pulse potential waveform of (A) triple pulsed amper-
ometry and (B) differential pulsed amperometry.
effectively obviated in this fashion, since such a pulse waveform
allows the anodic charge formed in the positive scan to be com-
pensated by the cathodic dissolution in the negative scan during
the detection period [17]. However, considerably high background
current in this mode remains problematic.
Also, there are some other pulse amperometric methods used for
separation techniques, including square wave voltammetry (SWV)
[18] and sinusoidal voltammetry (SV) [19]. Brazill [20] presented SV
detection on MCE to the determination of catecholamines. By com-
bining the digital lock-in technique, and optimizing the excitation
and sampling frequency, a detection limit of 0.47 ␮M for dopamine
was achieved with gold film electrode.
In this paper, we report an initial research of differential pulsed
amperometry (DPA) that is performed on microfluidic chip with
platinum disk electrode. This method is based on a two-step
sampling following a preceding oxidation cleaning for working
electrode in a pulse cycle (Fig. 1B), and the differential current sig-
nal is recorded from the two-sampling events. By using three model
analytes, we examined the correlative parameters influencing the
detection sensitivity and peaks shape of electrophoretic elutes.
2. Experimental
2.1. Reagents and instrumentation
All reagents were of analytical grade and used as-received.
Poly(dimethylsiloxane) (PDMS) elastomer and Sylgard 184 cur-
ing agent were both from Dow Corning (MI, USA). A square glass
(Changsha Shaoguang Chrome Blank Co., Changsha, China) with
predeposit 145 nm thick chromium layer and 570 nm thick S-1805
photoresist was used for the fabrication of glass chip. Tyramine
hydrochloride (Tym), tryptophan (Trp), and p-aminobenzoic acid
(PABA), were obtained from Aladdin Reagent (Shanghai, China).
The running buffer was 20 mM borate (pH 9.2), and prepared in
175
ultrapure water (Millipore, MA, USA). All of solutions used for elec-
trophoresis analysis were filtered using 0.22 ␮m polypropylene
membrane (Membrana, Germany) prior to use.
A laboratory-built high voltage power supply (HVPS) device
providing adjustable voltage from 0 to 3000 V for electrophore-
sis separation, and constant voltage of 500 V for electrokinetic
injection, was used. The HVPS device can be easily functioned by
switching the voltage output between injection and separation.
2.2. Electrochemical measurement
Electrochemical detection for microchip electrophoresis was
performed on a CHI 810C electrochemical analyzer (Shanghai Chen-
hua Instrument Co., China). A three-electrode system consisted
of a platinum wire auxiliary electrode, a Ag/AgCl reference elec-
trode, and a platinum disc working electrode (300 ␮m). As to
constant potential detection, the “amperometric i–t curve” mode
was adopted. Pulsed amperometric techniques including TPA and
DPA were described infra. End-column electrochemical detection
was used, and electrochemical measurements were performed in
ambient condition.
2.3. Electrophoresis procedures
A glass/PDMS hybrid chip was fabricated as our previous work
[21,22], which contained 75-mm-long separation channel, and 10-
mm-long injection channel with a 250 ␮m double-tee region for
sample admittance.
Prior to use, the microchip channels were flushed successively
with 0.1 M NaOH, water and running buffer, each for 10 min. Elec-
trokinetic injection was utilized with the operating voltage of 500 V,
and the injection time was 5 s. Electrophoretic separation was initi-
ated by applying high voltage to the separation channel. Detection
signal was recorded after the baseline was stable.
3. Results and discussion
3.1. Baseline noise
Background noise has a vital impact on detection signal. In
general, the charging current stemming from the electrochemi-
cal double layer mainly contributes to the background signal [23].
In this regard, a comparative investigation was performed. As to
TPA, tens to hundreds of milliseconds of sampling time is usually
used [13,24]. Generally, longer sampling time results in lower base-
line current. As can be seen in Fig. 2, when sampling time changed
from 20 ms to 80 ms, the noise current decreased from 0.22 nA to
0.042 nA. Whereas, sampling potential is a crucial consideration
for DPA detection, because a sequential sampling procedure in a
cycle is carried out. And, it can be perceived that such two-step
sampling would cause background subtraction effect, which is ben-
eficial to analytical sensitivity. Practically, when the difference of
sampling potential (defined as E) was tested from 0.2 V to 0.8 V,
the noise current gradually increased from 12 pA to 66 pA, probably
due to the restraining background-subtraction effect when using
much greater E. Comparatively, more favorable baseline level was
obtained at DPA mode.
3.2. DPA detection
In this work, three model analytes were used to estimate the
present pulse electrochemical method. Tryptophan (Trp), tyramine
(Tym), and p-aminobenzoic acid (PABA) represent distinct elec-
troactive functional groups, corresponding respectively to indole,
aminophenol, and arylamine. The property of DPA was prelimi-
narily investigated by analyzing such model compounds. We first
176 X. Li et al. / J. Chromatogr. A 1291 (2013) 174–178
Fig. 2. Effects of sampling time (tsam ) and the difference of detection potential (E)
on the baseline noise of TPA (square) and DPA (triangle), respectively. The data was
averaged on triple measurements.
inspected the effect of sampling time (tsam ) on signal response and
peak shape. The oxidation cleaning potential was set 1.2 V; accord-
ingly the time was 100 ms. Besides, two sampling potentials (E1
and E2 ) of 0.8 V and 0 V, respectively, were adopted. As shown in
Fig. 3, the selected analytes generally displayed ever-increasing
current response and broadening electrophoretic peaks along with
the sampling time. For example, the half peak width (w1/2 ) of PABA
was 5.8 s at the tsam of 40 ms (t1 = t2 = 20 ms), and turned to be
11.1 s when the sampling time of 200 ms was used (t1 = t2 = 100 ms).
In addition, the signal-to-noise ratios (S/N) enhanced likewise,
and reached the apex almost at tsam of 160 ms. Even longer tsam
however, resulted in declined S/N values, owing mainly to the con-
currently amplified background noise.
Then, we investigated the effect of detection potential on elec-
trochemical response. According to the voltammetric behaviors of
the test compounds (data not shown), the first sampling potential
Fig. 3. Sampling time as a key factor influencing (A) half peak width, (B) response current, and (C) signal-to-noise ratio upon the analysis of Tym (100 ␮M, square), Trp
(100 ␮M, circle), PABA (200 ␮M, triangle) by DPA (n = 3). In this experiment, the total sampling time was equally occupied by t1 and t2 . Applied potential: E0 = 1.2 V, E1 = 0.8 V,
and E2 = 0 V.
(E1 ) was fixed at 0.8 V, then the second detection potential (E2 ) was
subject to alteration. Fig. 4A describes the details. All of the analytes
obtained gradually improved peak response when E2 continued to
decrease, suggesting that greater E can bring about higher detec-
tion current. Nevertheless, when E2 was less than −0.1 V, baseline
drift was observed, presumably due to the reduction of dissolved
oxygen in the buffer [25,26].
Furthermore, a comprehensive examination on detection
potential was implemented, as illustrated in Fig. 4B. By changing
E2 , two sets of comparative plots on signal-to-noise ratios for the
three analytes were recorded, corresponding to E1 of 0.6 V and 0.8 V,
respectively. It can be inferred that larger E1 and E synergistically
produced more favorable S/N values. In view of the absence of elec-
trode regeneration at DPA mode, no extensive attempt was made
to test more positive detection potential.
3.3. Effect of electrode cleaning time
As noted above, two sampling in a cycle is carried out after a
preceding oxidation cleaning for working electrode. Such a proce-
dure would cause momentous effect on electrochemical detection.
Therefore, electrode cleaning time (tcle ) was examined. In the time
course from 50 ms to 250 ms, the peak current of analytes mani-
fested a nearly positive tendency (Fig. 5A). This could be explicated
that sufficient oxidative cleaning improves electrocatalytic capa-
bility toward the detecting electrode. Additionally, such an anodic
polarization process may mediate the formation of oxidation film
of platinum electrode, which further promotes the oxide-catalyzed
electrochemistry of amines on metal electrode, thus increasing the
response current of the test analytes [27].
Peak shape directly correlates to electrophoresis efficiency and
analytical capacity. Fig. 5B depicts the relationship of w1/2 with
tcle . On the whole, the three analytes formed much wider peaks
when the cleaning time was raised. This is because longer clean-
ing time leads to diminished sampling frequency, which delays the
signals recording. In other words, time response constant of the
detector is prolonged, which consequently creates broader elec-
trophoretic peaks [28]. In this work, the optimal cleaning time
was 100 ms.
 X. Li et al. / J. Chromatogr. A 1291 (2013) 174–178
Fig. 4. (A) Serial electropherograms depicting the variations of peak response of
single component injection with DPA detection. The detection potential differences
(E) of 0.2 V, 0.4 V, 0.6 V, 0.8 V, respectively, were tested (top → bottom). In this
case, E1 was set at 0.8 V, while E2 was subjected to altering, and the total sampling
time was 160 ms (t1 = t2 = 80 ms). (B) Signal-to-noise ratios of the model analytes
vary along with E1 and E at DPA mode.
3.4. Repeatability
We next examined the repeatability of DPA detection. In this
experiment, consecutive injections and electrochemical detection
of Trp were conducted. As demonstrated in Fig. 6, the current
Fig. 5. Dependence of peak current (A) and half peak width (B) of Tym (square),
Trp (circle) and PABA (triangle) on the electrode cleaning time (tcle ) preceding the
current signals acquisition.
177
Fig. 6. Regeneration of platinum electrode at DPA detection. The arrow indicated
the decline of response current of 100 ␮M Trp on continuous injections, and the
gray rectangle represented an off-line treatment for working electrode by cyclic
voltammetry in 0.5 M H2 SO4 solution in the potential range of −0.2–1.2 V, then the
treated electrode was used for another sequential determinations.
response was almost repeatable on four successive injections, with
RSD of 5.3%. However, the peak current inclined to reduce on the
subsequent determinations. This phenomenon can be essentially
ascribed to the absence of reactivation procedure for the working
electrode, which results in gradually abated electrode activity. Prac-
tically, the electrode can be resuscitated in 0.5 M H2 SO4 solution by
cyclic voltammetry.
3.5. Analytical performance
The present pulse electrochemical technique was further used
for the simultaneous analysis of the three model compounds.
To obtain an overall perspective, constant potential amperom-
etry and triple pulsed amperometry were also employed. The
analytes exhibited similar electrophoretic outcome at DPA, in
comparison with CPA and TPA modes (Fig. 7). Moreover, the
well-shaped electrophoretic peaks confirmed that such a post-
column pulse electrochemical scheme would not impair separation
efficiency. We should note that the decreased peak current of
Trp was observed compared to Tym in DPA detection. Perhaps,
the deficiency of electrode regeneration in the present electro-
chemical technique is responsible for this effect. In addition, the
electrode behavior may alter in different electrochemical modes,
which causes variant current response to specific compound. Still,
Fig. 7. Electropherograms of the three model analytes detected by triple pulsed
amperometry (a), differential pulsed amperometry (b), and constant potential
detection (c) in the optimal conditions. Analytical concentration as in Fig. 3.
178 X. Li et al. / J. Chromatogr. A 1291 (2013) 174–178

Table 1 samples. Related research on DPA will be further expanded. For

Pulse parameters for TPA and DPA detection on MCE.
instance, other working electrodes (such as gold and copper) should
TPA DPA be tested. Besides, more analytes will be examined to demonstrate
E1 (V) 1.2 t1 (ms) 40 E0 (V) 1.2 t0 (ms) 100 the applicability. To conclude, the present pulse scheme offers
E2 (V) −0.2 t2 (ms) 100 E1 (V) 0.8 t1 (ms) 80 an opportunity for electrochemical detection, and broadens the
E3 (V) 0.8 t3 (ms) 60 E2 (V) 0.0 t2 (ms) 80 analytical strategy and applicative realm as well. Yet, this electro-
chemical pulse technique can also be transferred to HPLC, FIA and
Table 2
CE.
Data of limit of detection (LOD), and half peak width (w1/2 ) of the selected analytes
by TPA, DPA, and CPA detection on MCE (n = 3).a Acknowledgement
TPA DPA CPA
This work is financially supported by National Natural Science
LOD (␮M)b w1/2 (s) LOD (␮M)b w1/2 (s) LOD (␮M)b w1/2 (s)
Foundation of China (Nos. 20727006 and 21075139).
Tym 0.18 4.7 0.27 5.0 0.98 2.6
Trp 0.20 4.8 0.32 5.3 1.5 3.3
References
PABA 1.2 7.2 1.1 7.7 5.9 5.2
a
Obtained by determination of a mixture solution containing 100 ␮M Tym, [1] C.M. Kang, S. Joo, J.H. Bae, Y.R. Kim, Y. Kim, T.D. Chung, Anal. Chem. 84 (2012)
100 ␮M Trp, and 200 ␮M PABA. 901.
b
Based on S/N = 3:1. [2] M.H. Ghanim, M.Z. Abdullah, Talanta 85 (2011) 28.
[3] A. Fernández-la-Villa, D.F. Pozo-Ayuso, M. Castaño-Álvarez, Electrophoresis 31
(2010) 2641.
considerably high signal-to-noise ratios of the test compounds [4] M. Castano-Alvarez, M.T. Fernandez-Abedul, A. Costa-Garcia, J. Chromatogr. A
1109 (2006) 291.
could be gained, due to the more favorable baseline level.
[5] M.K. Hulvey, C.N. Frankenfeld, S.M. Lunte, Anal. Chem. 82 (2010) 1608.
In the optimal condition (Table 1), comparatively sensitive [6] J.J.P. Mark, R. Scholz, F.M. Matysik, J. Chromatogr. A 1267 (2012) 45.
detection was achieved by DPA, and the detection limit (S/N = 3) [7] J. Wang, Electroanalysis 17 (2005) 1133.
of Tym, Trp, and PABA was 0.27 ␮M, 0.32 ␮M and 1.1 ␮M, respec- [8] U. Backofen, F.M. Matysik, C.E. Lunte, Anal. Chem. 74 (2002) 4054.
[9] D.P. Manica, Y. Mitsumori, A.G. Ewing, Anal. Chem. 75 (2003) 4572.
tively. Correspondingly, the detection limits obtained at CPA, were [10] C. García, Anal. Chim. Acta 508 (2004) 1.
0.98 ␮M, 1.5 ␮M, and 5.9 ␮M, respectively. Clearly, the sensitivity [11] C.D. Garcia, C.S. Henry, Anal. Chem. 75 (2003) 4778.
of DPA was superior to CPA, and approximately comparable to TPA [12] S. Altunata, R.L. Earley, D.M. Mossman, L.E. Welch, Talanta 42 (1995) 17.
[13] D.C. Johnson, W.R. LaCourse, Anal. Chem. 62 (1990) 589A.
(Table 2). [14] J.C. Fanguy, C.S. Henry, Analyst 127 (2002) 1021.
[15] J.A. Vickers, C.S. Henry, Electrophoresis 26 (2005) 4641.
4. Conclusion [16] C.D. Garcia, C.S. Henry, Electroanalysis 17 (2005) 223.
[17] W.R. LaCourse, D.A. Mead, D.C. Johnson, Anal. Chem. 62 (1990) 220.
[18] G.C. Gerhardt, R.M. Cassidy, A.S. Baranski, Anal. Chem. 70 (1998) 2167.
We have demonstrated preliminarily the nature of differential [19] S.A. Brazill, P.H. Kim, W.G. Kuhr, Anal. Chem. 73 (2001) 4882.
pulse amperometry on microfluidic platform. By separating and [20] N.E. Hebert, W.G. Kuhr, S.A. Brazill, Anal. Chem. 75 (2003) 3301.
[21] X. Li, J. Pan, F. Yang, J. Feng, J. Mo, Z. Chen, Microchim. Acta 174 (2011) 123.
detecting three model compounds, the main parameters affect- [22] X. Li, Z. Chen, Y. Zhong, F. Yang, J. Pan, Y. Liang, Anal. Chim. Acta 710 (2012) 118.
ing peaks shape and electrochemical response were surveyed. [23] N.E. Hebert, W.G. Kuhr, S.A. Brazill, Electrophoresis 23 (2002) 3750.
This pulse technique is advantageous over conventional constant [24] R.E. Roberts, D.C. Johnson, Electroanalysis 7 (1995) 1015.
[25] T.J. O‘Shea, S.M. Lunte, W.R. LaCourse, Anal. Chem. 65 (1993) 948.
potential detection considering detection sensitivity. In addition,
[26] P. Kuban, P.C. Hauser, Electrophoresis 30 (2009) 3305.
it can create more satisfactory baseline level, compared to triple [27] C.D. García, C.S. Henry, Electroanalysis 17 (2005) 1125.
pulsed amperometry, which may be beneficial to complex-matrix [28] R.E. Roberts, D.C. Johnson, Electroanalysis 6 (1994) 269.