Electrochemical Detector Based on Nafion Membrane for Online ...

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY
Volume 40, Issue 5, May 2012
Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2012, 40(5), 675–680.
RESEARCH PAPER
<strong>Electrochemical</strong> <strong>Detector</strong> <strong>Based</strong> on Nafion Membrane for
Online Monitoring of Column Chromatography Purification
Process
TANG Yu-Hang 1 , LIU Wen-Han 1, *, TENG Yuan-Jie 1 , QIAN Jun-Qing 2 , MA Chun-An 1
1
College of Chemical Engineering and Material Science, Zhejiang University of Technology, Hangzhou 310014, China
2
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, China
Abstract: <strong>Electrochemical</strong> detection of eluent of column chromatography is considerably simple for monitoring the purification
process, In this detection system, electrolyte is needed to decrease the resistance of solution during the electrochemical detection.
However, in the case of natural medicine purification, it is highly challenging to added electrolyte into the purification process
without influencing the purification efficiency. Herein, on the basis of Paraffin-graphite Powder electrode, an online electrochemical
detector was developed without any additional electrolyte. The cyclic voltammograms of myricetin in ethanol-water (80%, V/V) as
mobile phase is obtained directly by using the solid acid characteristic of Nafion N-324 membrane in the micro-gap flow cell.
Peristaltic pump stimulates the chromatographic process, and then it realizes the online detection of eletroactive substances in the
eluent at 0.53 V. Peak area is linear with the myricetin concentration in the range of 2.0 × 10 –5 –1.0 × 10 –3 M, and the linear equation is
A = 2.7227c + 0.4341, r = 0.9976. Furthermore, the detector achieves the online detection of 30% AOB (antioxidant of bamboo leaves)
purified by macro porous resin without any supporting electrolyte in 80% (V/V) ethanol-water at 0.62 V, and the amperometric
time-current (t-i) curve during the purification process was presented. The result indicated that the detector is feasible for application.
Key Words: Natural medicine; Nafion membrane; Antioxidant of bamboo leaves; Cyclic Voltammetry; Micro-gap flow Cell;
Paraffin-graphite Powder electrode
1 Introduction
There is practical significance to separate and purify the
active ingredients of herbal medicine. At present, the
spectroscopy technology is generally selected for online
detecting liquid chromatography in most of pharmaceuticals
companies. However, because of high sensitivity and simple
instrument, also electrochemical detection can be used to
detect the insensitive matter, which cannot be distinguished by
spectroscopy.
Flavonoids belong to common active ingredients of herbal
medicine which can clear up the free radicals [13] , when the
reduction potential of free radicals is lower, the ability of
antioxidant and electrochemical activity are stronger [4,5] , thus
it is suitable for the electrochemical detection. The crude
flavonoids extracted from bamboo leaves are so complex that
it needs macroporous absorption resin to purify. Because the
carbon pasted electrode is sensitive for flavonoids [6–9] .
However, the traditional Cyclic Voltammetry (CV) method
does not fit for the online detection due to large amounts of
electrolytes. New electrochemical detection method should be
developed without electrolytes or less.
Nafion is a kind of polymer which consists of
tetrafluoroethylene and perfluoro-2-(sulfonic acid ethoxylate)
Received 1 August 2011; accepted 9 November 2011
* Corresponding author. Email: liuwh@zjut.edu.cn
This work was supported by the Science and Technology Project of Zhejiang Province/Analysis Test Foundation of China (No. 2009F70001), and Zhejiang Province
Key Scientific and Technological Innovation Team of China (No. 2011R09002-12).
Copyright © 2012, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
DOI: 10.1016/S1872-2040(11)60546-X

TANG Yu-Hang et al. / Chinese Journal of Analytical Chemistry, 2012, 40(5): 675–680
propylvinyl ether. Teflon is used as framework and SO 3– M +
is attached as the side chain, while M + usually is proton or
alkali metal ion, the center of acidity is single and has strong
acidity. The side chain present the surface acidity in water or
organic solution [10] . Other research groups have designed a
kind of flow cell that the noble metal is deposited on the film
of Nafion which serves as working electrode using the solid
acidity character. The organic contents of hydroquinone,
4-butyl ferrocene, cysteine have been detected without
electrolytes [11–13] .
In this paper, a new flow electrochemical detector was
designed using the solid acidity character of the Nafion film to
directly online detect the flavonoids without electrolytes, the
carbon pasted electrode serving as working electrode. And this
method possesses sensitive dectection capability, simple
experimental setup and reproducible results. Furthermore, it is
cost-effective for online detecting the active ingredients of
herbal medicine during the separation and purification
procession.
2 Experimental
2.1 Apparatus, materials and reagents
CHI660B electrochemical workstation (Shanghai CHI
Instruments Inc.), VC9808+3 1/2 figure multimeter (Shenzhen
Victor Hi-tech Co., Ltd); KQ-100DB ultrasonic cleaner (Kun
Shan Ultrasonic Instruments Co., Ltd), PHSJ-4A PH meter
and DDS-307 conductivity meter (Shanghai REX instruments
factory), HL-2 Peristaltic pump with fixed flow rate (Shanghai
Qipuluxi instruments factory) are used in this experiment.
AgCl/Ag is as reference electrode and platinum is as counter
electrode (Changshu noble metal Co. Ltd.). Nafion N-324
cation exchange film was purchased from DuPont Company,
USA, H103 macroporous absorption resin and graphite were
purchased from Sinopharm Chemical Reagent Co., Ltd. The
pureness of myricetin was 98% (Ningbo Dekang biological
product Co., Ltd), and the pureness of Antioxidant of bamboo
leaves was 30% (Zhejiang Anji Shengshi Biological
Technology Co., Ltd). All other reagents were of analytical
grade and all aqueous solutions were prepared with ultra pure
water after deoxygenated by nitrogen.
2.2 Experimental method
As shown in Fig.1, the flow detection cell is formed by two
PVC pieces (about 5.0 cm × 5.0 cm), both the reference
electrode and the counter electrode are in the same
compartment called counter-reference room containing
electrolyte, which is separated by Nafion film from the
working electrode. Nafion film needs to be dipped in H 2 SO 4
solution (pH 2) for 8 h and washed by ultra pure water before
using.
Fig.1 Schematic view of the cell assembly
W, the carbon paste working electrode; R, the reference electrode and C: the
counter electrode
Graphite and liquid paraffin were mixed (1 g mL –1 ) in
proper proportion to keep plasticity. The carbon paste was
filled into the pore of working electrode, pressed and in close
contact with the copper down-lead. And then the surface of
electrode was grinded by filter paper until the surface of
carbon paste becomes shiny. The new made electrode was
dipped in anhydrous ethanol and ultrasonicated for 1 min and
washed by ultra pure water. All parts of instrument were fitted
together by screw. 1.0 mM HCl solution was filled into
counter-reference room, and then CV measurements were
implemented by scaning the voltages over potential range
from 0.2 V to 1.0 V successively until the curve was stable.
Finally, the electrode was washed repeatedly with anhydrous
ethanol and ultra pure water after every using. The carbon
paste electrode needed to be filled and treated again after one
week using.
The sample liquid flowed in the inlet and out from the
outlet which passed by the carbon paste electrode surface. The
distance of working electrode and Nafion film could be
adjusted by the thickness of gasket. The CVs of different
concentration of myricetin scanned from –0.2 V to 1.2 V at a
scan rate of 100 mV s –1 when the sample liquid was still. The
amperometric Time-Current (t-i) curves were tested at the
potential of 0.53 V with the 0.1 s sampling interval during the
flow detection. All the electrochemical tests were performed
at the room temperature.
3 Results and discussion
3.1 Proportion of liquid paraffin and graphite
Liquid paraffin serves as adhesive and does not
participate in conducting, so less in carbon paste electrode
will be better. However, many organic compounds not only
absorb on the surface of electrode, but also extracted into
the electrode by liquid paraffin, which has enrichment effect,
thus the proper proportion of liquid paraffin and graphite
should be selected.
A series of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 mL liquid

TANG Yu-Hang et al. / Chinese Journal of Analytical Chemistry, 2012, 40(5): 675–680
paraffin were added into 1.0 g graphite respectively. After
mixing well, the mixture was made as working electrode. The
CV was tested in 2 × 10 –5 M myricetin solution (pH 2.01) at a
scan rate of 100 mV s –1 . The relationship of current of
oxidation peak and the volume of liquid paraffin was recorded
at 0.43 V. The electric resistance across the two sides of
electrode was recorded at the same time (seen Fig.2).
The results show that the response current of carbon paste
electrode was stronger when more liquid paraffin was added,
because the extract effect may play a major role in enhancing
current. The curvature became smaller when the volume was
up to 0.8 mL, which illustrated that the electron transfer
resistance was stronger than extract effect of liquid paraffin in
electrode until the response reached to the strongest at 1.0 mL.
And then, the response decreased and the electric resistance
increased quickly until the response decreased to 0 at 1.6 mL,
illustrating that the liquid paraffin became a sticky film on the
surface of graphite to hinder electron transfer. Therefore, it is
best to mix 1.0 g graphite with 1.0 mL liquid paraffin to form
carbon paste electrode.
3.2 Direct electrochemical behavior of myricetin on
carbon pasted electrode
The CVs were scanned in ethanol solution (pH 2.0)
containing 2 × 10 –5 M myricetin from –0.2 V to 1.0 V at a
scan rate of 100 mV s –1 . As shown in Fig.3, there were three
oxidation peaks at 0.41 V (peak 1), 0.69 V (peak 2), 0.85 V
(peak 3) respectively in the first cycle and two reduction peaks
appeared at 0.36 V (peak 4) and –0.11 V (peak 5) in the
second cycle.
The reduction peak 4 and the small peak 5 appeared when
the CV reverse scaned after passing by peak 1. Among this,
peak 1 relateed to the hydroxyl oxidation of Bring [8,9] and the
peak 4 and peak 5 correspond to the reduction of hydroxyl of
B ring. The CV of myricetin is similar with rutin, but rutin has
no peak 2 [8] . Comparing with the structure of myricetin and
rutin, peak 2 may correspond to the third position of hydroxy
oxidation of C ring. According to the literatures [14–16] , the free
radicals are produced during the reaction of myricetin on
carbon paste electrode. The mechanism shows below:
The relationship of different terminal potential and the ratio
of peak 4 oxidation current to peak 1 reduction current (i op /i rp )
in order to prove that the step 1 is a reversible process and the
step 2 is not at the beginning potential of –0.2 V. With the
increasing of terminal potential from 0.6 V to 0.8 V, the i op /i rp
decreased from 0.934 to 0.694 gradually. However, when the
terminal potential exceeded to 0.8 V which is the position of
peak 3, the i op /i rp decreased to 0.368 quickly. If the terminal
potential further increased, the i op /i rp decreased to 0.1.
Therefore, peak 3 should be the second process of the third
mechanism, which refers to the disproportionation reaction
process that the free radical obtained one electron transfer to
the structure of p-quinone.
Volume of liquid paraffin (mL)
Fig.2 Effect of added liquid paraffin on the electric resistance of
working electrode and the current of main oxidation peak
3.3 Effect of proton from Nafion to background solution
The outside three electrodes system was used to detect the
CVs of liquid sample before and after flowing by the detection
instrument in order to observe the effect of proton diffusing
from Nafion to background solution. The result showed that
there was no distinct change. The conductivities of the liquid
sample cycled in the cell at different times were tested to
further confirmation. The conductivity of water used in this
experiment was 1.2 S cm –1 . 10.0 mL pure water and 80%
ethanol served as solvent. The results were presented in Table 1.
The results indicated that the conductivity increased only
1.2 S cm –1 when the 80% ethanol solution circulation flowed
Fig.3 Cyclic voltammograms of 2 × 10 –5 M myricetin in pH 2.0 B-R
buffer (1) the first scan, the second scan, scan rate 100 mV s –1

TANG Yu-Hang et al. / Chinese Journal of Analytical Chemistry, 2012, 40(5): 675–680
Table 1 Conductivity of the solvent flowing in the cell for different
time
Time (min) 1 2 5 10 15 30 60
Water
(S cm –1 )
80% Ethanol
(S cm –1 )
1.2 1.2 1.2 1.3 1.5 1.6 2.2
1.2 1.2 1.3 1.4 1.8 2.0 2.4
for 1 h. Because the detection time of flowing is so short that
the number of hydrogen ion from electrolyte room to flow
room can be ignored, because it almost has no effect to
background solution.
calculated after baseline correction (as shown in Table 3).
The relationship of concentration and peak area is A =
2.723c + 0.4341 (r = 0.9976), where A is peak area, c is
concentration. And the linear range is 2.0 × 10 –5 –1.0 × 10 –3 M.
3.7 <strong>Electrochemical</strong> online detection of AOB extracted by
macroporous absorption resin
70% AOB (2.0 × 10 –5 M) was filled into flow cell directly
without electrolyte and the CV was detected to select the
detection potential. There were two weak peaks at 0.37 V and
0.80 V respectively and a strong peak appeared at 0.62 V, also
3.4 CVs of myricetin under the still mobile phase
0.5 mL 80% ethanol solution containing myricetin (0.4 mM)
was filled into the flow room of the detection instrument. The
CV of myricetin was tested when the mobile phase was still
(solid curve, in Fig.4), which was compared with the CV
obtained after adding supporting electrolyte (Fig.3). The
background current increased and the shape of the peak was
retained. The position of peaks shifted. For example, the peak
1 transferred from 0.43 V to 0.53 V and the peak 5
disappeared.
The different distances (0.5, 1.0 and 1.5 mm) between
carbon paste electrode and Nafion film were adopted to
validate the acidity of Nafion film. The highness of peak
decreased quickly with the distance increased. When the
distance was 1.0 mm, there was no distinct difference between
the sample and background signal (dashed line, Fig.4). It
illustrates that the Nafion film has acidity character and the
diffusion of proton can be ignored in short time.
Fig.4 CVs of myricetin without electrolyte while the mobile phase
is still
Full line: The distant between WE and Nafion: 0.5 mm; Dashed line: 1 mm
3.5 Effect of flow rate to amperometric time-current (t-i)
curves
As shown in Fig.5, the amperometric t-i curves were tested
at different flow rate (1.0, 1.8 and 2.6 mL min –1 ) at 0.53 V,
while the constant flow pump simulated the situation of liquid
chromatography.
The baseline was corrected and the peak area was
calculated by Origin 8.1. The results are listed in Table 2.
As the decreasing of flow rate of mobile phase, the shape of
peak became wider, seeming like chromatography. Because
the liquid sample is in the state of flowing, the curve should be
affected by peristaltic pump, and the noises and relative
standard deviation increase correspondingly at low flow rate.
3.6 Relationship of concentration and peak area
The amperometric t-i curves of different concentrations of
myricetin in 0.5 mL 80% ethanol were tested at 0.53 V with
the flow rate of 1.8 mL s –1 . The integral peak areas were
Fig.5 Time-Current response upon injection of 0.5 mL of 0.4 mM
myricetin in 80% (V/V) ethanol-water, under the volume flow
rate of 1.0, 1.8, 2.6 mL min –1 respectively
Table 2 Peak area of time-current curve at different flow rate
Flow rate
(mL s –1 )
Appearance time
(s)
Peak area
RSD
(%)
2.6 65, 60, 64 3.28, 3.45, 3.32 2.6
1.8 93, 89, 90 3.44, 3.20, 3.32 3.6
1.0 116, 117, 113 3.18, 3.38, 3.60 6.2
Table 3 Integral peak area of the different concentrations of
myricetin
Concentration
(10 –4 M )
0.2 0.4 0.8 1.0 2.0 4.0 8.0 10
Peak area 0.55 1.23 2.79 3.48 5.90 11.54 23.56 26.48

TANG Yu-Hang et al. / Chinese Journal of Analytical Chemistry, 2012, 40(5): 675–680
two reduction peaks overlapped. Thus the final detection
potential should be 0.62 V.
This electrochemical chromatography detector was used for
the detection of AOB during the purification process by
macroporous absorption resin. The sample continuously
flowed into the detector by peristaltic pump at 0.62 V at the
flow rate of 1 mL min –1 , and the sampling interval was 0.5 s
(as shown in Fig.7). The peak appearing time, maximum time
and the tendency are similar with the elution curve of
nowadays detection method.
that of other solid electrodes.
3.8 Stability and repeatability
The working electrode was dipped in anhydrous ethanol,
0.1 M HCl and 0.1 M NaOH successively in one week. The
peak potential and the current of myricetin solution (2.0 × 10 –5
M, pH 2.01) were tested for three times. The mean values are
listed in Table 4. The data cannot be obtained after the
electrode dipping in ethanol and NaOH for 10 d, because the
surface of electrode has chapped. Combining with the results
in Table 4, it shows that the peak potential, current and shape
have not changed in different time, because the carbon paste
electrode has good stability.
However, the most stable potentials of carbon paste
electrode are changed at different pH. The increasing of
positive potential would make the carbon on the surface of
electrode loosen and detached under the conditions of pH 2.01,
3.13, 4.01, 5.02, 6.30 and 7.00, so the most stable potential is
1.37, 1.45, 1.50, 1.55, 1.65 and 1.78 V correspondingly. The
positive potential range of carbon paste electrode is wider than
Fig.6 CV of AOB in the cell
Fig.7
Time-Current response of spent regenerant during the
absorption and purification process of AOB
Table 4 Stability of carbon paste electrode in different media
1 d 2 d 4 d 8 d 10 d
Pp Pc Pp Pc Pp Pc Pp Pc Pp Pc
Alcohol (V, A) 0.43 6.5 0.45 6.3 0.45 5.9 0.46 5.3 - -
Acid (V, A) 0.43 5.8 0.43 5.7 0.43 5.4 0.44 5.0 0.43 5.3
Alkali (V, A) 0.43 5.9 0.43 5.4 0.45 5.2 0.50 4.3 - -
Notice: Pp, Peak potentia ; Pc, Peak current.
References
[1] Zhang J J, Cao S W, Yu Y Y. Chinese. J. Anal. Chem., 2009,
37(12): 1810–1814
[2] Jovanovic S V, Steenken S, Hara Y, Simic M G. Journal of the
Chemical Society, Perkin Transactions 2, 1996, (11): 2497–2504
[3] Pannala A S, Chan T S, O'Brien P J, Rice-Evans C J.
Biochemical and Biophysical Research Communications,
2001, 282(5): 1161–1168
[4] Jovanovic S V, Steenken S, Tosic M, Marjanovic B, Simic M
G. Journal of the American Chemical Society, 1994, 116(11):
4846–4851
[5] Jovanovic S V, Hara Y, Steenken S, Simic M G. Journal of the
American Chemical Society, 1995, 117(39): 9881–9888
[6] Xu X R, He J B, Cheng P. Food Science, 2010, 31(7): 6–9
[7] Yu C L, He J B. Chemistry & Bioengineering, 2006, 23(5):
55–57
[8] Ghica M E, Brett A M O. Electroanalysis, 2005, 17(4):
313–318
[9] Brett A M O, Ghica M E.. Electroanalysis, 2003, 15(22):
1745–1750
[10] Xu B Q, Qiu X Q, Zhu Q M. Chemistry Online, 1999, 62(13):
90091 (in Chinese)
[11] Toniolo R, Comisso N, Schiavon G, Bontempelli G. Anal.
Chem., 2004, 76(7): 2133~2137
[12] Kaaret T W, Evans D H. Anal. Chem., 1988, 60(7):657–662
[13] Prasad K S, Muthuraman G, Zen J M. Electroanalysis, 2008,
20(11): 1167–1174
[14] Krol E S, Bolton J L. Chemico-Biological Interactions, 1997,
104(1): 11–27

TANG Yu-Hang et al. / Chinese Journal of Analytical Chemistry, 2012, 40(5): 675–680
[15] Galati G, Moridani M Y, Chan T S, O’Brien P J. Free Radical
Biology & Medicine, 2001, 30(4): 370–382
[16] Wei Y L, Wang Y Z, Hu S S. Chem. J. Chinese Universities,
2004, 25(1): 148–150