- Email: [email protected]
Simultaneous determination of five metal ions by on-line complexion combined with micelle to solvent stacking in capillary electrophoresis
Journal Pre-proof Simultaneous determination of five metal ions by on-line complexion combined with micelle to solvent stacking in capillary electrophoresis Xiaoyu Song, Rui Zhang, Yue Wang, Mengqing Feng, Honghua Zhang, Shuling Wang, Jun Cao, Tian Xie PII:
S0039-9140(19)31211-1
DOI:
https://doi.org/10.1016/j.talanta.2019.120578
Reference:
TAL 120578
To appear in:
Talanta
Received Date: 27 August 2019 Revised Date:
18 November 2019
Accepted Date: 19 November 2019
Please cite this article as: X. Song, R. Zhang, Y. Wang, M. Feng, H. Zhang, S. Wang, J. Cao, T. Xie, Simultaneous determination of five metal ions by on-line complexion combined with micelle to solvent stacking in capillary electrophoresis, Talanta (2019), doi: https://doi.org/10.1016/j.talanta.2019.120578. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical abstract
1
Simultaneous determination of five metal ions by on-line complexion
2
combined with micelle to solvent stacking in capillary electrophoresis
3 4
Xiaoyu Songa, Rui Zhanga, Yue Wanga, Mengqing Fenga, Honghua Zhanga, Shuling Wanga,*, Jun Caoa,b,*, Tian Xiea,c,d,e*
5
a
6 7 8 9 10 11 12 13
Medical College, Hangzhou Normal University, Hangzhou 311121, P.R. China
b
College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, P.R. China c
Holistic Integrative Pharmacy Institutes, Hangzhou Normal University, Hangzhou 311121, P.R. China d
Key Laboratory of Elemene Anti-cancer Medicine of Zhejiang Province, Hangzhou 311121, P.R. China e
Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, Hangzhou 311121, P.R. China
14
E-mail: [email protected]; [email protected]; [email protected]
15
Fax: 86-571-28860237; 86-571-28867909
16 17 18 19 20 21 22 23 24 25 26 27
28
ABSTRACT
29
A direct on-line complexion combined with micelle to solvent stacking method
30
was proposed for simultaneous determination of metal ions by capillary
31
electrophoresis coupled diode array detector. During the experiment, a plug of
32
complexing agent was first injected to the inlet of capillary, followed by introducing
33
the micelle-bound metal ions. Then the metal ions produced a micelle-to-solvent
34
stacking effect and interacted with the complexing agent under a positive voltage.
35
Continued application of voltage, the analytes were effectively focused and separated
36
in the capillary zone electrophoresis. Repeatability was ranged from 1.89% to 1.94%
37
for the migration time. The detection limits were 2.66-27.9 ng mL-1 for Ni2+, Co2+,
38
Cu2+, Hg2+ and Cd2+. Furthermore, the developed method showed a great potential for
39
the determination of metal ions in the crayfish, beebread and Dendrobium officinale
40
samples.
41 42
Keywords: Metal ions; On-line complexion; Micelle to solvent stacking; Capillary
43
electrophoresis
44 45 46 47 48 49
1
50
1. Introduction
51
Metal ions were widely found in organisms and natural environments. Some
52
metal ions, closely related to human life activities, are an important part of the body
53
composition for maintaining the osmotic balance of multiphase systems. However, the
54
high-dose metal ions pose a potential hazard to ecosystems and human health due to
55
their bioaccumulation and non-biodegradability [1]. For example, nickel (Ni) is an
56
essential trace element with potential toxicity. The most usual symptoms caused by Ni
57
are respiratory cancer, respiratory disorders and dermatitis. The Ni compounds even
58
have been the carcinogens [2]. Cobalt (Co), a component of vitamin B12 [3],
59
contributes to the synthesis of hemoglobin and increases the number of red blood cells
60
by stimulating the hematopoietic system of the human bone marrow. However, the
61
main influence of Co on the skin is allergic or irritating dermatitis, which also affects
62
the respiratory system. Copper (Cu) is one of the indispensable metal elements of the
63
human body. According to previous studies [4], Cu has a function of promoting bone
64
metabolism and has certain antibacterial activities, but excess Cu takes potential risks
65
to the kidney, gastrointestinal tract, movement and sensory nerves [5]. The highly
66
toxic nature of mercury (Hg) is now known to the public. Exposure to high Hg likely
67
causes brain damage, inflammation, autoimmunity, and affects the development of the
68
nervous system [6,7]. Exposure to cadmium (Cd) which was partly produced by
69
industrial emissions or smoking causes kidney and liver toxicity and influences the
70
normal growth of the embryos [8,9]. Therefore, the issue of metal pollution has
71
increasingly attracted the attention of analysts. 2
72
At present, various analytical techniques including colorimetric determination
73
[10], fluorescent sensor array voltammetry [11], flame atomic absorption
74
spectrometry [12,13] and resonance Raman [14] were developed for the determination
75
of metal ions. However, the wider applications of these techniques are limited due to
76
the requirements of sophisticated instrument and cumbersome pre-processing program.
77
In recent years, capillary electrophoresis equipped with diode array detector
78
(CE-DAD) was increasingly popular and had been used to detect metal ions with high
79
efficiency, easy controlling, and low solvent consumption [15,16]. Moreover, the use
80
of complexing agents, pre- or on-capillary, is necessary because some metals do not
81
have chromophores under ultraviolet visible (UV) [17]. The complexing agents that
82
have been reported were imidazole [18], 1,10-phenanthroline [19,20], 18-crown
83
ether-6 [21], L-cysteine [22,23] and so on. The complexing agents used in these
84
reports were prepared in background solution (BGS), the preparation method was
85
complicated, and the amount of complexing agents actually combined with the
86
analytes was not easy to calculate. Therefore, in this study, the complexing agent was
87
prepared separately, and the on-line complexation reaction was completed by only
88
twice simple injection. However, the limited injection amount and short optical path
89
of CE lead to its low determination sensitivity. The application of on-line
90
preconcentration strategies can increase the sensitivity of CE [24].
91
Micelle to solvent stacking (MSS), a new stacking technique, was first
92
introduced by Joselito P. Quirino in 2009 [25]. The focus of MSS is based on the
93
reversion of the effective electrophoretic mobility of charged analytes at the MSS
3
94
boundary (MSSB) where separates the sample solution (S) and BGS at the inlet end.
95
The essential conditions for MSS are micelle-containing S and organic
96
solvent-containing BGS. It is required that the micelle in the S has an opposite charge
97
with the charged analytes for binding and transporting analytes to MSSB. The organic
98
solvent in the BGS must be in an amount sufficient to reduce the binding of micelle to
99
analytes after passing through the MSSB, thereby releasing the analytes. In order to
100
confirm that the focus of the analyte is caused by MSS, rather than other field
101
amplification effects, it is desirable that the concentration of electrolyte in the S is
102
consistent with the BGS [26]. Currently, the MSS has been used to detect multiple
103
analytes, such as anticancer drugs [27], antipsychotic drugs [28], alkaloids [29,30]
104
and nitroimidazoles [31], and all the application showed an excellent concentration
105
effect. According to the researches already reported, MSS has not been used for the
106
concentration of metal ions.
107
In this work, five metal ions were studied by on-line complexion combined with
108
MSS in capillary zone electrophoresis (CZE). The five metal ions were Ni2+, Co2+,
109
Cu2+, Hg2+, Cd2+ and they were detected by DAD. As far as our knowledge goes, the
110
MSS was first combined with the complexion approach and it has not been reported
111
for the determination of metal ions. The model of on-line complexaion combined with
112
MSS was explained and the concentration effect of MSS was validated. Additionally,
113
several main influence factors were investigated, such as the type and concentration
114
of complexing agents, the amount of SDS and sodium acetate in S, the methanol
115
content in BGS and so on. Furthermore, the repeatability, linearity, limit of detection 4
116
(LOD) and limit of quantification (LOQ) were evaluated under the selected conditions.
117
Finally, sample matrix effect on the developed method was demonstrated by spike
118
recovery studies in real samples of crayfish, beebread and Dendrobium officinale.
119
2. Materials and methods
120
2.1. Reagents and materials
121
Nickel nitrate hexahydrate (Ni2+), cobaltous nitrate hexahydrate (Co2+), copper
122
nitrate trihydrate (Cu2+), mercury nitrate monohydrate (Hg2+), cadmium nitrate
123
tetrahydrate (Cd2+), imidazole (
124
and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
125
1,10-phenanthroline (99%) was provided by Alfa Aesar (China) Chemical Co., Ltd.
126
(Tianjin, China). L-Cysteine (99%) was obtained from Saan Chemical Technology
127
(Shanghai) Co., Ltd. (Shanghai, China). 18-Crown-6 (99%) was purchased from
128
Aladdin Industrial Corporation (Shanghai, China). Sodium acetate (HPLC,
129
was acquired from Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). The
130
stock solutions of Ni2+, Co2+, Cu2+, Cd2+ were prepared in ultrapure water at 1000 µg
131
mL-1 and Hg2+ was at 100 µg mL-1 due to the low solubility of mercury nitrate
132
monohydrate. The working solutions were diluted with ultrapure water. The stock
133
solution of 100 mmol L-1 1,10-phenanthroline was prepared in methanol and diluted
134
with methanol before use. Imidazole, L-cysteine and 18-crown-6 were prepared in
135
ultrapure water. BGS was prepared by sodium acetate in ultrapure water and the pH
136
was adjusted to 5.5 with acetic acid. Working solutions of complexing agent and BGS
99.0%) and nitric acid (HNO3) all were AR grade
5
99.0%)
137
were prepared daily. The samples of crayfish, beebread and Dendrobium officinale
138
were purchased from local market of Hangzhou (Hangzhou, China), Beijing (Beijing,
139
China) and Zhuji (Zhuji, China), respectively. The methanol and ultrapure water used
140
throughout the experiment were HPLC grade and produced by Tedia Company, Inc.
141
(Fairfield, USA). All solutions before analysis were filtered through 0.22 µm
142
disposable filter (Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China)).
143
2.2. Instrument and CE injection procedures
144
An Agilent CE system (7100, Palo Alto, CA, USA), equipped with a DAD
145
detector, was used for the detection of metal ions. The separation of five analytes was
146
achieved in fused-silica capillary of 41.5 cm (33 cm of effective length) × 50 µm i.d.
147
obtained from Agilent Technologies. The detection wavelength of target analytes was
148
set at 214 nm without reference wavelength. Before it was first used, the capillary was
149
activated with 1.0 mol L-1 NaOH (10 min), 0.1 mol L-1 NaOH (10 min), ultrapure
150
water (5 min) and BGS (5 min), successively. Between two runs, the capillary was
151
flushed with 0.1 mol L-1 NaOH (3 min), ultrapure water (3 min) and BGS (2 min) to
152
ensure the accuracy of the data. In the end of the daily experiment, the capillary was
153
rinsed with 0.1 mol L-1 NaOH (10 min) and ultrapure water (5 min) to prevent residue
154
from adhering to the inner wall of the capillary. The MSS of this experiment was
155
injected as follows (initial condition): first, the complexing agent was injected at 50
156
mbar for 3 s after the capillary was filled with BGS. Next, the S containing micelle
157
was injected at 50 mbar for 60 s. Then the operation begins under the applied voltage
6
158
of 16 kV and the temperature in the whole experiment was maintained at 25
. The
159
other injection methods mentioned in this paper were illustrated in the part of 3.1.2.
160
2.3. Sample preparation
161
The preparation methods of samples were referred to previous studies [32,18,33]
162
with minor modifications. Briefly, two crayfish heads were placed in oven and dried
163
at 105 ℃. Weighed and recorded every 30 minutes until the weight maintained
164
constant. The dry crayfish head was ground into powder using agate mortar and pestle,
165
then 0.5 g of powder was weighed and placed into a clean little beaker. The samples
166
were mixed with 10 mL of HNO3. After stirring evenly and standing for 20 min, the
167
beaker containing the mixture was placed on a hot plate at 100℃ for digestion until
168
the solution was clear. The clear digested liquor was centrifuged at 4000 rpm for 10
169
min when it was cooled to room temperature, then took 1 mL into a 10 mL volumetric
170
flask and diluted to the mark with ultrapure water.
171
Similarly, 1 g of weighted beebread was put in a clean beaker and mixed fully
172
with 15 mL of NHO3. Then the mixture was heated until it was clear and transparent
173
after standing for 20 min. The digested liquor was centrifuged at 4000 rpm for 10 min
174
and took 1 mL of the supernatant into a volumetric flask and made up to 10 mL with
175
ultrapure water.
176
About 0.2 g of Dendrobium officinale powder (50 mesh) was weighed into a
177
clean beaker and added 8 mL of HNO3, waited for about 20 min after mixing
178
completely. The undigested powder was precipitated at 4000 rpm (10 min). The
7
179
supernatant was taken 1 mL into a 10 mL volumetric flask and diluted to the mark
180
with ultrapure water.
181
3. Results and discussion
182
3.1. The model and proof of on-line complexaion combined with MSS
183
3.1.1. Model
184
The program for simultaneous determination and separation of five metal ions by
185
on-line complexaion and MSS in CZE was presented in Fig. 1. After the new capillary
186
was activated with 1 mol L-1 NaOH, 0.1 mol L-1 NaOH and water, the entire capillary
187
was filled with BGS containing electrolyte and organic solvent by pressure injection.
188
Then the complexing agent and S were injected successively (Fig. 1A). The S was
189
prepared in electrolyte and anionic micelle, and the electrolyte in S had a conductivity
190
value close to BGS. When positive voltage was applied at the inlet end of the
191
capillary (Fig. 1B), the direction of electroosmotic flow (EOF) was from positive
192
electrode to negative electrode. Thus yielded an electric field and the uncharged
193
complexing agent migrated towards the detector slowly. Meanwhile, the
194
micelle-bound analytes migrated to the anode since the SDS was negatively charged,
195
so the effective electrophoretic velocity of analytes (µep*(a)) was got by Eq. (1) [34]:
196
µ ep ∗ (a )=
197
where µep(a) represents the electrophoretic mobility of the analyte (a), µep(mc) refers
198
to the electrophoretic mobility of the micelle, and k is retention factor.
k 1 µ ep (a ) + µ ep(mc) E 1+ k 1 + k
(1)
8
199
Continued to apply voltage at the inlet end (Fig. 1C), when micelle-bound
200
analytes reached the MSSB, µep*(a) was given by Eq. (2):
201
µ ep ∗ (a ) =
202
At MSSB, the organic solvent contained in BGS reduced the affinity of anionic
203
micelles to cationic analytes and then the micelles gradually collapsed, which resulted
204
in the µep(a) to reverse to the cathode. With more and more micelles passed through
205
MSSB, all the analytes were released and focused at the MSSB. At the that time, the
206
effect of micelle on the µep*(a) was almost negligible or k=0, the µep*(a) depended
207
only on µep (a). Therefore, Eq. (2) was changed to Eq. (3):
208
µ ep ∗ (a )MSSB = µ ep(a )
209
The positively charged metal ions moved faster than uncharged 1,10-phenanthroline
210
(Supplementary material, Fig. S1), moreover, 1,10-phenanthroline was a bidentate
211
N-donor ligand and was capable of forming stable complexes with a variety of
212
transition metals within seconds [19,20], so the complexation occurred between
213
analytes and complexing agent (Fig. 1D). Since 1,10-phenanthroline improved the
214
detection sensitivity and resolution of analytes, the focused analytes with different
215
ratio of charge to mass were effectively separated in CZE and moved toward the
216
detector.
217
3.1.2. Proof
(2)
1 k µ ep(a ) + µ ep (mc) 1+ k 1+ k
(3)
218
The effect of MSS was proved using 1,10-phenanthroline as complexing agent
219
and 50% methanol as organic modifier in BGS. The electropherograms illustrated in
9
220
Fig. 2 were obtained by typical injection (a), large volume injection (b), MSS (c),
221
on-line complexation and typical injection (d), on-line complexation and large volume
222
injection (e), on-line complexation and MSS (f) of metal cations in CZE. In Fig. 2c
223
and Fig. 2d, the concentration of analytes was 5 µg mL-1 (Ni2+, Co2+, Cu2+) or 10 µg
224
mL-1 (Hg2+, Cd2+) prepared in 7.2 mmol L-1 SDS and 180 mmol L-1 sodium acetate
225
solution to form micelles. In Figs. 2a, 2b, 2e and 2f, the metal ions were prepared in
226
sodium acetate solution without SDS. The typical injection was at 50 mbar for 5 s, the
227
large volume injection and MSS were at 50 mbar for 60 s, the on-line complexation
228
was performed by injecting complexing agent at 50 mbar for 3 s before injecting
229
sample. The 200 mmol L-1 sodium acetate solution containing 50% methanol was
230
used as the BGS for all injections.
231
The response of the analytes was low and almost undetectable in the typical
232
injection (Figs. 2a and 2d). The large volume injection of S without micelle yielded
233
broad peaks which caused by the absent stacking effect (Fig. 2b) [35]. It was observed
234
in Fig. 2c that the Ni2+, Cu2+, Hg2+ and Cd2+ were detected when MSS was performed,
235
but the response was low. However, the five metal ions were detected clearly when
236
the complexing agent was injected before the large volume injection (Fig. 2e).
237
Especially, as shown in Fig. 2f, the peaks of five analytes were tall and sharp when
238
on-line complexion combined with MSS. These results confirmed that metal ions
239
were sensitively detected by binding with complexing agent and effectively focused
240
by MSS.
241
3.2. Optimization of on-line complexion and micelle to solvent stacking for metal ions 10
242
3.2.1. Choice of complexing agents
243
The examined metal ions are in the form of its hydrate, nevertheless, most
244
hydrated metal ions do not exhibit significant UV absorption above 185 nm. In
245
addition, metal ions with the same electric charge have almost the same
246
electrophoretic mobility due to similar charge-to-mass ratios, which will result in poor
247
resolution. But the detection sensitivity and resolution can be improved by use of
248
complexing agents [21,36]. In order to determine and separate multiple metal ions
249
simultaneously, several complexing agents such as 1,10-phenanthroline, 18-crown
250
ether-6, L-cysteine and imidazole that have been reported for complexing metal ions,
251
were studied at the same condition (S was 4 or 8 µg mL-1 of each metal ions in 7.2
252
mmol L-1 of SDS and 180 mmol L-1 of sodium acetate, pH 5.5; BGS was 200 mmol
253
L-1 of sodium acetate containing 50% methanol, pH 5.5; 30 mmol L-1 of complexing
254
agent was injected at 50 mbar for 3 s followed by injecting the S at 50 mbar for 60 s).
255
The electropherograms are shown in Fig. S2 in Supplementary Material. It is obvious
256
that all five metal ions of Ni2+, Co2+, Cu2+, Hg2+ and Cd2+ could be determined by
257
complexing with 1,10-phenanthroline and 18-crown ether-6. However, only the peaks
258
of Ni2+, Co2+, Cu2+, Hg2+ and Ni2+, Co2+, Cu2+, Cd2+ appeared when injecting
259
L-cysteine and imidazole as the complexing agent, respectively. Furthermore,
260
compared with the other three complexing agent, the response of five metal ions
261
complexed by 1,10-phenanthroline was higher. Accordingly, 1,10-phenanthroline was
262
adopted as the complexing agent to enhance the detection sensitivity and change the
263
electrophoretic mobility of five metal ions. 11
264
3.2.2. Effect of the amount and injection time of complexing agent
265
The determination and separation of five metal ions were enhanced by the
266
complexation of complexing agent to metal ions. Therefore, the amount of
267
complexing agent was an important parameter that influences the level of
268
complexation. The effect of 1,10-phenanthroline amount was investigated in the range
269
of 10-50 mmol L-1. From the result shown in Fig. 3A, the peak area of Ni2+, Co2+ and
270
Cu2+ increased as the concentration value increased from 10 to 30 mmol L-1, and
271
subsequently decreased from 30 to 50 mmol L-1. However, the peak area of Hg2+ and
272
Cd2+ was increased as the amount of 1,10-phenanthroline up to 40 mmol L-1 and then
273
remained basically constant. The phenomenon was ascribed to the different UV
274
absorption of Ni2+, Co2+, Cu2+ and Hg2+, Cd2+. In order to achieve high response
275
simultaneously for five metal ions, 30 mmol L-1 was chosen as the optimum
276
complexing agent concentration. Differ from the complexing agent concentration, the
277
injection time of complexing agent is a vital factor affecting the interaction time of the
278
complexing agent with the metal ions. So the injection time varied from 2 to 10 s was
279
evaluated. The Fig. 3B revealed that the peak area of all five metal ions increased with
280
the increase of complexing agent injection time, but the peak area grew slowly when
281
the injection time up to 6 s. It is possibly due to the effect of the methanol contained
282
in the complexing agent on the MSS. Consequently, an injection time of 8 s was
283
selected for complexing with metal ions.
284
3.2.3. Effect of the amount of SDS in sample solution
12
285
The concentration of SDS added into the sample solvent needs to reach its
286
critical micelle concentration (CMC) to form micelle and transport the metal ions to
287
the stacking boundary. On the other hand, SDS micelles should be prone to collapse in
288
the buffer. Series concentrations of SDS (4.2, 7.2, 10.2, 13.2, 16.2 mmol L-1) were
289
prepared in 180 mmol L-1 of NaAc (pH 5.5) to research the impact of the amount of
290
SDS in S (Fig. 3C). It can be observed that the increase of SDS concentration yielded
291
inconspicuous enhancement for the peak area of Ni2+, Co2+ and Cu2+. However, the
292
response of Hg2+ and Cd2+ was improved when the concentration of SDS was lower
293
than 7.2 mmol L-1, and succeeding decreased gradually from 7.2 mmol L-1 to 16.2
294
mmol L-1. Additionally, the tailing peaks of Hg2+ and Cd2+ were improved as the
295
increase of the SDS amount in the S. It was probable attributed to the low detection
296
sensitivity of Hg2+ and Cd2+, the accumulated effect of MSS was more visible. But
297
once the amount of SDS was large enough, it was hard to be diluted by the organic
298
modifier in BGS, thereby hindered the release of the metal ions from the micelle. In
299
summary, 7.2 mmol L-1 SDS was used for the next experiment.
300
3.2.4. Effect of the amount of sodium acetate in sample solution
301
The relative conductivity of S decided by the amount of electrolyte to BGS
302
affects the mobility reversal of the target analytes, thus influences the stacking of
303
analytes [37]. The effect of the amount of NaAc in S was studied and the line chart
304
was exhibited in Fig. 3D. The S was composed of 7.2 mmol L-1 SDS and varying
305
amounts of NaAc (140, 160, 180, 200, 220 mmol L-1). The buffer was 200 mmol L-1
13
306
NaAc contained 50% methanol. The result illustrated the increase of NaAc amount in
307
the range of 140-180 mmol L-1 contributed to the detection of metal ions. However,
308
the detection sensitivity decreased as the NaAc amount in S increased to equal to or
309
greater than the NaAc amount in buffer. Moreover, the peak shape of Ni2+ and Hg2+
310
deteriorated with the increase of NaAc amount (Fig. S3 in Supplementary Material),
311
especially at 220 mmol L-1. The phenomenon was due to the fact that as the increase
312
of NaAc concentration in the S, the discrepancy of conductivity between S and BGS
313
regions increased. This caused the destacking of metal ions at the solution zone and
314
the unexpected distorted peaks. So 180 mmol L-1 NaAc in the sample matrix was
315
adopted.
316
3.2.5. Effect of methanol content in BGS
317
The content of organic modifier in BGS affects the viscosity and dielectric
318
constants of the micelle, and then further influence the interaction of metal ions with
319
micelle until the micelle collapse. Therefore, it is essential to investigate the influence
320
of methanol content on the focusing and separation efficiency of metal ions. The
321
results shown in Fig. 3E and Fig. S4 were obtained by changing the percentage of
322
methanol (30, 40, 50, 60, and 70 %) in the BGS while keeping the amount of NaAc at
323
200 mmol L-1. It was clear that the peak area increased firstly and then gradually
324
decreased with the increase of methanol content. In the electropherogram, Ni2+, Co2+
325
and Cu2+ showed low detection sensitivity at 30% methanol, and even Hg2+, Cd2+
326
cannot be detected. When the methanol content exceeded 50%, the resolution between
14
327
the metal ions and the 1,10-phenanthroline was diminished, thus affected the
328
detection of metal ions that appeared later. This can be explained by the fact that low
329
methanol content is not enough to lower the affinity between micelle and metal ions,
330
and the metal ions cannot be released. However, when higher content of organic
331
solvents was added in the BGS, the viscosity of the BGS was increased and led to the
332
decrease of the signal intensities [38]. Based on the research results, 50% methanol
333
was used as the organic modifier in MSS.
334
3.2.6. Effect of the injection time of sample solution
335
Increasing the sample injection time can improve the enrichment factor. On the
336
other hand, the injection time of S decides the length of the introduced micelle in
337
sample matrix, which affects the disintegration degree of micelle, and then further
338
influences the detection limit. The effect of the injection time of S on the detection
339
sensitivity was studied in the range of 40-100 s. As be observed from Fig. 3F, the peak
340
area was increased with the increase of injection time from 40 to 80 s. However, the
341
peak area was almost constant or decreased when the injection time was longer than
342
80 s. In addition, Fig. S5 showed that the peak height for five metal ions increased
343
with the increase of injection time, but the separation efficiency was poor when
344
sample was injected for 100 s. The above phenomenon was explained by that higher
345
focusing efficiency could be obtained when prolonged the S injection time, whereas
346
overloaded sample failed to be fully complexed by the injected complexing agent,
347
resulting in a decrease of resolution. Taking the focusing efficiency and resolution
15
348
into account, the 80 s of S injection time was selected for further research.
349
3.3. Method verification and application
350
To further evaluate the proposed method, repeatability, calibration curve, LOD,
351
LOQ and sensitivity enhancement factor (SEF) were verified under optimal
352
conditions. The obtained data are summarized in Table 1. The repeatability was
353
analyzed by calculating the relative standard deviation (RSD) of three continuously
354
injections in the same capillary column. Repeatability RSD (%, n = 3) in the
355
migration time was ranged from 1.89% to 1.94% and in the peak area was ranged
356
from 2.87% to 4.73%, respectively. The calibration curves were obtained by plotting
357
the ratio of peak area of five metal ions against the metal ions concentration ranging
358
from 0.25-5 µg mL-1 or 0.5-10 µg mL-1. The linear equations were listed in Table 1
359
and good linearities were obtained with the determination coefficient (R2) ranged
360
from 0.9916-0.9933 for five metal ions. The LODs, on the basis of three times of
361
signal-to-noise, were calculated to examine the sensitivity of the method and the
362
obtained results were in the range of 2.66-27.9 ng mL-1. The LOQs, calculated as ten
363
times of signal-to-noise, were 8.87-93.02 ng mL-1. The SEF obtained by Eq. (4) [39]:
364
SEF =
365
Where the P and Po are peak areas of proposed stacking method and other types of
366
injection, respectively. The C and Co are concentration in proposed stacking method
367
and other types of injection, respectively. In this work, C=Co. According to Eq. (4),
368
the SEFs, obtained from on-line complexation and MSS method comparing with other
(4)
P Co × Po C
16
369
different types of injection, were listed in Table S1. Additionally, some SEFs were not
370
calculated because metal ions no obvious absorption peaks in some types of injection.
371
The developed on-line complexation and MSS method was applied to determine
372
metal residues in real samples. The real samples selected in the study included
373
crayfish, beebread and Dendrobium officinale. The typical electropherograms are
374
shown in Fig. 4. It was obvious that the blank shrimp sample was detected with
375
several peaks, only Cu2+ was detected about 0.26 µg mL-1 (1.16 µg g-1) according to
376
the comparison with standard sample, other peaks were still uncertain. In addition,
377
only Co2+ was detected about 0.10 µg mL-1 (0.74 µg g-1) in the Dendrobium officinale
378
sample. However, the target analytes were not detected in the beebread sample. To
379
further evaluate the effect of sample matrix on the determination of metal ions,
380
recoveries were studied by analyzing the target analytes in spiked samples and the
381
recoveries (%) were listed in Table 2. The recoveries were calculated by Eq. (5) [40]:
382
R% = (
383
where R% is recovery (%), Ass is the amount found in the spiked sample, As is the
384
amount found in the sample, and Sd is the amount added. The recoveries of all five
385
metal ions in three sample matrix were between 83.29% and 115.51%, which
386
indicated that the on-line complexation and MSS method was not affected by the
387
complex sample matrices.
388
3.4. Comparison with other method
389
(5)
Ass - As ) ×100% Sb
In order to highlight the advantages of the proposed method, several techniques 17
390
used for the determination of metal ions in capillary electrophoresis were compared,
391
and the results were tabulated in Table 3. According to the comparison, the new
392
method was easy-operating and did not require complex pre-processing compared to
393
other methods [16,17,18]. In addition, high SEF and low LODs were obtained by
394
MSS in contrast to other types of injection [20-21]. Therefore, the developed method
395
is simple, sensitive and effective.
396
4. Conclusion
397
In this work, simultaneous determination and separation of five metal ions, Ni2+,
398
Co2+, Cu2+, Hg2+ and Cd2+, were achieved by on-line complexation combined with
399
MSS in CZE. The complexing agent was used to enhance the UV absorption of metal
400
ions. The MSS, based on the reversal of the effective electrophoretic mobility of the
401
analytes at the MSSB, was used to focus the analytes and increase the detection
402
sensitivity. The basic requirement of MSS is that the sample matrix contains a
403
surfactant which is capable of forming micelle and has an opposite charge to the
404
target analytes. Meanwhile, the BGS must contain an appropriate proportion of
405
organic modifier. According to the main influencing factors of on-line complexation
406
and MSS, several parameters were studied. Then the stability, reliability and
407
sensitivity of this method was confirmed with the repeatability ranged from 1.89% to
408
4.73% and the LODs were 2.66-27.9 ng mL-1 for all analytes. In addition, the
409
determination of the five metal ions was completed by this method with only twice
410
pressure
injections,
which
was
sample,
18
highly-automated,
easy-operating,
411
environment-friendly and efficient. Furthermore, it is expected to develop a new
412
complexing agent or a mixture of various complexing agents to achieve simultaneous
413
determination of more metal ions, especially harmful metal ions. It will have great
414
development prospects in the fields of analytical chemistry, agronomy, environmental
415
studies, hylology and so on. It also has great potential application by replacing micelle
416
or organic solvent in MSS with other appropriate reagents.
417
Conflicts of interest
418
419
The authors declare no conflicts of interest in relation to this research.
Acknowledgements
420
This study was supported by the Key project of National Natural Science
421
Foundation of China (81730108); Key project of Zhejiang Province Ministry of
422
Science and Technology (2015C03055); Key project of Hangzhou Ministry of
423
Science and Technology (20162013A07); Hangzhou Social Development of Scientific
424
Research projects (20191203B17).
425 426 427 428 429 430
19
431
References
432
[1] F.-L. Fu, Q. Wang, Removal of heavy metal ions from wastewaters: a review, J.
433
Environ. Manage. 92 (2011) 407-418.
434
[2] M. Amjadi, A. Samadi, Modified ionic liquid-coated nanometer TiO2 as a new
435
solid phase extraction sorbent for preconcentration of trace nickel, Colloid. Surface. A
436
434 (2013) 171-177.
437
[3] M.R. Pourjavid, M. Arabieh, S.R. Yousefi, M.R. Jamali, M. Rezaee, M.H.
438
Hosseini, A.A. Sehat, Study on column SPE with synthesized graphene oxide and
439
FAAS for determination of trace amount of Co(II) and Ni(II) ions in real samples,
440
Mat. Sci. Eng. C-Mater. 47 (2015) 114-122.
441
[4] K.-Q. Li, C. Xia, Y.-Q. Qiao, X.-Y. Liu, Dose-response relationships between
442
copper and its biocompatibility/antibacterial activities, J. Trace Elem. Med. Bio. 55
443
(2019) 127-135.
444
[5] X.-M. Wu, H. Wang, S.-X. Yang, H.-Y. Tian, Y.-G. Liu, B.-G. Sun, A novel
445
coumarin-based fluorescent probe for sensitive detection of copper(II) in wine, Food
446
Chem. 284 (2019) 23-27.
447
[6] L.T. Budnik, L. Casteleyn, Mercury pollution in modern times and its
448
socio-medical consequences, Sci. Total Environ. 654 (2019) 720-734.
449
[7] K.M. Pollard, D.M. Cauvi, C.B. Toomey, P. Hultman, D.H. Kono,
450
Mercury-induced
451
https://doi.org/10.1016/j.bbagen. 2019. 02. 001.
452
[8] H. Zhang, M. Reynolds, Cadmium exposure in living organisms: A short review,
inflammation
and
autoimmunity,
20
BBA-Gen.
Subjects
453
Sci. Total Environ. 678 (2019) 761-767.
454
[9] H.-X. Geng, L. Wang, Cadmium: Toxic effects on placental and embryonic
455
development, Environ. Toxicol. Phar. 67 (2019) 102-107.
456
[10] A. Shahat, E.A. Ali, M.F. El Shahat, Colorimetric determination of some toxic
457
metal ions in post-mortem biological samples, Sensor. Actuat. B-Chem. 221 (2015)
458
1027-1034.
459
[11] Y.-P. Wu, X. Liu, Q.-H. Wu, J. Yi, G.-L. Zhang, Differentiation and determination
460
of metal ions using fluorescent sensor array based on carbon nanodots, Sensor. Actuat.
461
B-Chem. 246 (2017) 680-685.
462
[12] C. Arpa, I. Aridasir, Ultrasound assisted ion pair based surfactant-enhanced
463
liquid-liquid microextraction with solidification of floating organic drop combined
464
with flame atomic absorption spectrometry for preconcentration and determination of
465
nickel and cobalt ions in vegetable and herb samples, Food Chem. 284 (2019) 16-22.
466
[13] T. Dasbasi, S. Sacmaci, A. Ulgen, S. Kartal, Determination of some metal ions in
467
various meat and baby food samples by atomic spectrometry, Food Chem. 197 (2016)
468
107-113.
469
[14] Z. Yu, L.A. Bracero, L. Chen, W. Song, X. Wang, B. Zhao, Sensitive metal ions
470
(II) determination with resonance Raman method, Spectrochim. Acta A 105 (2013)
471
52-56.
472
[15] N. Shakulashvili, T. Faller, H. Engelhardt, Simultaneous determination of alkali,
473
alkaline earth and transition metal ions by capillary electrophoresis with indirect UV
474
detection, J. Chromatogr. A 895 (2000) 205–212.
21
475
[16] J. Bi, T. Li, H. Ren, R. Ling, Z.-L. Wu, W.-D. Qin, Capillary electrophoretic
476
determination of heavy-metal ions using 11-mercaptoundecanoic acid and
477
6-mercapto-1-hexanol co-functionalized gold nanoparticle as colorimetric probe, J.
478
Chromatogr. A 1594 (2019) 208-215.
479
[17] S. Almeda, H.E. Gandolfi, L. Arce, M. Valcárcel, Potential of porphyrins as
480
chromogenic reagents for determining metals in capillary electrophoresis, J.
481
Chromatogr. A 1216 (2009) 6256–6258.
482
[18] L.-Q. Peng, L.-H. Ye, J. Cao, L.-J. Du, J.-J. Xu, Q.-D. Zhang, Separation of metal
483
ions via capillary electrophoresis using a pseudostationary phase microfunctionalized
484
with carbon nanotubes, Microchim. Acta 184 (2017) 1747-1754.
485
[19] Y.S. Fung, H.S. Tung, Application of capillary electrophoresis for trace ion
486
analysis in rain water, J. Microcolumn Sep. 12 (2000) 337-344.
487
[20] S. Boonchiangma, C. Kukusamude, W. Ngeontae, S. Srijaranai, In-capillary
488
derivatization and preconcentration for CE of metal ions as their phenanthroline
489
complexes, Chromatographia 77 (2014) 277-286.
490
[21] J. Petr, S. Gerstmann, H. Frank, Determination of some heavy metal cations in
491
molten snow by transient isotachophoresis/capillary zone electrophoresis, J. Sep. Sci.
492
29 (2006) 2256-2260.
493
[22] S.L. Wang, X.Y. Song, J.D. Hu, R. Zhang, L.H. Men, M.M. Wei, T. Xie, J. Cao,
494
Direct speciation analysis of organic mercury in fish and kelp by on-line
495
complexation and stacking using capillary electrophoresis, Food Chem. 281 (2019)
496
41-48.
22
497
[23] F. Yang, J. Li, W. Lu, Y. Wen, X. Cai, J. You, J. Ma, Y. Ding, L. Chen, Speciation
498
analysis of mercury in water samples by dispersive liquid-liquid microextraction
499
coupled to capillary electrophoresis, Electrophoresis 35(4) (2014) 474-81.
500
[24] A.R. Timerbaev, Recent advances and trends in capillary electrophoresis of
501
inorganic ions, Electrophoresis 23 (2002) 3884–3906.
502
[25] J.P. Quirino, Micelle to solvent stacking of organic cations in capillary zone
503
electrophoresis with electrospray ionization mass spectrometry, J Chromatogr. A 1216
504
(2009) 294-299.
505
[26] J.P. Quirino, A.T. Aranas, Simultaneous electrokinetic and hydrodynamic
506
injection with on-line sample concentration via micelle to solvent stacking in micellar
507
electrokinetic chromatography, Anal. Chim. Acta 733 (2012) 84-89.
508
[27] L.Y. Thang, H.H. See, J.P. Quirino, Field-enhanced sample injection-micelle to
509
solvent stacking in nonaqueous capillary electrophoresis, Talanta 161 (2016) 165-169.
510
[28] H.R. Rabanes, A.T. Aranas, N.L. Benbow, J.P. Quirino, Synergistic effect of field
511
enhanced sample injection on micelle to solvent stacking in capillary electrophoresis,
512
J. Chromatogr. A 1267 (2012) 74-79.
513
[29] H.-D. Zhu, W.-J. Lü, H.-H. Li, Y.-H. Ma, S.-Q. Hu, H.-L. Chen, X.-G. Chen,
514
Micelle to solvent stacking of two alkaloids in nonaqueous capillary electrophoresis, J.
515
Chromatogr. A 1218 (2011) 5867-5871.
516
[30] H.-D. Zhu, C.-L. Ren, S.-Q. Hu, X.-M. Zhou, H.-L. Chen, X.-G. Chen, Thousand
517
fold concentration of an alkaloid in capillary zone electrophoresis by micelle to
518
solvent stacking, J. Chromatogr. A 1218 (2011) 733-738.
23
519
[31] X.-P. Yang, X.-M. Cheng, Y.-Y. Lin, Z.-J. Tan, L.-X. Xie, M.M. Choi,
520
Determination of three nitroimidazoles in rabbit plasma by two-step stacking in
521
capillary zone electrophoresis featuring sweeping and micelle to solvent stacking, J.
522
Chromatogr. A 1325 (2014) 227-233.
523
[32] L.I. Ezemonye, P.O. Adebayo, A.A. Enuneku, I. Tongo, E. Ogbomida, Potential
524
health risk consequences of heavy metal concentrations in surface water, shrimp
525
(Macrobrachium macrobrachion) and fish (Brycinus longipinnis) from Benin River,
526
Nigeria, Toxicol. Rep. 6 (2019) 1-9.
527
[33] S. Tokalioglu, Determination of trace elements in commonly consumed
528
medicinal herbs by ICP-MS and multivariate analysis, Food Chem. 134 (2012)
529
2504-2508.
530
[34] J. Vindevogel, P. Sandra, Introduction to micellar electrokinetic chromatography,
531
Chromatogra. Phie. Methods, eds. W. Bertsch, H. Frank, WG Jennings en P. Sandra,
532
Hüthig Buch Verlag Heidelberg (1992).
533
[35] J.P. Quirino, A.T. Aranas, On-line sample concentration via micelle to solvent
534
stacking of cations prepared with aqueous organic solvents in capillary
535
electrophoresis, Electrophoresis 33 (2012) 2167-2175.
536
[36] Evaldas Naujalis, A. Padarauskas, Development of capillary electrophoresis for
537
the determination of metal ions using mixed partial and complete complexation
538
techniques, J. Chromatogr. A 977 (2002) 135–142.
539
[37] J.P. Quirino, A.T. Aranas, Micelle to solvent stacking of organic cations in
540
micellar electrokinetic chromatography with sodium dodecyl sulfate, J. Chromatogr. A
24
541
1218 (2011) 7377-7383.
542
[38] Z. Aturki, S. Fanali, A. Rocco, Online sample concentration and analysis of drugs
543
of abuse in human urine by micelle to solvent stacking in capillary zone
544
electrophoresis, Electrophoresis 37 (2016) 2875-2881.
545
[39] X.F. Yin, Z. Li, S.H. Zhang, C.X. Wu, C. Wang, Z. Wang, Determination of
546
strychnine and brucine in traditional Chinese medicine preparations by capillary zone
547
electrophoresis with micelle to solvent stacking, Chinese Chem. Lett. 22(3) (2011)
548
330-333.
549
[40] P. Li, M. He, B. Chen, B. Hu, Automated dynamic hollow fiber
550
liquid-liquid-liquid microextraction combined with capillary electrophoresis for
551
speciation of mercury in biological and environmental samples, J. Chromatogr. A
552
1415 (2015) 48-56.
553 554 555 556 557 558 559 560 561 562
25
563
Figure captions
564
Fig. 1. The model of on-line complexaion and MSS. (A) Starting situation: the
565
complexing agent and S were injected into the capillary successively. (B) Application
566
of voltage: the direction of EOF was toward to negative electrode and the direction of
567
complexing agent was toward to anode. Meanwhile, the micelle-bound analytes were
568
migrated to the anode due to the negatively charged micelle. (C-D) Continued
569
application of voltage: when the micelle-bound analytes were transported to the
570
MSSB, the organic solvent contained in BGS reduced the affinity of anionic micelles
571
to cationic analytes and the analytes were released, which resulted in the
572
accumulation of analytes and the reversal of µep*(a) to the cathode. Then the
573
positively charged analytes caught up and complexed with uncharged complexing
574
agent, and were separated in CZE. More explanation was in the text.
575
Fig. 2. Experimental verification of on-line complexation based MSS in CZE. (a)
576
typical injection, S was injected at 50 mbar for 5 s. The S were prepared in 180 mmol
577
L-1 sodium acetate solution; (b) large volume injection, S was injected at 50 mbar for
578
60 s. The S were prepared in 180 mmol L-1 sodium acetate solution; (c) MSS, S was
579
injected at 50 mbar for 60 s. The S was prepared in 7.2 mmol L-1 SDS and 180 mmol
580
L-1 sodium acetate solution; (d) simple on-line complexation, complexing agent was
581
injected at 50 mbar for 3 s first, then S was injected at 50 mbar for 5 s. The S were
582
prepared in 180 mmol L-1 sodium acetate solution; (e) on-line complexation and large
583
volume injection, complexing agent was injected at 50 mbar for 3 s first, then S was
584
injected at 50 mbar for 60 s. The S were prepared in 180 mmol L-1 sodium acetate 26
585
solution; (f) on-line complexation and MSS, complexing agent was injected at 50
586
mbar for 3 s first, then S was injected at 50 mbar for 60 s. The S was prepared in 7.2
587
mmol L-1 SDS and 180 mmol L-1 sodium acetate solution. Among all the types of
588
injection, BGS was 200 mmol L-1 sodium acetate containing 50% methanol.
589
Separation voltage was 16 kV and detection wavelength was 214 nm.
590
Fig. 3. Effect of the amount of complexing agent (A), injection time of complexing
591
agent (B), SDS amount in S (C), sodium acetate amount in S (D), methanol content in
592
BGS (E) and injection time of sample (F) on the peak area of metal ions. The initial
593
conditions were as follow: 7.2 mmol L-1 of SDS and 180 mmol L-1 of sodium acetate
594
as sample matrix, pH 5.5; 200 mmol L-1 of sodium acetate containing 50% methanol
595
as BGS, pH 5.5; 30 mmol L-1 of complexing agent, 50 mbar for 3 s; S containing 4 or
596
8 µg mL-1 metal ions, 50 mbar for 60 s.
597
Fig. 4. Typical electropherograms of metal ions in blank crayfish (a), beebread (b),
598
Dendrobium officinale (c).
27
Table 1 Analytical performance of the investigated analytes. Repeatabilitya ( n=3)
Linear range
Linear equation
Determination coefficient
LODb
LOQb
(R2)
(ng mL-1)
(ng mL-1)
Analytes
Migration time
Peak area
(µg mL-1)
Ni
2.85
4.54
0.25-5
y = 77.80 x + 9.37
0.9933
2.66
8.87
Co
2.94
3.03
0.25-5
y = 71.58 x + 13.49
0.9916
3.95
13.16
Cu
2.71
4.61
0.25-5
y = 127.85 x - 5.01
0.9929
3.14
10.48
Hg
1.89
2.87
0.5-10
y = 51.78 x - 4.99
0.9926
17.0
56.56
Cd
2.04
4.73
0.5-10
y = 29.72 x - 14.28
0.9921
27.9
93.02
a
Repeatability is defined as the RSD (%).
b
LOD and LOQ are calculated on the basis of the signal-to-noise ratio of 3 and 10, respectively.
Table 2 Recovery studies of five metal ions in real samples. Analytes
Ni
Co
Cu
Hg
Cd
Beebread
Added
Crayfish
(ug/mL)
Found (ug/mL)
Recoverya (%)
Found (ug/mL)
Recoverya (%)
Found (ug/mL)
Recoverya (%)
0
NDb
-
ND
-
ND
-
0.5
0.44
87.49
0.52
104.69
0.46
92.85
4
3.78
94.48
4.57
114.19
4.02
100.49
0
ND
-
ND
-
0.10
-
0.5
0.45
89.71
0.49
97.13
0.58
96.67
4
3.38
84.50
4.22
105.48
4.15
101.22
0
0.26
-
ND
-
ND
-
0.5
0.70
87.07
0.52
103.71
0.47
93.36
4
4.22
99.10
4.62
115.51
4.10
102.39
0
ND
-
ND
-
ND
-
1 8
0.91 7.36
91.47 91.94
0.97 9.08
96.65 113.51
0.94 7.82
94.25 97.80
0
ND
-
ND
-
ND
-
1
0.93
92.96
1.00
100.19
0.95
95.48
8
6.66
83.29
8.20
102.49
7.81
97.58
Dendrobium officinale
a
Recovery (%) = (the amount found in the spiked sample - the amount found in the sample) × 100 / the amount added.
b
ND, not detect.
Table 3 Comparison of present method with reported methods for the determination of metal ions in capillary electrophoresis. Analytes
Samples
Detection method
LOD (ng/mL)
EFa
Method evaluation
Ref.
Cd2+, Pb2+, and Hg2+
Cosmetics
CE-AuNP/ABCDb
21.4-214
-
Complex synthesis and functionalization of AuNPs,
[16]
long preparation time, expensive material, complex device Co(II),
Ni(II),
Zn(II)
and
River water
CZE-photometric detection
0.012-260
-
Complex formation of complexes,
Honey
pseudostationary phase based
18.5-124
-
Complex
[17]
Mn(II) K(I),
Ba(II),
Ca(II),
Na(I),
Mg(II), Co(II), Ni(II),Zn(II),
CZE-DAD
functionalization
of
MWCNTsc,
long
[18]
preparation time, low sensitivity
Li(I) and Cd(II) Fe(II), Zn(II), Cu(II)
Wine samples
Large volume sample injection
and Cd(II)
50-200
12.7-21.1
Low sensitivity, low enrichment factor
[20]
40-120
42-74
Complex preparation of BGS, the use of sulfuric acid,
[21]
-photodiode array detection
Cd(II), Pb(II), Cu(II), Ni(II),
tITPd-DAD
Snow
and Zn(II) 2+
2+
low sensitivity 2+
2+
Ni , Co , Cu , Hg and Cd
2+
Crayfish,
beebread,
e
MSS -DAD
2.66-27.9
45
Dendrobium officinale a
EF: enrichment factor.
b
CE-AuNP/ABCD: capillary electrophoresis-gold nanoparticle aggregation based-colorimetric detection.
c
MWCNTs: multi-walled carbon nanotubes.
d
tITP: transient isotachophoretic.
e
MSS: micelle to solvent stacking.
High sensitivity, high enrichment factor, simple
This
operation, no extra reagent and instrument
work
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Highlights: MSS combined with the on-line complexion method was presented. The proposed method was used for the analysis of metal ions. The method was sensitive, efficient, easy-operating and environment-friendly. The method was applied to the determination of real samples.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
S0039-9140(19)31211-1
DOI:
https://doi.org/10.1016/j.talanta.2019.120578
Reference:
TAL 120578
To appear in:
Talanta
Received Date: 27 August 2019 Revised Date:
18 November 2019
Accepted Date: 19 November 2019
Please cite this article as: X. Song, R. Zhang, Y. Wang, M. Feng, H. Zhang, S. Wang, J. Cao, T. Xie, Simultaneous determination of five metal ions by on-line complexion combined with micelle to solvent stacking in capillary electrophoresis, Talanta (2019), doi: https://doi.org/10.1016/j.talanta.2019.120578. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical abstract
1
Simultaneous determination of five metal ions by on-line complexion
2
combined with micelle to solvent stacking in capillary electrophoresis
3 4
Xiaoyu Songa, Rui Zhanga, Yue Wanga, Mengqing Fenga, Honghua Zhanga, Shuling Wanga,*, Jun Caoa,b,*, Tian Xiea,c,d,e*
5
a
6 7 8 9 10 11 12 13
Medical College, Hangzhou Normal University, Hangzhou 311121, P.R. China
b
College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, P.R. China c
Holistic Integrative Pharmacy Institutes, Hangzhou Normal University, Hangzhou 311121, P.R. China d
Key Laboratory of Elemene Anti-cancer Medicine of Zhejiang Province, Hangzhou 311121, P.R. China e
Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, Hangzhou 311121, P.R. China
14
E-mail: [email protected]; [email protected]; [email protected]
15
Fax: 86-571-28860237; 86-571-28867909
16 17 18 19 20 21 22 23 24 25 26 27
28
ABSTRACT
29
A direct on-line complexion combined with micelle to solvent stacking method
30
was proposed for simultaneous determination of metal ions by capillary
31
electrophoresis coupled diode array detector. During the experiment, a plug of
32
complexing agent was first injected to the inlet of capillary, followed by introducing
33
the micelle-bound metal ions. Then the metal ions produced a micelle-to-solvent
34
stacking effect and interacted with the complexing agent under a positive voltage.
35
Continued application of voltage, the analytes were effectively focused and separated
36
in the capillary zone electrophoresis. Repeatability was ranged from 1.89% to 1.94%
37
for the migration time. The detection limits were 2.66-27.9 ng mL-1 for Ni2+, Co2+,
38
Cu2+, Hg2+ and Cd2+. Furthermore, the developed method showed a great potential for
39
the determination of metal ions in the crayfish, beebread and Dendrobium officinale
40
samples.
41 42
Keywords: Metal ions; On-line complexion; Micelle to solvent stacking; Capillary
43
electrophoresis
44 45 46 47 48 49
1
50
1. Introduction
51
Metal ions were widely found in organisms and natural environments. Some
52
metal ions, closely related to human life activities, are an important part of the body
53
composition for maintaining the osmotic balance of multiphase systems. However, the
54
high-dose metal ions pose a potential hazard to ecosystems and human health due to
55
their bioaccumulation and non-biodegradability [1]. For example, nickel (Ni) is an
56
essential trace element with potential toxicity. The most usual symptoms caused by Ni
57
are respiratory cancer, respiratory disorders and dermatitis. The Ni compounds even
58
have been the carcinogens [2]. Cobalt (Co), a component of vitamin B12 [3],
59
contributes to the synthesis of hemoglobin and increases the number of red blood cells
60
by stimulating the hematopoietic system of the human bone marrow. However, the
61
main influence of Co on the skin is allergic or irritating dermatitis, which also affects
62
the respiratory system. Copper (Cu) is one of the indispensable metal elements of the
63
human body. According to previous studies [4], Cu has a function of promoting bone
64
metabolism and has certain antibacterial activities, but excess Cu takes potential risks
65
to the kidney, gastrointestinal tract, movement and sensory nerves [5]. The highly
66
toxic nature of mercury (Hg) is now known to the public. Exposure to high Hg likely
67
causes brain damage, inflammation, autoimmunity, and affects the development of the
68
nervous system [6,7]. Exposure to cadmium (Cd) which was partly produced by
69
industrial emissions or smoking causes kidney and liver toxicity and influences the
70
normal growth of the embryos [8,9]. Therefore, the issue of metal pollution has
71
increasingly attracted the attention of analysts. 2
72
At present, various analytical techniques including colorimetric determination
73
[10], fluorescent sensor array voltammetry [11], flame atomic absorption
74
spectrometry [12,13] and resonance Raman [14] were developed for the determination
75
of metal ions. However, the wider applications of these techniques are limited due to
76
the requirements of sophisticated instrument and cumbersome pre-processing program.
77
In recent years, capillary electrophoresis equipped with diode array detector
78
(CE-DAD) was increasingly popular and had been used to detect metal ions with high
79
efficiency, easy controlling, and low solvent consumption [15,16]. Moreover, the use
80
of complexing agents, pre- or on-capillary, is necessary because some metals do not
81
have chromophores under ultraviolet visible (UV) [17]. The complexing agents that
82
have been reported were imidazole [18], 1,10-phenanthroline [19,20], 18-crown
83
ether-6 [21], L-cysteine [22,23] and so on. The complexing agents used in these
84
reports were prepared in background solution (BGS), the preparation method was
85
complicated, and the amount of complexing agents actually combined with the
86
analytes was not easy to calculate. Therefore, in this study, the complexing agent was
87
prepared separately, and the on-line complexation reaction was completed by only
88
twice simple injection. However, the limited injection amount and short optical path
89
of CE lead to its low determination sensitivity. The application of on-line
90
preconcentration strategies can increase the sensitivity of CE [24].
91
Micelle to solvent stacking (MSS), a new stacking technique, was first
92
introduced by Joselito P. Quirino in 2009 [25]. The focus of MSS is based on the
93
reversion of the effective electrophoretic mobility of charged analytes at the MSS
3
94
boundary (MSSB) where separates the sample solution (S) and BGS at the inlet end.
95
The essential conditions for MSS are micelle-containing S and organic
96
solvent-containing BGS. It is required that the micelle in the S has an opposite charge
97
with the charged analytes for binding and transporting analytes to MSSB. The organic
98
solvent in the BGS must be in an amount sufficient to reduce the binding of micelle to
99
analytes after passing through the MSSB, thereby releasing the analytes. In order to
100
confirm that the focus of the analyte is caused by MSS, rather than other field
101
amplification effects, it is desirable that the concentration of electrolyte in the S is
102
consistent with the BGS [26]. Currently, the MSS has been used to detect multiple
103
analytes, such as anticancer drugs [27], antipsychotic drugs [28], alkaloids [29,30]
104
and nitroimidazoles [31], and all the application showed an excellent concentration
105
effect. According to the researches already reported, MSS has not been used for the
106
concentration of metal ions.
107
In this work, five metal ions were studied by on-line complexion combined with
108
MSS in capillary zone electrophoresis (CZE). The five metal ions were Ni2+, Co2+,
109
Cu2+, Hg2+, Cd2+ and they were detected by DAD. As far as our knowledge goes, the
110
MSS was first combined with the complexion approach and it has not been reported
111
for the determination of metal ions. The model of on-line complexaion combined with
112
MSS was explained and the concentration effect of MSS was validated. Additionally,
113
several main influence factors were investigated, such as the type and concentration
114
of complexing agents, the amount of SDS and sodium acetate in S, the methanol
115
content in BGS and so on. Furthermore, the repeatability, linearity, limit of detection 4
116
(LOD) and limit of quantification (LOQ) were evaluated under the selected conditions.
117
Finally, sample matrix effect on the developed method was demonstrated by spike
118
recovery studies in real samples of crayfish, beebread and Dendrobium officinale.
119
2. Materials and methods
120
2.1. Reagents and materials
121
Nickel nitrate hexahydrate (Ni2+), cobaltous nitrate hexahydrate (Co2+), copper
122
nitrate trihydrate (Cu2+), mercury nitrate monohydrate (Hg2+), cadmium nitrate
123
tetrahydrate (Cd2+), imidazole (
124
and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
125
1,10-phenanthroline (99%) was provided by Alfa Aesar (China) Chemical Co., Ltd.
126
(Tianjin, China). L-Cysteine (99%) was obtained from Saan Chemical Technology
127
(Shanghai) Co., Ltd. (Shanghai, China). 18-Crown-6 (99%) was purchased from
128
Aladdin Industrial Corporation (Shanghai, China). Sodium acetate (HPLC,
129
was acquired from Sigma-Aldrich Shanghai Trading Co., Ltd. (Shanghai, China). The
130
stock solutions of Ni2+, Co2+, Cu2+, Cd2+ were prepared in ultrapure water at 1000 µg
131
mL-1 and Hg2+ was at 100 µg mL-1 due to the low solubility of mercury nitrate
132
monohydrate. The working solutions were diluted with ultrapure water. The stock
133
solution of 100 mmol L-1 1,10-phenanthroline was prepared in methanol and diluted
134
with methanol before use. Imidazole, L-cysteine and 18-crown-6 were prepared in
135
ultrapure water. BGS was prepared by sodium acetate in ultrapure water and the pH
136
was adjusted to 5.5 with acetic acid. Working solutions of complexing agent and BGS
99.0%) and nitric acid (HNO3) all were AR grade
5
99.0%)
137
were prepared daily. The samples of crayfish, beebread and Dendrobium officinale
138
were purchased from local market of Hangzhou (Hangzhou, China), Beijing (Beijing,
139
China) and Zhuji (Zhuji, China), respectively. The methanol and ultrapure water used
140
throughout the experiment were HPLC grade and produced by Tedia Company, Inc.
141
(Fairfield, USA). All solutions before analysis were filtered through 0.22 µm
142
disposable filter (Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China)).
143
2.2. Instrument and CE injection procedures
144
An Agilent CE system (7100, Palo Alto, CA, USA), equipped with a DAD
145
detector, was used for the detection of metal ions. The separation of five analytes was
146
achieved in fused-silica capillary of 41.5 cm (33 cm of effective length) × 50 µm i.d.
147
obtained from Agilent Technologies. The detection wavelength of target analytes was
148
set at 214 nm without reference wavelength. Before it was first used, the capillary was
149
activated with 1.0 mol L-1 NaOH (10 min), 0.1 mol L-1 NaOH (10 min), ultrapure
150
water (5 min) and BGS (5 min), successively. Between two runs, the capillary was
151
flushed with 0.1 mol L-1 NaOH (3 min), ultrapure water (3 min) and BGS (2 min) to
152
ensure the accuracy of the data. In the end of the daily experiment, the capillary was
153
rinsed with 0.1 mol L-1 NaOH (10 min) and ultrapure water (5 min) to prevent residue
154
from adhering to the inner wall of the capillary. The MSS of this experiment was
155
injected as follows (initial condition): first, the complexing agent was injected at 50
156
mbar for 3 s after the capillary was filled with BGS. Next, the S containing micelle
157
was injected at 50 mbar for 60 s. Then the operation begins under the applied voltage
6
158
of 16 kV and the temperature in the whole experiment was maintained at 25
. The
159
other injection methods mentioned in this paper were illustrated in the part of 3.1.2.
160
2.3. Sample preparation
161
The preparation methods of samples were referred to previous studies [32,18,33]
162
with minor modifications. Briefly, two crayfish heads were placed in oven and dried
163
at 105 ℃. Weighed and recorded every 30 minutes until the weight maintained
164
constant. The dry crayfish head was ground into powder using agate mortar and pestle,
165
then 0.5 g of powder was weighed and placed into a clean little beaker. The samples
166
were mixed with 10 mL of HNO3. After stirring evenly and standing for 20 min, the
167
beaker containing the mixture was placed on a hot plate at 100℃ for digestion until
168
the solution was clear. The clear digested liquor was centrifuged at 4000 rpm for 10
169
min when it was cooled to room temperature, then took 1 mL into a 10 mL volumetric
170
flask and diluted to the mark with ultrapure water.
171
Similarly, 1 g of weighted beebread was put in a clean beaker and mixed fully
172
with 15 mL of NHO3. Then the mixture was heated until it was clear and transparent
173
after standing for 20 min. The digested liquor was centrifuged at 4000 rpm for 10 min
174
and took 1 mL of the supernatant into a volumetric flask and made up to 10 mL with
175
ultrapure water.
176
About 0.2 g of Dendrobium officinale powder (50 mesh) was weighed into a
177
clean beaker and added 8 mL of HNO3, waited for about 20 min after mixing
178
completely. The undigested powder was precipitated at 4000 rpm (10 min). The
7
179
supernatant was taken 1 mL into a 10 mL volumetric flask and diluted to the mark
180
with ultrapure water.
181
3. Results and discussion
182
3.1. The model and proof of on-line complexaion combined with MSS
183
3.1.1. Model
184
The program for simultaneous determination and separation of five metal ions by
185
on-line complexaion and MSS in CZE was presented in Fig. 1. After the new capillary
186
was activated with 1 mol L-1 NaOH, 0.1 mol L-1 NaOH and water, the entire capillary
187
was filled with BGS containing electrolyte and organic solvent by pressure injection.
188
Then the complexing agent and S were injected successively (Fig. 1A). The S was
189
prepared in electrolyte and anionic micelle, and the electrolyte in S had a conductivity
190
value close to BGS. When positive voltage was applied at the inlet end of the
191
capillary (Fig. 1B), the direction of electroosmotic flow (EOF) was from positive
192
electrode to negative electrode. Thus yielded an electric field and the uncharged
193
complexing agent migrated towards the detector slowly. Meanwhile, the
194
micelle-bound analytes migrated to the anode since the SDS was negatively charged,
195
so the effective electrophoretic velocity of analytes (µep*(a)) was got by Eq. (1) [34]:
196
µ ep ∗ (a )=
197
where µep(a) represents the electrophoretic mobility of the analyte (a), µep(mc) refers
198
to the electrophoretic mobility of the micelle, and k is retention factor.
k 1 µ ep (a ) + µ ep(mc) E 1+ k 1 + k
(1)
8
199
Continued to apply voltage at the inlet end (Fig. 1C), when micelle-bound
200
analytes reached the MSSB, µep*(a) was given by Eq. (2):
201
µ ep ∗ (a ) =
202
At MSSB, the organic solvent contained in BGS reduced the affinity of anionic
203
micelles to cationic analytes and then the micelles gradually collapsed, which resulted
204
in the µep(a) to reverse to the cathode. With more and more micelles passed through
205
MSSB, all the analytes were released and focused at the MSSB. At the that time, the
206
effect of micelle on the µep*(a) was almost negligible or k=0, the µep*(a) depended
207
only on µep (a). Therefore, Eq. (2) was changed to Eq. (3):
208
µ ep ∗ (a )MSSB = µ ep(a )
209
The positively charged metal ions moved faster than uncharged 1,10-phenanthroline
210
(Supplementary material, Fig. S1), moreover, 1,10-phenanthroline was a bidentate
211
N-donor ligand and was capable of forming stable complexes with a variety of
212
transition metals within seconds [19,20], so the complexation occurred between
213
analytes and complexing agent (Fig. 1D). Since 1,10-phenanthroline improved the
214
detection sensitivity and resolution of analytes, the focused analytes with different
215
ratio of charge to mass were effectively separated in CZE and moved toward the
216
detector.
217
3.1.2. Proof
(2)
1 k µ ep(a ) + µ ep (mc) 1+ k 1+ k
(3)
218
The effect of MSS was proved using 1,10-phenanthroline as complexing agent
219
and 50% methanol as organic modifier in BGS. The electropherograms illustrated in
9
220
Fig. 2 were obtained by typical injection (a), large volume injection (b), MSS (c),
221
on-line complexation and typical injection (d), on-line complexation and large volume
222
injection (e), on-line complexation and MSS (f) of metal cations in CZE. In Fig. 2c
223
and Fig. 2d, the concentration of analytes was 5 µg mL-1 (Ni2+, Co2+, Cu2+) or 10 µg
224
mL-1 (Hg2+, Cd2+) prepared in 7.2 mmol L-1 SDS and 180 mmol L-1 sodium acetate
225
solution to form micelles. In Figs. 2a, 2b, 2e and 2f, the metal ions were prepared in
226
sodium acetate solution without SDS. The typical injection was at 50 mbar for 5 s, the
227
large volume injection and MSS were at 50 mbar for 60 s, the on-line complexation
228
was performed by injecting complexing agent at 50 mbar for 3 s before injecting
229
sample. The 200 mmol L-1 sodium acetate solution containing 50% methanol was
230
used as the BGS for all injections.
231
The response of the analytes was low and almost undetectable in the typical
232
injection (Figs. 2a and 2d). The large volume injection of S without micelle yielded
233
broad peaks which caused by the absent stacking effect (Fig. 2b) [35]. It was observed
234
in Fig. 2c that the Ni2+, Cu2+, Hg2+ and Cd2+ were detected when MSS was performed,
235
but the response was low. However, the five metal ions were detected clearly when
236
the complexing agent was injected before the large volume injection (Fig. 2e).
237
Especially, as shown in Fig. 2f, the peaks of five analytes were tall and sharp when
238
on-line complexion combined with MSS. These results confirmed that metal ions
239
were sensitively detected by binding with complexing agent and effectively focused
240
by MSS.
241
3.2. Optimization of on-line complexion and micelle to solvent stacking for metal ions 10
242
3.2.1. Choice of complexing agents
243
The examined metal ions are in the form of its hydrate, nevertheless, most
244
hydrated metal ions do not exhibit significant UV absorption above 185 nm. In
245
addition, metal ions with the same electric charge have almost the same
246
electrophoretic mobility due to similar charge-to-mass ratios, which will result in poor
247
resolution. But the detection sensitivity and resolution can be improved by use of
248
complexing agents [21,36]. In order to determine and separate multiple metal ions
249
simultaneously, several complexing agents such as 1,10-phenanthroline, 18-crown
250
ether-6, L-cysteine and imidazole that have been reported for complexing metal ions,
251
were studied at the same condition (S was 4 or 8 µg mL-1 of each metal ions in 7.2
252
mmol L-1 of SDS and 180 mmol L-1 of sodium acetate, pH 5.5; BGS was 200 mmol
253
L-1 of sodium acetate containing 50% methanol, pH 5.5; 30 mmol L-1 of complexing
254
agent was injected at 50 mbar for 3 s followed by injecting the S at 50 mbar for 60 s).
255
The electropherograms are shown in Fig. S2 in Supplementary Material. It is obvious
256
that all five metal ions of Ni2+, Co2+, Cu2+, Hg2+ and Cd2+ could be determined by
257
complexing with 1,10-phenanthroline and 18-crown ether-6. However, only the peaks
258
of Ni2+, Co2+, Cu2+, Hg2+ and Ni2+, Co2+, Cu2+, Cd2+ appeared when injecting
259
L-cysteine and imidazole as the complexing agent, respectively. Furthermore,
260
compared with the other three complexing agent, the response of five metal ions
261
complexed by 1,10-phenanthroline was higher. Accordingly, 1,10-phenanthroline was
262
adopted as the complexing agent to enhance the detection sensitivity and change the
263
electrophoretic mobility of five metal ions. 11
264
3.2.2. Effect of the amount and injection time of complexing agent
265
The determination and separation of five metal ions were enhanced by the
266
complexation of complexing agent to metal ions. Therefore, the amount of
267
complexing agent was an important parameter that influences the level of
268
complexation. The effect of 1,10-phenanthroline amount was investigated in the range
269
of 10-50 mmol L-1. From the result shown in Fig. 3A, the peak area of Ni2+, Co2+ and
270
Cu2+ increased as the concentration value increased from 10 to 30 mmol L-1, and
271
subsequently decreased from 30 to 50 mmol L-1. However, the peak area of Hg2+ and
272
Cd2+ was increased as the amount of 1,10-phenanthroline up to 40 mmol L-1 and then
273
remained basically constant. The phenomenon was ascribed to the different UV
274
absorption of Ni2+, Co2+, Cu2+ and Hg2+, Cd2+. In order to achieve high response
275
simultaneously for five metal ions, 30 mmol L-1 was chosen as the optimum
276
complexing agent concentration. Differ from the complexing agent concentration, the
277
injection time of complexing agent is a vital factor affecting the interaction time of the
278
complexing agent with the metal ions. So the injection time varied from 2 to 10 s was
279
evaluated. The Fig. 3B revealed that the peak area of all five metal ions increased with
280
the increase of complexing agent injection time, but the peak area grew slowly when
281
the injection time up to 6 s. It is possibly due to the effect of the methanol contained
282
in the complexing agent on the MSS. Consequently, an injection time of 8 s was
283
selected for complexing with metal ions.
284
3.2.3. Effect of the amount of SDS in sample solution
12
285
The concentration of SDS added into the sample solvent needs to reach its
286
critical micelle concentration (CMC) to form micelle and transport the metal ions to
287
the stacking boundary. On the other hand, SDS micelles should be prone to collapse in
288
the buffer. Series concentrations of SDS (4.2, 7.2, 10.2, 13.2, 16.2 mmol L-1) were
289
prepared in 180 mmol L-1 of NaAc (pH 5.5) to research the impact of the amount of
290
SDS in S (Fig. 3C). It can be observed that the increase of SDS concentration yielded
291
inconspicuous enhancement for the peak area of Ni2+, Co2+ and Cu2+. However, the
292
response of Hg2+ and Cd2+ was improved when the concentration of SDS was lower
293
than 7.2 mmol L-1, and succeeding decreased gradually from 7.2 mmol L-1 to 16.2
294
mmol L-1. Additionally, the tailing peaks of Hg2+ and Cd2+ were improved as the
295
increase of the SDS amount in the S. It was probable attributed to the low detection
296
sensitivity of Hg2+ and Cd2+, the accumulated effect of MSS was more visible. But
297
once the amount of SDS was large enough, it was hard to be diluted by the organic
298
modifier in BGS, thereby hindered the release of the metal ions from the micelle. In
299
summary, 7.2 mmol L-1 SDS was used for the next experiment.
300
3.2.4. Effect of the amount of sodium acetate in sample solution
301
The relative conductivity of S decided by the amount of electrolyte to BGS
302
affects the mobility reversal of the target analytes, thus influences the stacking of
303
analytes [37]. The effect of the amount of NaAc in S was studied and the line chart
304
was exhibited in Fig. 3D. The S was composed of 7.2 mmol L-1 SDS and varying
305
amounts of NaAc (140, 160, 180, 200, 220 mmol L-1). The buffer was 200 mmol L-1
13
306
NaAc contained 50% methanol. The result illustrated the increase of NaAc amount in
307
the range of 140-180 mmol L-1 contributed to the detection of metal ions. However,
308
the detection sensitivity decreased as the NaAc amount in S increased to equal to or
309
greater than the NaAc amount in buffer. Moreover, the peak shape of Ni2+ and Hg2+
310
deteriorated with the increase of NaAc amount (Fig. S3 in Supplementary Material),
311
especially at 220 mmol L-1. The phenomenon was due to the fact that as the increase
312
of NaAc concentration in the S, the discrepancy of conductivity between S and BGS
313
regions increased. This caused the destacking of metal ions at the solution zone and
314
the unexpected distorted peaks. So 180 mmol L-1 NaAc in the sample matrix was
315
adopted.
316
3.2.5. Effect of methanol content in BGS
317
The content of organic modifier in BGS affects the viscosity and dielectric
318
constants of the micelle, and then further influence the interaction of metal ions with
319
micelle until the micelle collapse. Therefore, it is essential to investigate the influence
320
of methanol content on the focusing and separation efficiency of metal ions. The
321
results shown in Fig. 3E and Fig. S4 were obtained by changing the percentage of
322
methanol (30, 40, 50, 60, and 70 %) in the BGS while keeping the amount of NaAc at
323
200 mmol L-1. It was clear that the peak area increased firstly and then gradually
324
decreased with the increase of methanol content. In the electropherogram, Ni2+, Co2+
325
and Cu2+ showed low detection sensitivity at 30% methanol, and even Hg2+, Cd2+
326
cannot be detected. When the methanol content exceeded 50%, the resolution between
14
327
the metal ions and the 1,10-phenanthroline was diminished, thus affected the
328
detection of metal ions that appeared later. This can be explained by the fact that low
329
methanol content is not enough to lower the affinity between micelle and metal ions,
330
and the metal ions cannot be released. However, when higher content of organic
331
solvents was added in the BGS, the viscosity of the BGS was increased and led to the
332
decrease of the signal intensities [38]. Based on the research results, 50% methanol
333
was used as the organic modifier in MSS.
334
3.2.6. Effect of the injection time of sample solution
335
Increasing the sample injection time can improve the enrichment factor. On the
336
other hand, the injection time of S decides the length of the introduced micelle in
337
sample matrix, which affects the disintegration degree of micelle, and then further
338
influences the detection limit. The effect of the injection time of S on the detection
339
sensitivity was studied in the range of 40-100 s. As be observed from Fig. 3F, the peak
340
area was increased with the increase of injection time from 40 to 80 s. However, the
341
peak area was almost constant or decreased when the injection time was longer than
342
80 s. In addition, Fig. S5 showed that the peak height for five metal ions increased
343
with the increase of injection time, but the separation efficiency was poor when
344
sample was injected for 100 s. The above phenomenon was explained by that higher
345
focusing efficiency could be obtained when prolonged the S injection time, whereas
346
overloaded sample failed to be fully complexed by the injected complexing agent,
347
resulting in a decrease of resolution. Taking the focusing efficiency and resolution
15
348
into account, the 80 s of S injection time was selected for further research.
349
3.3. Method verification and application
350
To further evaluate the proposed method, repeatability, calibration curve, LOD,
351
LOQ and sensitivity enhancement factor (SEF) were verified under optimal
352
conditions. The obtained data are summarized in Table 1. The repeatability was
353
analyzed by calculating the relative standard deviation (RSD) of three continuously
354
injections in the same capillary column. Repeatability RSD (%, n = 3) in the
355
migration time was ranged from 1.89% to 1.94% and in the peak area was ranged
356
from 2.87% to 4.73%, respectively. The calibration curves were obtained by plotting
357
the ratio of peak area of five metal ions against the metal ions concentration ranging
358
from 0.25-5 µg mL-1 or 0.5-10 µg mL-1. The linear equations were listed in Table 1
359
and good linearities were obtained with the determination coefficient (R2) ranged
360
from 0.9916-0.9933 for five metal ions. The LODs, on the basis of three times of
361
signal-to-noise, were calculated to examine the sensitivity of the method and the
362
obtained results were in the range of 2.66-27.9 ng mL-1. The LOQs, calculated as ten
363
times of signal-to-noise, were 8.87-93.02 ng mL-1. The SEF obtained by Eq. (4) [39]:
364
SEF =
365
Where the P and Po are peak areas of proposed stacking method and other types of
366
injection, respectively. The C and Co are concentration in proposed stacking method
367
and other types of injection, respectively. In this work, C=Co. According to Eq. (4),
368
the SEFs, obtained from on-line complexation and MSS method comparing with other
(4)
P Co × Po C
16
369
different types of injection, were listed in Table S1. Additionally, some SEFs were not
370
calculated because metal ions no obvious absorption peaks in some types of injection.
371
The developed on-line complexation and MSS method was applied to determine
372
metal residues in real samples. The real samples selected in the study included
373
crayfish, beebread and Dendrobium officinale. The typical electropherograms are
374
shown in Fig. 4. It was obvious that the blank shrimp sample was detected with
375
several peaks, only Cu2+ was detected about 0.26 µg mL-1 (1.16 µg g-1) according to
376
the comparison with standard sample, other peaks were still uncertain. In addition,
377
only Co2+ was detected about 0.10 µg mL-1 (0.74 µg g-1) in the Dendrobium officinale
378
sample. However, the target analytes were not detected in the beebread sample. To
379
further evaluate the effect of sample matrix on the determination of metal ions,
380
recoveries were studied by analyzing the target analytes in spiked samples and the
381
recoveries (%) were listed in Table 2. The recoveries were calculated by Eq. (5) [40]:
382
R% = (
383
where R% is recovery (%), Ass is the amount found in the spiked sample, As is the
384
amount found in the sample, and Sd is the amount added. The recoveries of all five
385
metal ions in three sample matrix were between 83.29% and 115.51%, which
386
indicated that the on-line complexation and MSS method was not affected by the
387
complex sample matrices.
388
3.4. Comparison with other method
389
(5)
Ass - As ) ×100% Sb
In order to highlight the advantages of the proposed method, several techniques 17
390
used for the determination of metal ions in capillary electrophoresis were compared,
391
and the results were tabulated in Table 3. According to the comparison, the new
392
method was easy-operating and did not require complex pre-processing compared to
393
other methods [16,17,18]. In addition, high SEF and low LODs were obtained by
394
MSS in contrast to other types of injection [20-21]. Therefore, the developed method
395
is simple, sensitive and effective.
396
4. Conclusion
397
In this work, simultaneous determination and separation of five metal ions, Ni2+,
398
Co2+, Cu2+, Hg2+ and Cd2+, were achieved by on-line complexation combined with
399
MSS in CZE. The complexing agent was used to enhance the UV absorption of metal
400
ions. The MSS, based on the reversal of the effective electrophoretic mobility of the
401
analytes at the MSSB, was used to focus the analytes and increase the detection
402
sensitivity. The basic requirement of MSS is that the sample matrix contains a
403
surfactant which is capable of forming micelle and has an opposite charge to the
404
target analytes. Meanwhile, the BGS must contain an appropriate proportion of
405
organic modifier. According to the main influencing factors of on-line complexation
406
and MSS, several parameters were studied. Then the stability, reliability and
407
sensitivity of this method was confirmed with the repeatability ranged from 1.89% to
408
4.73% and the LODs were 2.66-27.9 ng mL-1 for all analytes. In addition, the
409
determination of the five metal ions was completed by this method with only twice
410
pressure
injections,
which
was
sample,
18
highly-automated,
easy-operating,
411
environment-friendly and efficient. Furthermore, it is expected to develop a new
412
complexing agent or a mixture of various complexing agents to achieve simultaneous
413
determination of more metal ions, especially harmful metal ions. It will have great
414
development prospects in the fields of analytical chemistry, agronomy, environmental
415
studies, hylology and so on. It also has great potential application by replacing micelle
416
or organic solvent in MSS with other appropriate reagents.
417
Conflicts of interest
418
419
The authors declare no conflicts of interest in relation to this research.
Acknowledgements
420
This study was supported by the Key project of National Natural Science
421
Foundation of China (81730108); Key project of Zhejiang Province Ministry of
422
Science and Technology (2015C03055); Key project of Hangzhou Ministry of
423
Science and Technology (20162013A07); Hangzhou Social Development of Scientific
424
Research projects (20191203B17).
425 426 427 428 429 430
19
431
References
432
[1] F.-L. Fu, Q. Wang, Removal of heavy metal ions from wastewaters: a review, J.
433
Environ. Manage. 92 (2011) 407-418.
434
[2] M. Amjadi, A. Samadi, Modified ionic liquid-coated nanometer TiO2 as a new
435
solid phase extraction sorbent for preconcentration of trace nickel, Colloid. Surface. A
436
434 (2013) 171-177.
437
[3] M.R. Pourjavid, M. Arabieh, S.R. Yousefi, M.R. Jamali, M. Rezaee, M.H.
438
Hosseini, A.A. Sehat, Study on column SPE with synthesized graphene oxide and
439
FAAS for determination of trace amount of Co(II) and Ni(II) ions in real samples,
440
Mat. Sci. Eng. C-Mater. 47 (2015) 114-122.
441
[4] K.-Q. Li, C. Xia, Y.-Q. Qiao, X.-Y. Liu, Dose-response relationships between
442
copper and its biocompatibility/antibacterial activities, J. Trace Elem. Med. Bio. 55
443
(2019) 127-135.
444
[5] X.-M. Wu, H. Wang, S.-X. Yang, H.-Y. Tian, Y.-G. Liu, B.-G. Sun, A novel
445
coumarin-based fluorescent probe for sensitive detection of copper(II) in wine, Food
446
Chem. 284 (2019) 23-27.
447
[6] L.T. Budnik, L. Casteleyn, Mercury pollution in modern times and its
448
socio-medical consequences, Sci. Total Environ. 654 (2019) 720-734.
449
[7] K.M. Pollard, D.M. Cauvi, C.B. Toomey, P. Hultman, D.H. Kono,
450
Mercury-induced
451
https://doi.org/10.1016/j.bbagen. 2019. 02. 001.
452
[8] H. Zhang, M. Reynolds, Cadmium exposure in living organisms: A short review,
inflammation
and
autoimmunity,
20
BBA-Gen.
Subjects
453
Sci. Total Environ. 678 (2019) 761-767.
454
[9] H.-X. Geng, L. Wang, Cadmium: Toxic effects on placental and embryonic
455
development, Environ. Toxicol. Phar. 67 (2019) 102-107.
456
[10] A. Shahat, E.A. Ali, M.F. El Shahat, Colorimetric determination of some toxic
457
metal ions in post-mortem biological samples, Sensor. Actuat. B-Chem. 221 (2015)
458
1027-1034.
459
[11] Y.-P. Wu, X. Liu, Q.-H. Wu, J. Yi, G.-L. Zhang, Differentiation and determination
460
of metal ions using fluorescent sensor array based on carbon nanodots, Sensor. Actuat.
461
B-Chem. 246 (2017) 680-685.
462
[12] C. Arpa, I. Aridasir, Ultrasound assisted ion pair based surfactant-enhanced
463
liquid-liquid microextraction with solidification of floating organic drop combined
464
with flame atomic absorption spectrometry for preconcentration and determination of
465
nickel and cobalt ions in vegetable and herb samples, Food Chem. 284 (2019) 16-22.
466
[13] T. Dasbasi, S. Sacmaci, A. Ulgen, S. Kartal, Determination of some metal ions in
467
various meat and baby food samples by atomic spectrometry, Food Chem. 197 (2016)
468
107-113.
469
[14] Z. Yu, L.A. Bracero, L. Chen, W. Song, X. Wang, B. Zhao, Sensitive metal ions
470
(II) determination with resonance Raman method, Spectrochim. Acta A 105 (2013)
471
52-56.
472
[15] N. Shakulashvili, T. Faller, H. Engelhardt, Simultaneous determination of alkali,
473
alkaline earth and transition metal ions by capillary electrophoresis with indirect UV
474
detection, J. Chromatogr. A 895 (2000) 205–212.
21
475
[16] J. Bi, T. Li, H. Ren, R. Ling, Z.-L. Wu, W.-D. Qin, Capillary electrophoretic
476
determination of heavy-metal ions using 11-mercaptoundecanoic acid and
477
6-mercapto-1-hexanol co-functionalized gold nanoparticle as colorimetric probe, J.
478
Chromatogr. A 1594 (2019) 208-215.
479
[17] S. Almeda, H.E. Gandolfi, L. Arce, M. Valcárcel, Potential of porphyrins as
480
chromogenic reagents for determining metals in capillary electrophoresis, J.
481
Chromatogr. A 1216 (2009) 6256–6258.
482
[18] L.-Q. Peng, L.-H. Ye, J. Cao, L.-J. Du, J.-J. Xu, Q.-D. Zhang, Separation of metal
483
ions via capillary electrophoresis using a pseudostationary phase microfunctionalized
484
with carbon nanotubes, Microchim. Acta 184 (2017) 1747-1754.
485
[19] Y.S. Fung, H.S. Tung, Application of capillary electrophoresis for trace ion
486
analysis in rain water, J. Microcolumn Sep. 12 (2000) 337-344.
487
[20] S. Boonchiangma, C. Kukusamude, W. Ngeontae, S. Srijaranai, In-capillary
488
derivatization and preconcentration for CE of metal ions as their phenanthroline
489
complexes, Chromatographia 77 (2014) 277-286.
490
[21] J. Petr, S. Gerstmann, H. Frank, Determination of some heavy metal cations in
491
molten snow by transient isotachophoresis/capillary zone electrophoresis, J. Sep. Sci.
492
29 (2006) 2256-2260.
493
[22] S.L. Wang, X.Y. Song, J.D. Hu, R. Zhang, L.H. Men, M.M. Wei, T. Xie, J. Cao,
494
Direct speciation analysis of organic mercury in fish and kelp by on-line
495
complexation and stacking using capillary electrophoresis, Food Chem. 281 (2019)
496
41-48.
22
497
[23] F. Yang, J. Li, W. Lu, Y. Wen, X. Cai, J. You, J. Ma, Y. Ding, L. Chen, Speciation
498
analysis of mercury in water samples by dispersive liquid-liquid microextraction
499
coupled to capillary electrophoresis, Electrophoresis 35(4) (2014) 474-81.
500
[24] A.R. Timerbaev, Recent advances and trends in capillary electrophoresis of
501
inorganic ions, Electrophoresis 23 (2002) 3884–3906.
502
[25] J.P. Quirino, Micelle to solvent stacking of organic cations in capillary zone
503
electrophoresis with electrospray ionization mass spectrometry, J Chromatogr. A 1216
504
(2009) 294-299.
505
[26] J.P. Quirino, A.T. Aranas, Simultaneous electrokinetic and hydrodynamic
506
injection with on-line sample concentration via micelle to solvent stacking in micellar
507
electrokinetic chromatography, Anal. Chim. Acta 733 (2012) 84-89.
508
[27] L.Y. Thang, H.H. See, J.P. Quirino, Field-enhanced sample injection-micelle to
509
solvent stacking in nonaqueous capillary electrophoresis, Talanta 161 (2016) 165-169.
510
[28] H.R. Rabanes, A.T. Aranas, N.L. Benbow, J.P. Quirino, Synergistic effect of field
511
enhanced sample injection on micelle to solvent stacking in capillary electrophoresis,
512
J. Chromatogr. A 1267 (2012) 74-79.
513
[29] H.-D. Zhu, W.-J. Lü, H.-H. Li, Y.-H. Ma, S.-Q. Hu, H.-L. Chen, X.-G. Chen,
514
Micelle to solvent stacking of two alkaloids in nonaqueous capillary electrophoresis, J.
515
Chromatogr. A 1218 (2011) 5867-5871.
516
[30] H.-D. Zhu, C.-L. Ren, S.-Q. Hu, X.-M. Zhou, H.-L. Chen, X.-G. Chen, Thousand
517
fold concentration of an alkaloid in capillary zone electrophoresis by micelle to
518
solvent stacking, J. Chromatogr. A 1218 (2011) 733-738.
23
519
[31] X.-P. Yang, X.-M. Cheng, Y.-Y. Lin, Z.-J. Tan, L.-X. Xie, M.M. Choi,
520
Determination of three nitroimidazoles in rabbit plasma by two-step stacking in
521
capillary zone electrophoresis featuring sweeping and micelle to solvent stacking, J.
522
Chromatogr. A 1325 (2014) 227-233.
523
[32] L.I. Ezemonye, P.O. Adebayo, A.A. Enuneku, I. Tongo, E. Ogbomida, Potential
524
health risk consequences of heavy metal concentrations in surface water, shrimp
525
(Macrobrachium macrobrachion) and fish (Brycinus longipinnis) from Benin River,
526
Nigeria, Toxicol. Rep. 6 (2019) 1-9.
527
[33] S. Tokalioglu, Determination of trace elements in commonly consumed
528
medicinal herbs by ICP-MS and multivariate analysis, Food Chem. 134 (2012)
529
2504-2508.
530
[34] J. Vindevogel, P. Sandra, Introduction to micellar electrokinetic chromatography,
531
Chromatogra. Phie. Methods, eds. W. Bertsch, H. Frank, WG Jennings en P. Sandra,
532
Hüthig Buch Verlag Heidelberg (1992).
533
[35] J.P. Quirino, A.T. Aranas, On-line sample concentration via micelle to solvent
534
stacking of cations prepared with aqueous organic solvents in capillary
535
electrophoresis, Electrophoresis 33 (2012) 2167-2175.
536
[36] Evaldas Naujalis, A. Padarauskas, Development of capillary electrophoresis for
537
the determination of metal ions using mixed partial and complete complexation
538
techniques, J. Chromatogr. A 977 (2002) 135–142.
539
[37] J.P. Quirino, A.T. Aranas, Micelle to solvent stacking of organic cations in
540
micellar electrokinetic chromatography with sodium dodecyl sulfate, J. Chromatogr. A
24
541
1218 (2011) 7377-7383.
542
[38] Z. Aturki, S. Fanali, A. Rocco, Online sample concentration and analysis of drugs
543
of abuse in human urine by micelle to solvent stacking in capillary zone
544
electrophoresis, Electrophoresis 37 (2016) 2875-2881.
545
[39] X.F. Yin, Z. Li, S.H. Zhang, C.X. Wu, C. Wang, Z. Wang, Determination of
546
strychnine and brucine in traditional Chinese medicine preparations by capillary zone
547
electrophoresis with micelle to solvent stacking, Chinese Chem. Lett. 22(3) (2011)
548
330-333.
549
[40] P. Li, M. He, B. Chen, B. Hu, Automated dynamic hollow fiber
550
liquid-liquid-liquid microextraction combined with capillary electrophoresis for
551
speciation of mercury in biological and environmental samples, J. Chromatogr. A
552
1415 (2015) 48-56.
553 554 555 556 557 558 559 560 561 562
25
563
Figure captions
564
Fig. 1. The model of on-line complexaion and MSS. (A) Starting situation: the
565
complexing agent and S were injected into the capillary successively. (B) Application
566
of voltage: the direction of EOF was toward to negative electrode and the direction of
567
complexing agent was toward to anode. Meanwhile, the micelle-bound analytes were
568
migrated to the anode due to the negatively charged micelle. (C-D) Continued
569
application of voltage: when the micelle-bound analytes were transported to the
570
MSSB, the organic solvent contained in BGS reduced the affinity of anionic micelles
571
to cationic analytes and the analytes were released, which resulted in the
572
accumulation of analytes and the reversal of µep*(a) to the cathode. Then the
573
positively charged analytes caught up and complexed with uncharged complexing
574
agent, and were separated in CZE. More explanation was in the text.
575
Fig. 2. Experimental verification of on-line complexation based MSS in CZE. (a)
576
typical injection, S was injected at 50 mbar for 5 s. The S were prepared in 180 mmol
577
L-1 sodium acetate solution; (b) large volume injection, S was injected at 50 mbar for
578
60 s. The S were prepared in 180 mmol L-1 sodium acetate solution; (c) MSS, S was
579
injected at 50 mbar for 60 s. The S was prepared in 7.2 mmol L-1 SDS and 180 mmol
580
L-1 sodium acetate solution; (d) simple on-line complexation, complexing agent was
581
injected at 50 mbar for 3 s first, then S was injected at 50 mbar for 5 s. The S were
582
prepared in 180 mmol L-1 sodium acetate solution; (e) on-line complexation and large
583
volume injection, complexing agent was injected at 50 mbar for 3 s first, then S was
584
injected at 50 mbar for 60 s. The S were prepared in 180 mmol L-1 sodium acetate 26
585
solution; (f) on-line complexation and MSS, complexing agent was injected at 50
586
mbar for 3 s first, then S was injected at 50 mbar for 60 s. The S was prepared in 7.2
587
mmol L-1 SDS and 180 mmol L-1 sodium acetate solution. Among all the types of
588
injection, BGS was 200 mmol L-1 sodium acetate containing 50% methanol.
589
Separation voltage was 16 kV and detection wavelength was 214 nm.
590
Fig. 3. Effect of the amount of complexing agent (A), injection time of complexing
591
agent (B), SDS amount in S (C), sodium acetate amount in S (D), methanol content in
592
BGS (E) and injection time of sample (F) on the peak area of metal ions. The initial
593
conditions were as follow: 7.2 mmol L-1 of SDS and 180 mmol L-1 of sodium acetate
594
as sample matrix, pH 5.5; 200 mmol L-1 of sodium acetate containing 50% methanol
595
as BGS, pH 5.5; 30 mmol L-1 of complexing agent, 50 mbar for 3 s; S containing 4 or
596
8 µg mL-1 metal ions, 50 mbar for 60 s.
597
Fig. 4. Typical electropherograms of metal ions in blank crayfish (a), beebread (b),
598
Dendrobium officinale (c).
27
Table 1 Analytical performance of the investigated analytes. Repeatabilitya ( n=3)
Linear range
Linear equation
Determination coefficient
LODb
LOQb
(R2)
(ng mL-1)
(ng mL-1)
Analytes
Migration time
Peak area
(µg mL-1)
Ni
2.85
4.54
0.25-5
y = 77.80 x + 9.37
0.9933
2.66
8.87
Co
2.94
3.03
0.25-5
y = 71.58 x + 13.49
0.9916
3.95
13.16
Cu
2.71
4.61
0.25-5
y = 127.85 x - 5.01
0.9929
3.14
10.48
Hg
1.89
2.87
0.5-10
y = 51.78 x - 4.99
0.9926
17.0
56.56
Cd
2.04
4.73
0.5-10
y = 29.72 x - 14.28
0.9921
27.9
93.02
a
Repeatability is defined as the RSD (%).
b
LOD and LOQ are calculated on the basis of the signal-to-noise ratio of 3 and 10, respectively.
Table 2 Recovery studies of five metal ions in real samples. Analytes
Ni
Co
Cu
Hg
Cd
Beebread
Added
Crayfish
(ug/mL)
Found (ug/mL)
Recoverya (%)
Found (ug/mL)
Recoverya (%)
Found (ug/mL)
Recoverya (%)
0
NDb
-
ND
-
ND
-
0.5
0.44
87.49
0.52
104.69
0.46
92.85
4
3.78
94.48
4.57
114.19
4.02
100.49
0
ND
-
ND
-
0.10
-
0.5
0.45
89.71
0.49
97.13
0.58
96.67
4
3.38
84.50
4.22
105.48
4.15
101.22
0
0.26
-
ND
-
ND
-
0.5
0.70
87.07
0.52
103.71
0.47
93.36
4
4.22
99.10
4.62
115.51
4.10
102.39
0
ND
-
ND
-
ND
-
1 8
0.91 7.36
91.47 91.94
0.97 9.08
96.65 113.51
0.94 7.82
94.25 97.80
0
ND
-
ND
-
ND
-
1
0.93
92.96
1.00
100.19
0.95
95.48
8
6.66
83.29
8.20
102.49
7.81
97.58
Dendrobium officinale
a
Recovery (%) = (the amount found in the spiked sample - the amount found in the sample) × 100 / the amount added.
b
ND, not detect.
Table 3 Comparison of present method with reported methods for the determination of metal ions in capillary electrophoresis. Analytes
Samples
Detection method
LOD (ng/mL)
EFa
Method evaluation
Ref.
Cd2+, Pb2+, and Hg2+
Cosmetics
CE-AuNP/ABCDb
21.4-214
-
Complex synthesis and functionalization of AuNPs,
[16]
long preparation time, expensive material, complex device Co(II),
Ni(II),
Zn(II)
and
River water
CZE-photometric detection
0.012-260
-
Complex formation of complexes,
Honey
pseudostationary phase based
18.5-124
-
Complex
[17]
Mn(II) K(I),
Ba(II),
Ca(II),
Na(I),
Mg(II), Co(II), Ni(II),Zn(II),
CZE-DAD
functionalization
of
MWCNTsc,
long
[18]
preparation time, low sensitivity
Li(I) and Cd(II) Fe(II), Zn(II), Cu(II)
Wine samples
Large volume sample injection
and Cd(II)
50-200
12.7-21.1
Low sensitivity, low enrichment factor
[20]
40-120
42-74
Complex preparation of BGS, the use of sulfuric acid,
[21]
-photodiode array detection
Cd(II), Pb(II), Cu(II), Ni(II),
tITPd-DAD
Snow
and Zn(II) 2+
2+
low sensitivity 2+
2+
Ni , Co , Cu , Hg and Cd
2+
Crayfish,
beebread,
e
MSS -DAD
2.66-27.9
45
Dendrobium officinale a
EF: enrichment factor.
b
CE-AuNP/ABCD: capillary electrophoresis-gold nanoparticle aggregation based-colorimetric detection.
c
MWCNTs: multi-walled carbon nanotubes.
d
tITP: transient isotachophoretic.
e
MSS: micelle to solvent stacking.
High sensitivity, high enrichment factor, simple
This
operation, no extra reagent and instrument
work
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Highlights: MSS combined with the on-line complexion method was presented. The proposed method was used for the analysis of metal ions. The method was sensitive, efficient, easy-operating and environment-friendly. The method was applied to the determination of real samples.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.