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

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Determination of three nitroimidazoles in rabbit plasma by two-step stacking in

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capillary zone electrophoresis featuring sweeping and micelle to solvent stacking, J.

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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.