Nano-electromembrane extraction

Accepted Manuscript

Title: Nano-Electromembrane Extraction

Author: Marıa D. Ramos Payan Bin Li Nickolaj JacobPetersen Henrik Jensen Steen Honore Hansen StigPedersen-Bjergaard

PII: S0003-2670(13)00618-1DOI: http://dx.doi.org/doi:10.1016/j.aca.2013.04.055Reference: ACA 232558

To appear in: Analytica Chimica Acta

Received date: 10-12-2012Revised date: 25-4-2013Accepted date: 28-4-2013

Please cite this article as: M.D.R. Payan, B. Li, N.J. Petersen, H. Jensen, S.H. Hansen,S. Pedersen-Bjergaard, Nano-Electromembrane Extraction, Analytica Chimica Acta(2013), http://dx.doi.org/10.1016/j.aca.2013.04.055

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http://dx.doi.org/doi:10.1016/j.aca.2013.04.055

http://dx.doi.org/10.1016/j.aca.2013.04.055

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Manuscript for Analytica Chimica Acta1

Nano-Electromembrane Extraction2

3

María D. Ramos Payána, Bin Lib, Nickolaj Jacob Petersenb*, Henrik Jensenb, 4

Steen Honoré Hansenb, and Stig Pedersen-Bjergaardb,c5

a Department of Analytical Chemistry, Faculty of Chemistry, University of Seville, P.O. Box 6

41012, Seville, Spain7

b School of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 8

2100 Copenhagen, Denmark9

c School of Pharmacy, University of Oslo, P.O Box 1068 Blindern, 0316 Oslo, Norway10

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Keywords: Sample preparation13

Nano-extraction14

Membrane extraction15

Nano-electromembrane extraction16

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* Corresponding author at: School of Pharmacy, Faculty of Health and Medical Sciences, 18

University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark, Tel.: +4519

35336184; E-mail address: [email protected]

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Abstract21

The present work has for the first time described nano-electromembrane extraction (nano-22

EME). In nano-EME, five basic drugs substances were extracted as model analytes from 200 23

µL acidified sample solution, through a supported liquid membrane (SLM) of 2-nitrophenyl 24

octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor 25

phase. The driving force for the extraction was an electrical potential sustained over the 26

SLM. The acceptor phase was located inside a fused silica capillary, and this capillary was 27

also used for the final analysis of the acceptor phase by capillary electrophoresis (CE). In that 28

way the sample preparation performed by nano-EME was coupled directly with a CE 29

separation. Separation performance of 42.000-193.000 theoretical plates could easily be 30

obtained by this direct sample preparation and injection technique that both provided31

enrichment as well as extraction selectivity. Compared with conventional EME, the acceptor 32

phase volume in nano-EME was down-scaled by a factor of more than 1000. This resulted in 33

a very high enrichment capacity. With loperamide as an example, an enrichment factor 34

exceeding 500 was obtained in only 5 minutes of extraction. This corresponded to 100-times 35

enrichment per minute of nano-EME. Nano-EME was found to be a very soft extraction 36

technique, and about 99.2-99.9 % of the analytes remained in the sample volume of 200 µL. 37

The SLM could be reused for more than 200 nano-EME extractions, and memory effects in 38

the membrane were avoided by effective electro-assisted cleaning, where the electrical 39

potential was actively used to clean the membrane.40

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1. Introduction41

Recently, electromembrane extraction (EME) was proposed as a new concept for42

analytical sample preparation [1]. In EME, charged analytes are extracted from an aqueous 43

sample, through an organic solvent (supported liquid membrane) immobilized in the pores 44

of a thin polymeric membrane, and into a µL volume of an aqueous acceptor phase. The 45

driving force for the extraction is a dc electrical potential sustained over the supported liquid 46

membrane (SLM). For extraction of cationic analytes, the cathode is located in the acceptor 47

phase, and the anode is placed in the sample. Both the sample and the acceptor phase are48

kept under pH control to ensure that the analytes are positively charged during EME. For 49

acidic analytes, the polarity of the electrical potential is reversed, with the anode located in 50

the acceptor phase. Because the analytes have to pass the SLM during EME, which is an 51

organic phase immiscible with water, high extraction selectivity can be obtained [2,3]. The 52

selectivity can easily be tuned by optimizing the chemical composition of the SLM, the 53

magnitude of the electrical potential, and by the pH conditions on both side of the SLM [2,3]. 54

In addition, because the analytes normally are transferred from a relatively large sample 55

volume (typically 0.1-10 mL) and into a small volume of acceptor phase (typically 10-25 µL), 56

EME also provides excellent pre-concentration [2,3].57

EME has been shown to give very clean extracts from complicated real samples like 58

human plasma [1,4], whole blood [5,6], urine [1,4], saliva [7], breast milk [8], waste water 59

[9,10], and drinking water [11]. Since the acceptor phase is aqueous, EME is directly 60

compatible with high-performance liquid chromatography (HPLC) [12], liquid 61

chromatography – mass spectrometry (LC-MS) [13], and capillary electrophoresis (CE) [1]. 62

Typically, less than 25 µL of organic solvent is consumed per sample to establish the SLM. 63

Extraction recoveries in EME are normally in the range 25-75 % after 5 minutes of extraction 64

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[2,3], and enrichment factors up to 190 times have been reported [2,3]. Several applications 65

of EME have been published, among others basic drug substances [1,5,7,8,14-18], acidic 66

drug substances [10,19], lithium [20], peptides [12,13,21], lead [22], heavy metals [23], 67

alkylphosphonic acid derivatives [24], and chlorophenols [25].68

EME is essentially a micro-extraction technique, where analytes are transferred to a µL 69

volume of acceptor phase. Extractions are often close to being exhaustive, and enter steady-70

state conditions after 5-10 minutes. Both these characteristics are highly beneficial for the 71

majority of analytical routine applications. However, for more specialized studies, soft 72

extraction may be important where only small amounts (<1 %) of analyte are removed from 73

the sample. Soft extractions are important in cases where target analytes are in equilibrium 74

with components of the sample matrix, and where the free concentration of the target 75

analyte is requested. This can be the case with drugs interacting with proteins in biological 76

fluids as an example. This paper reports on experiments with down-scaling conventional 77

EME to nano-EME. The idea was to develop a nano-extraction technique providing both soft 78

extraction and high analyte enrichment, and to couple the sample preparation directly with 79

a capillary electrophoresis (CE) separation. This was realized by reducing the volume of the 80

acceptor phase, from typically 10-25 µL in conventional EME, to approximately 8 nL in the 81

present nano-EME work. A preliminary technical solution to nano-EME is presented, and 82

proof-of-principle studies are reported. The proof-of-principle studies included 83

experimentally measured enrichment factors, and were also focused on sample depletion, 84

acceptor phase saturation, and electro-assisted cleaning of the SLM which was reused for 85

more than 200 extractions. The intention of the paper is thus to present a new concept 86

which can be utilized in new analytical procedures in the future. The nano-EME system is 87

also described theoretically by a simple model which provides a good qualitative description 88

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of the system, and therefore may be helpful in optimising performance in future applications 89

of the methodology. The nano-EME system was realized in a crack in a fused silica CE 90

capillary, which was surrounded by a SLM. 91

Systems with some relation to the present work have been published previously [26-28]. 92

In one paper, analytes were transferred from the sample and through a dialysis membrane 93

directly into a CE capillary [26]. Here, the principle for mass transfer was dialysis. In another94

paper, analytes were transferred from the sample and through a SLM and into a CE capillary 95

[27]. In this case, the driving force for the extraction was a pH gradient, and the acceptor 96

phase volume was 280 nL compared to 8 nL in the present work. Very recently, a system 97

close to the current set-up was presented [28]. In this system, analytes were extracted 98

through a SLM and into a CE capillary by using the power supply of the CE-instrument, and 99

this system was used for analysis of perchlorate in biological samples.100

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2. Experimental101

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2.1. Chemicals and reagents103

Analytical grades of pethidine (C15H21NO2; pKa=8.2; log P=2.2), nortriptyline (C19H21N; 104

pKa=9.7; log P=4.3), methadone (C21H27NO; pKa=9.1; log P=3.9), haloperidol (C21H23ClFNO2; 105

pKa=8.1; log P=3.8), and loperamide (C29H33ClN2O2; pKa=9.4; log P=4.1) were all obtained 106

from Sigma-Aldrich (St. Louis, MO, USA). Except for haloperidol, all these basic drugs were 107

used as hydrochlorides. 2-nitrophenyl octyl ether (NPOE) was obtained from Fluka (Buchs, 108

Switzerland). Stock solutions containing 1 mg mL-1 of each compound were prepared in 10% 109

(v/v) ethanol and stored protected from light at 277 K (4oC). Aqueous working solutions of 110

each compound were daily prepared by adequate dilutions from the 1 mg mL-1 stock 111

solutions by 10 mM HCl. Ultra-high purity water (18 MΩ cm) was used to prepare stock 112

solutions, aqueous working solutions, and separation buffer for capillary electrophoresis.113

114

2.2. Capillary electrophoresis115

Capillary electrophoresis (CE) was performed using an Agilent Technologies HP3D-CE 116

instrument (Waldbronn, Germany) equipped with an UV detector operated at 200 nm, and a117

fused-silica capillary (TSP050375, Polymicro Technologies, Phoenix, AZ, USA) of 50 μm i.d, 118

and with an effective length of 41.0 cm and a total length of 61.5 cm. The separation buffer 119

was 60 mM sodium dihydrogen phosphate adjusted to pH 2.7 with ortho-phosphoric acid. 120

The buffer was degased with helium for 10 min to avoid bubble generation during injection 121

and separation. A separation voltage of 15 kV was used, and the temperature of the capillary 122

was set at 298 K (25°C).123

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2.3. System and procedure for nano-EME 126

The nano-EME set up is illustrated in Fig. 1. The system comprised a fused silica capillary, 127

a piece of (porous) hollow fibre, a small polypropylene vial as sample reservoir, two 128

electrodes, and a power supply. The power supply was a model HV5448 3000V (LabSmith, 129

Livermore, CA, USA) with programmable voltage in the range up to 3000 V. The electrodes 130

for the extraction consisted of two 0.5 mm OD platinum wires. A 200 µL PCR tube (VWR 131

International) was used as the sample compartment. The piece of hollow fibre used for 132

immobilization of the supported liquid membrane was a Plasmaphan P1LX polypropylene 133

hollow fibre (330 µm internal diameter, 150 µm wall thickness, and 0.4 µm pore size) 134

purchased from Membrana (Wuppertal, Germany). A fused silica capillary (TSP050375, 135

Polymicro Technologies) of 50 µm internal diameter, 363 µm outside diameter, and a total 136

length of 61.5 cm was used for both nano-EME and capillary electrophoresis (CE).137

First, a narrow crack was made in the fused silica capillary approximately 2 cm from the 138

inlet. This crack was made with a tool for cutting optical fibres (T 108, Biccotest, Cheshunt, 139

UK). The optical fiber cleaver is originally meant for cutting optical fibers for 140

telecommunication at a perfect 90o angle, but can be used for fused silica capillaries as well. 141

The tool clamps onto the capillary thereby allowing exact positioning of the cut (crack). The 142

clamping generates a small stress in the capillary and combined with a sapphire cutting knife 143

slightly touches the capillary surface a fracture in the capillary is generated. The crack144

(fracture) was formed across the whole fused silica capillary at a perfect 90o angle. The 145

polyimide coating covering the fused silica had the same perfect cut as the fused silica but it 146

remained intact opposite to where the knife of the optical fibre cutter touched the capillary. 147

The polyimide therefore maintained the perfect alignment of the cleaved fused silica148

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capillary and assured that when the capillary was held straight the crack in the capillary was 149

closed thereby forming a sub micron channel with close to zero dead volume. When making 150

the crack on the capillary not too much force should be added on the cutter to keep capillary 151

integrity. Doing successfully, a crack was made in the fused silica capillary and the two pieces 152

were still held together by the external polyimide coating. Then, a 5 mm piece of hollow 153

fibre was placed on the outside of the fused silica capillary covering the crack. To facilitate 154

this, the piece of hollow fibre was treated with ethanol that made it slide easily onto the 155

capillary. Covering the crack, the ends of the hollow fibre were carefully melted in order to 156

fix the membrane to the fused silica capillary and to create a leak-tight connection, Fig. 1b.157

For the heat fixing, a PTFE tube was applied to control the heated length and protect the 158

other part. The heat fixing also prevented the sample from migrating into the capillary 159

without passing through the membrane. The heat fixed system was stable for long periods 160

(months) of operations. In addition, the heat fixing reduced the membrane length thereby 161

reducing the carry-over effect caused by a large volume of the SLM. 1 µL of 2-nitrophenyl 162

octyl ether (NPOE) were delivered by a micro-pipette to the piece of hollow fibre. NPOE 163

served as the supported liquid membrane (SLM) and was immediately immobilized into the 164

polypropylene membrane by capillary forces. This process was visually inspected as the 165

appearance of the membrane changed from white to transparent during immobilization, Fig. 166

1c. Excess of NPOE was removed with a medical wipe. The same NPOE was used several days 167

before a new portion of NPOE was added to the membrane. The fused silica capillary with 168

the SLM was passed through holes in the side of the sample vial and the SLM was then 169

located in the sample reservoir. From the sample reservoir, the fused silica capillary was170

directed into the CE-instrument, and a detection window for the UV detector was burned 171

20.5 cm from the outlet. The CE instrument was operated in CE-MS mode, where the electric 172

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current was connected to ground outside the instrument, with a negative potential applied 173

to the buffer reservoir inside the CE instrument during the separation. The height of outlet 174

capillary was adjusted in the same level as the reservoir inside the CE, which minimized the 175

siphoning effect and thereby assuring that no flow was present in the capillary during the 176

injection. Nano-EME and injection was being performed at the end of the capillary placed 177

outside the instrument.178

179

Prior to nano-EME, the sample reservoir was filled with 200 µL of sample consisting of 0.5 180

µg/mL pethidine, nortriptyline, methadone, haloperidol, and loperamide in 10 mM HCl. A 181

buffer vial was located at the inlet of the fused silica capillary and connected to ground with 182

a Pt electrode, and this vial contained the CE separation buffer (60 mM sodium dihydrogen 183

phosphate buffer adjusted to pH 2.7 with ortho-phosphoric acid). The Pt electrode served 184

both as the anode during the CE separation and as the cathode during nano-EME. Prior to an 185

analysis, the separation buffer was flushed into the entire capillary from the buffer vial 186

placed inside the CE-instrument. Finally, the anode for nano-EME was placed in the sample 187

solution and extraction was accomplished by application of extraction voltage. The cationic 188

model analytes were extracted through the SLM and the crack in the fused silica capillary, 189

and into a small segment of the buffer inside the capillary. After nano-EME, the analytes 190

were separated in the fused silica capillary by applying -15 kV at the reservoir placed inside 191

the CE instrument. The sample vial (with sample) was left in the same position during the 192

analysis. The electric potential applied at the sample reservoir was removed during the 193

separation and no sample was leaking into the separation channel during the CE analysis. For 194

both the nano-EME and the separation, the electrode in the buffer reservoir outside the CE 195

instrument was grounded.196

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197

2.4. Calculations of extraction efficiency and enrichment factor198

The enrichment factor ( iEF ) for the analyte i was calculated according to the following 199

equation:200

201

i

i

s

a

i C

CEF (1)202

203

iaC is the concentration of the analyte i in the acceptor phase after extraction and isC is the 204

initial concentration of the analyte in the sample. iaC was determined by capillary 205

electrophoresis and UV-detection using external calibration.206

The extraction efficiency or recovery ( iR ) for the analyte i was defined as the fraction of 207

analyte i in the sample that was transferred to the acceptor phase by the following equation:208

209

%100

i

i

ss

aai CV

CVR (2)210

where Va is the volume of the acceptor phase and Vs is the volume of the sample.211

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3. Results and discussion212

3.1. Technical set-up for nano-extraction213

The technical set-up for nano-electromembrane extraction (nano-EME) is illustrated in 214

Fig. 1. This preliminary set-up was built around a fused silica capillary. A narrow crack was 215

made in the capillary. A small piece of a porous polypropylene hollow fibre was treaded onto 216

the capillary and placed over this crack. The mechanical stability of this arrangement was 217

acceptable for the current proof-of-principle studies, but a more robust design should be 218

developed in the future. The piece of hollow fibre was treated with 2-nitrophenyl octyl ether 219

(NPOE). NPOE served as the supported liquid membrane (SLM), and was immobilized in the 220

pores of the hollow fibre by capillary forces. NPOE was selected without further optimization 221

based on earlier experience from conventional EME [1-3]. The SLM was located in the 222

sample reservoir, and sample was filled into this reservoir. The anode for nano-EME was 223

placed in the sample, whereas the cathode was placed in a buffer reservoir at the “inlet” end 224

of the capillary. The inlet buffer reservoir was in liquid contact with buffer inside the 225

capillary. By application of voltage to the anode in the sample reservoir, extraction of 226

cationic model analytes was accomplished from the sample, through the SLM and the crack 227

in the capillary and into the phosphate buffer solution (pH 2.7) located inside the capillary. 228

This buffer solution also served as the running buffer in the subsequent analysis by capillary 229

electrophoresis (CE), which was accomplished inside the same capillary. Thus, after nano-230

EME, the cationic model analytes were directly separated, detected, and quantified by CE.231

A small zone of the buffer solution inside the fused silica capillary, located around the 232

crack, served as acceptor phase. The volume of this active acceptor phase was 233

approximately 8 nL when extraction was performed for 30 seconds. This volume was 234

measured by comparing the peak widths from the nano-EME / CE-system with peak widths 235

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from conventional CE with the same capillary. The model analytes were in the latter case 236

dissolved in CE buffer to avoid stacking, and injection volumes from conventional CE were 237

calculated with a computer program (CE-Expert, Beckman Coulter, Brea, CA). Since the 238

acceptor phase volume was far below 0.05 µL, we defined the extraction set-up as a nano-239

extraction system and termed it “nano-EME”.240

The experiments conducted with nano-EME in this work were all based on the basic drug 241

substances pethidine, nortriptyline, methadone, haloperidol, and loperamide as model 242

analytes. These substances were selected based on extensive experience from conventional 243

EME [1]. The sample solution contained 10 mM HCl as sample electrolyte. The acidic 244

conditions ensured full protonation of the basic model analytes, which was important for 245

their electro-kinetic migration during nano-EME. Fig. 2 shows a typical electropherogram 246

after nano-EME and CE. In this experiment the sample contained 0.5 µg mL-1 pethidine, 247

nortriptyline, methadone, haloperidol, and loperamide in 10 mM HCl, the sample volume 248

was 200 µL, the supported liquid membrane was 2-nitrophenyl octyl ether, the voltage was 249

200 V and the extraction time was 30 seconds. In spite of the small crack in the capillary, the 250

electrophoretic separation efficiency was high. Some peak tailing was observed, most 251

probably due to trace leakage of analyte from the SLM during the initial stage of the CE 252

analysis, but overall peak shapes were found satisfactory for the proof-of-principle studies 253

(Fig. 2).254

255

3.2. Extraction performance256

In a first series of practical experiment, enrichment factor (EF) was measured as function 257

of applied voltage. The results are summarized in Fig. 3. In these experiments, with a nL-258

volume of acceptor phase, the sample volume was 200 µL, NPOE was used as SLM, and the 259

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extraction time was 30 seconds. As seen from Fig. 3, enrichment factors for all of the model 260

analytes increased with increasing voltage from 0 to 500 V. With increasing voltage, both the 261

magnitude of the sample-to-SLM distribution coefficient and the electrophoretic migration 262

across the SLM increased. A similar behaviour has been reported for conventional EME [29]. 263

The extraction current was in the range of 14-16 µA for a 200 V extraction. Above 200 V, our 264

preliminary system for nano-EME became less stable. Relative standard deviations from 265

replicate extractions increased, and the current during the subsequent CE-analysis became 266

less stable. This indicated that gas bubbles were formed due to electrolysis at high voltages 267

with nano-EME, and this was the reason for the instability. Therefore, it was decided to 268

operate the system at 200 V for the rest of this study. At 200 V, the voltage drop over the 269

SLM was estimated to about 20-40 V. The rest of the voltage drop was in the buffer in the 270

fused silica capillary between the acceptor phase zone and the inlet buffer reservoir with the 271

cathode.272

In another experiment, the enrichment factor (EF) was measured as function of extraction 273

time. The results are summarized in Fig. 4. In these experiments, the sample volume was 200 274

µL, NPOE was used as SLM, and the extractions were carried out at 200 V. As seen from Fig.275

4, enrichment factors for all of the model analytes increased linearly with increasing 276

extraction time from 0 to 300 seconds. The enrichment factors were compound dependent, 277

due to differences in the magnitude of the distribution coefficient and due to differences in278

electrokinetic mobility in the SLM.279

Fig. 4 illustrates a very important point with the nano-EME system. Enrichment per unit 280

time was extremely high for the nano-EME system. With loperamide as an example, an 281

enrichment factor exceeding 500 was obtained in only 5 minutes of extraction. This 282

corresponded to 100-times enrichment per minute of nano-EME. For comparison, a recent 283

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work on EME in a micro-chip format, demonstrated a 107 times enrichment of loperamide 284

after 30 minutes of extraction [30]. In the latter case, the acceptor phase volume was 0.6 µL, 285

and the enrichment per minute was 3.6. In addition to very high enrichment per unit time, 286

the nano-EME system also provided high extraction selectivity. This is illustrated in Fig. 5a+b, 287

where human urine was spiked with pethidine, nortriptyline, methadone, haloperidol, and 288

loperamide, and subsequently processed by nano-EME. Although the urine sample 289

contained a broad range of matrix components, as illustrated by direct CE analysis in Fig. 5a, 290

most matrix components were removed when nano-EME was combined with CE analysis 291

(Fig. 5b). This high selectivity has been discussed previously for conventional EME [1-3].292

The influence of the ionic strength of the sample was investigated with the nano-EME 293

system by comparing extractions performed from pure water samples, 2 M sodium chloride, 294

and 1 M sodium sulphate, and all acidified to pH 2.0 using HCl. As illustrated in Table 1, 295

enrichment factors for the given model analytes were independent of the ionic strength. 296

Also, as seen from Table 1, extractions were repeatable within 1.4 % RSD. Detection limits 297

were 15, 3, 2, 4, and 0.2 ng mL-1 for pethidine, nortriptyline, methadone, haloperidol, and 298

loperamide, respectively.299

300

3.3. Sample depletion301

In a subsequent experiment, the level of sample depletion was investigated. The results 302

are summarized in Table 2. In these experiments, the sample volume was 200 µL, NPOE was 303

used as SLM, the extractions were carried out at 200 V, and the extraction time was 120 304

seconds. As seen from Table 2, recoveries were in the range 0.10 %-0.79 %. Although small 305

amounts of analyte also were trapped inside the SLM (discussed below), nano-EME served as 306

a very soft extraction technique, extremely small amounts of analyte were removed from 307

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the sample, and about 99.2-99.9 % of the analytes remained in the sample. Although 308

recoveries were low, the nano-EME system still provided high enrichment due to the very 309

small volume of acceptor phase (Table 2). This is a unique feature of nano-EME.310

In another experiment, nano-EME was conducted with stirring of the sample. This 311

experiment provided results (recoveries) comparable with data reported above without 312

stirring. The observation supported that extraction in the current nano-EME system occurred 313

only in the boundary layer of the SLM, while the rest of the sample remained relatively 314

undisturbed. This conclusion was further supported by an experiment where the sample 315

volume was varied between 50 and 300 µL. From this experiment, the absolute amount of 316

extracted analyte was unaffected by the sample volume. Thus, whereas conventional EME 317

can extract exhaustively [2,3], nano-EME may provide a very interesting alternative for very 318

soft extractions in a strongly limited sample volume segment.319

320

3.4. Acceptor phase saturation321

With a nL-volume of acceptor phase, the extraction capacity was expected to be limited. 322

This was confirmed by linearity measurements as illustrated in Fig. 6. In these experiments, 323

the sample volume was 200 µL, NPOE was used as SLM, the extractions were carried out at 324

200 V, and the extraction time was 120 seconds. As illustrated in Fig. 6, the nano-EME 325

system provided linear data up to sample concentrations of about 2.5 µg mL-1 for 326

haloperidol, and up to 1 µg mL-1 for pethidine, nortriptyline, and methadone. For the most 327

hydrophobic substance among the model analytes, namely loperamide, which also provided 328

the highest enrichment factor, deviation from linearity was observed at even lower 329

concentration (between 0.25 and 0.5 µg mL-1). For loperamide, the enrichment factor was 330

approximately 200 after 120 seconds. With a concentration of 0.25 µg mL-1 in the sample, 331

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this corresponded to a final concentration of about 50 µg mL-1 in the acceptor phase 332

segment. At higher analyte concentrations (or with longer extraction times) the amount of 333

analyte extracted into the acceptor phase approached the capacity of the system, and 334

significant deviation from linearity was observed. 335

336

3.5. Membrane cleaning337

With the current preliminary set-up for nano-EME, re-use of the SLM for many extractions 338

was mandatory to avoid time-consuming changing of the small SLM in-between each 339

extraction. Although nano-EME may be developed into other technical configurations in the 340

future, re-use of the very small SLM will probably remain as an important issue because very 341

small membranes are difficult to handle. In the current nano-EME system, traces of analyte 342

were trapped inside the SLM after each extraction. This is illustrated in Fig. 7, where a 343

sample free of analytes (pure 10 mM HCl) was extracted right after a sample containing the 344

analytes. As seen from Fig. 7a), carry-over was observed in the system due to trapping in the 345

SLM. However, this carry-over was eliminated by immersing the SLM in pure 10 mM HCl, 346

applying the potential (200 V) for 2 minutes, while flushing the CE capillary with separation 347

buffer. In this electro-assisted cleaning procedure, the SLM was efficiently rinsed using the 348

electrical potential. With the electro-assisted cleaning, the SLM was re-used for more than 349

200 extractions over a period of 2 months before replacement. During this time period, both 350

water samples (low complexity) and urine samples (moderate complexity) were extracted. 351

Highly complex samples like human plasma were not extracted in these experiments. Also, 352

with electro-assisted cleaning, all nano-EME experiments were highly repeatable with RSD 353

values less than 15 %.354

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3.6. Theoretical considerations356

The nano-EME system has several unique features that do not allow previous EME theory 357

to be applied directly. In the present setup, the volume of the acceptor phase was much 358

smaller than the volumes of sample and SLM. As discussed in section 3.3, the experimental 359

results showed that stirring (convective mass transport in the donor solution) had no effect 360

on the extractions efficiency. In order to develop a theoretical model for nano-EME, we 361

assume the following:362

363

1) No significant analyte depletion occurs in the sample (as reported in section 3.3).364

2) After a lag time, tlag, the limiting step to mass transport is the overall electro-365

migration (related to analyte concentration and mobility) in the SLM. Although there 366

may be a small diffusive contribution to mass transport, we shall omit it in the 367

present description. The validity of this assumption has been addressed in the 368

literature [1]. 369

3) The steady state concentration in the SLM is governed by the sample-to-SLM 370

distribution coefficient, Kd,i*, which in turn is related to the Galvani potential 371

difference across the sample-SLM interface. 372

4) The performance is not limited by the solubility in the acceptor phase. This 373

assumption is obviously not valid if very high enrichment factors are achieved (as 374

discussed in section 3.4). In such a situation deviations from the theoretical 375

predictions are likely to occur. 376

377

Under these assumptions, the analyte (i) concentration in the SLM, imc , is governed by the 378

distribution coefficient: 379

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380

t> tlag: *,,, idiDim Kcc (3)381

382

where cD,I is the analyte concentration in the sample. Kd,i* can be estimated as previously 383

described [29] and is dependent on the standard distribution potential of the ion and the 384

Galvani potential difference across the sample-SLM interface, which in turn is related to the 385

overall applied extraction potential.386

387

The analyte flux across the SLM (NPOE membrane), NPOEiJ , can be expressed as: 388

389

dEKcudEcuJ memidiDNPOEimemim

NPOEi

NPOEi // *

,,, (4)390

391

where NPOEiu is the electrokinetic mobility of analyte i in NPOE, memE is the potential drop 392

across the SLM, and d is the SLM thickness. We now infer that at t> tlag we have equality of 393

fluxes from analyte leaving the SLM and of analyte entering the acceptor phase, AiJ :394

395

NPOEiJ = A

iJ (5)396

397

From the definition of flux, the amount of analyte transport into the acceptor phase as a 398

function of time (t> tlag) , niA(t), can be expressed as:399

400

AtJAtJtn NPOEi

Ai

Ai )( (6)401

402

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where t is time and A is the "area of extraction" between the SLM and the acceptor phase. 403

Combining equation (4) and (6) above gives us: 404

405

AtdEKcutn memidiDNPOEi

Ai /)( *

,, (7)406

407

From equation (7) the enrichment factor can be obtained (t> tlag) : 408

tdEKuV

AcVtntEF memid

NPOEi

aiDa

Aii ///)()( *

,,

(8)409

The enrichment factor is independent on the analyte concentration in the sample solution410

provided that the assumptions are valid. Equation (8) predicts a linear relation between 411

enrichment factor and time, which also was observed during the practical experiments 412

discussed in section 3.2. Equation (8) also shows that the most lipophilic compounds (high 413

Kd,i*) are extracted with the highest efficiency, and this was in agreement with the 414

experimental results in section 3.2. It should be noted that the model is not valid for very 415

high analyte concentrations, where the extraction efficiency becomes limited by the 416

solubility of the analyte in the SLM or in the acceptor phase. In the latter case the 417

enrichment factor will level off to a constant value after a long extraction time as discussed 418

in section 3.4.419

420

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4. Conclusions420

The present work has for the first time described nano-electromembrane extraction 421

(nano-EME) using an electrical field as the driving force for mass transfer. Attention was 422

focused on both practical experiments and theoretical considerations. Essentially, nano-EME423

was accomplished by down-scaling conventional EME, reducing the volume of the acceptor 424

phase approximately by a factor of 1000 to only 8 nL. Although the technical set-up used for 425

the nano-EME experiments was preliminary in nature and requires further development, the 426

results clearly demonstrated several very interesting features of nano-EME. First, nano-EME 427

provided extremely high analyte enrichment factors per unit time. Secondly, nano-EME was 428

found to be a very soft extraction technique. Thus, in nano-EME, extraction was performed 429

only in the boundary layer around the supported liquid membrane (SLM), and less than 0.8 430

% of the analyte molecules were extracted from the 200 µL sample. The combination of very 431

soft extraction, high enrichment from very small samples volumes, and transfer of the 432

analytes into a nL aqueous solution, is unique for nano-EME. This should be interesting for 433

the future, among others for non-disturbing investigation of biochemical reactions taking 434

place in very small compartments. 435

436

5. Acknowledgement437

The Danish Council for Independent Research, Technology and Production Sciences (11-438

106647) is acknowledged for financial support.439

440

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References441

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486

Fig. 1. a) Schematic illustration of the nano-EME system in nano-extraction mode and photo of a 375 487

µm OD fused silica capillary with crack and SLM b) before and c) after filling the pores with NPOE.488

489

490

Fig. 2. Electropherogram after nano-EME and capillary electrophoresis. Sample: 0.5 µg mL-1 of 491

pethidine, nortriptyline, methadone, haloperidol, and loperamide in 10 mM HCl; Sample volume: 200 492

µL; Supported liquid membrane: 2-nitrophenyl octyl ether; Extraction voltage: 200 V; Extraction time: 493

30 seconds.494

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495

Fig. 3. Analyte enrichment versus extraction voltage. Sample: 0.5 µg mL-1 of pethidine, nortriptyline, 496

methadone, haloperidol, and loperamide in 10 mM HCl; Sample volume: 200 µL; Supported liquid 497

membrane: 2-nitrophenyl octyl ether; Extraction time: 30 seconds.498

499

500

Fig. 4. Analyte enrichment versus extraction time. Sample: 0.5 µg mL-1 of pethidine, nortriptyline, 501

methadone, haloperidol, and loperamide in 10 mM HCl; Sample volume: 200 µL; Supported liquid 502

membrane: 2-nitrophenyl octyl ether; Voltage: 200 V.503

504

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505

Fig. 5. Electropherograms showing a) blank urine injected without nano-EME, b) urine containing 5 506

µg mL-1 of model analytes without nano-EME, and c) urine containing 5 µg mL-1 of model analytes 507

after nano-EME. 1) pethidine, 2) nortriptyline, 3) methadone, 4 ) haloperidol and 5 ) loperamide. 508

Sample volume: 200 µL; Supported liquid membrane: 2-nitrophenyl octyl ether; Voltage: 200 V; 509

Extraction time: 15 seconds.510

511

512

Fig. 6 Peak area divided by concentration as function of concentration (µg mL-1). Sample: pethidine, 513

nortriptyline, methadone, haloperidol, and loperamide in 10 mM HCl; Sample volume: 200 µL; 514

Supported liquid membrane: 2-nitrophenyl octyl ether; Voltage: 200 V; Extraction time: 120 seconds. 515

Left hand axis is for pethidine, nortriptyline and methadone. Right hand axis is for haloperidol and 516

loperamide.517

518

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519

Fig. 7. Electropherogram after nano-EME and capillary electrophoresis of pure 10 mM HCl (no 520

analytes), following a sample containing 0.5 µg mL-1 of pethidine 1), nortriptyline 2), methadone 3), 521

haloperidol 4), and loperamide 5) in 10 mM HCl. a) No cleaning of the SLM, b) after cleaning of the 522

SLM by 200 V for 2 minutes. Sample volume: 200 µL; Supported liquid membrane: 2-nitrophenyl 523

octyl ether; Voltage: 200 V; Extraction time: 120 seconds.524

525

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527

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Table 1 Enrichment factor obtained with different sample matrix 527

Sample matrix 528

Water 2 M NaCl 1 M Na2SO4529

Analyte (n=3) (n=3) (n=3)530

531

Pethidine 4.9 (1.3) 4.7 (1.2) 4.6 (0.9)532

Nortriptyline 12.6 (1.1) 12.3 (0.9) 12.4 (1.3)533

Methadone 32.2 (1.4) 30.1 (1.1) 30.1 (1.0)534

Haloperidol 6.0 (1.3) 5.4 (1.2) 5.7 (1.3)535

Loperamide 58.1 (0.9) 56.4 (1.0) 57.9 (1.3)536

537

Sample: 0.5 µg mL-1 of pethidine, nortriptyline, methadone, haloperidol, and loperamide in water, 2 538

M NaCl and 1 M sodium sulphate, all adjusted to pH 2 with HCl; Sample volume: 200 µL; Supported 539

liquid membrane: 2-nitrophenyl octyl ether; Extraction voltage: 200 V; Extraction time: 60 seconds. 540

RSD (in %) are given in parentheses.541

542

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Table 2 Amount of analyte extracted into the acceptor phase544

Analyte Recovery in Amount left Enrichment545

acceptor phase in sample factor546

(n=3) (n=3) (n=3)547

548

Pethidine 0.10 % ~99.9 25549

Nortriptyline 0.27 % ~99.7 69550

Methadone 0.46 % ~99.5 114551

Haloperidol 0.28 % ~99.7 63552

Loperamide 0.79 % ~99.2 196553

554

Sample: 0.5 µg mL-1 of pethidine, nortriptyline, methadone, haloperidol, and loperamide in 10 mM 555

HCl; Sample volume: 200 µL; Supported liquid membrane: 2-nitrophenyl octyl ether; Extraction 556

voltage: 200 V; Extraction time: 120 seconds.557

558

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EME (electromembrane extraction) was coupled directly with a CE separation561capillary.562

The acceptor volume was only in the order of 8 nL 563 High efficient CE separations could be obtained without the need of sample stacking. 564 The system presented high enrichment factors exceeding 500.565 The supported liquid membrane (SLM) was stable for more than 200 extractions.566

567

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*Graphical Abstract

Nano-electromembrane extraction

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