Salt Removal from Microliter Sample Volumes by Multiple Phase

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Salt removal from microliter sample volumes by multiple phase micro-electromembrane extractions across free liquid membranes Pavel Kuban Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02017 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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Analytical Chemistry

Salt removal from microliter sample volumes by multiple phase micro-electromembrane extractions across free liquid membranes

Pavel Kubáň

Institute of Analytical Chemistry of the Czech Academy of Sciences, v. v. i., Veveří 97, CZ60200 Brno, Czech Republic

1 ACS Paragon Plus Environment


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ABSTRACT: A new concept for rapid and efficient salt removal from minute volumes of saline samples is presented by using multiple phase micro-electromembrane extraction (µEME). Disposable µ-EME unit is filled with five consecutive plugs of immiscible aqueous and organic solutions; the aqueous sample forms the central phase and is encompassed by two free liquid membranes (FLMs) and two extraction solutions. Salt cations and anions migrate in opposite directions from the sample solution on application of 300 V d.c. electric potential, cross the neighboring FLM and are transferred to the corresponding extraction solution. At the same time, the two FLMs selectively eliminate µ-EME transfers of target analytes, which are retained in the sample, and the resulting salt-free sample is then used for subsequent analysis. At optimized conditions (FLMs consisted of 1-pentanol and extraction solutions of DI water and 100 mM NH4OH), more than 99.3% NaCl was removed from samples containing physiological NaCl concentrations. Simultaneously, more than 95% of model biochemical species (human serum albumin, neurotensin, creatinine, glycine and alanine) were retained in the samples for subsequent analyses. Good quality CE-C4D electropherograms and interference-free ESI-MS spectra of selected biochemical species were obtained after µ-EME salt removal from samples containing µM concentrations of the target analytes and 150 mM of NaCl.

KEYWORDS: Capillary electrophoresis; Contactless conductivity detection; Electrospray ionization mass spectrometry; Micro-extractions; Salt removal


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INTRODUCTION. Analyses of complex samples are usually hampered by the presence of high concentrations of matrix components, which may interfere with separation process and with detection of target analytes. Matrix components may also be deposited at the analytical instrumentation inlet and induce its reversible or even irreversible damage. Sample pretreatment is thus applied before injections of complex samples, which is traditionally carried out by liquid-liquid extraction (LLE) or solid phase extraction (SPE). Despite their wide acceptance and apart from having uncontentious advantages, LLE and SPE have also some disadvantages, which have been discussed previously1-4, and alternative techniques for sample treatment have been extensively developed during the last two decades. A number of novel microextraction techniques, which offer faster, cheaper and “greener” pretreatment of complex samples, have been described as alternatives to LLE and SPE, of which solid-phase microextraction (SPME)4-10 and liquid-phase microextraction (LPME)4,11-16 are the two most successful successors. In SPME and LPME, target analytes are extracted from large volumes of complex samples onto SPME fibers and into low volumes of acceptor solutions, respectively, and simultaneous sample clean-up and analyte preconcentration is achieved. SPME and LPME are predominantly used for extractions of low molecular weight (MW) analytes from complex matrices where inorganic salts and various macromolecular compounds are major interfering constituents4-16. In addition to clean-up and preconcentration of low MW analytes, micropreparative techniques can be also applied to aid the opposite purpose, i.e. to remove low MW species (salts and other small ions) from saline samples in order to analyze macromolecules retained in the original solution. This is especially important in MS characterization of biomolecules using the two soft ionization modes (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization), which are most amenable for these purposes but significantly suffer from the presence of high salt contents in the samples17,18. To ensure interference-free



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operation of ESI-MS, various salt removal techniques were employed. Pretreatment of saline samples was carried out by membrane clean-up17,18, application of gel filtration19,20, SPE21-23, microdialysis24,25 and other techniques, such as, use of solution additives26 and direct membrane-ESI27. Major difficulties with the reported techniques are rather high pretreatment time and significant analyte loss in gel filtration, application of additional organic solvents in SPE and dilution of samples in microdialysis. In addition, one drawback common to all above mentioned preparative techniques is the difficulty in handling and pretreatment of minute volumes (in µL range) of complex samples. Electromembrane extraction (EME) was developed as a novel microextraction technique in 200628. In EME, ionic species are transferred from aqueous sample across organic liquid membrane into aqueous acceptor solution by the action of electric potential. Selectivity of the EME transfer is given by the use of tailor-made liquid membrane, which usually enables transfer of selected ions only, and by the applied electric potential29-34. EMEs are exclusively used for clean-up and preconcentration of low MW ionizable organic species, whereas matrix components, such as salts, proteins and other macromolecular species are retained by the liquid membranes. In a few publications, EMEs were also applied to extractions of biochemical species, such as amino acids and peptides35-37. Recently, EME was miniaturized into micro-EME (µ-EME), which uses disposable polymeric capillaries as extraction devices and only sub-µL to µL volumes of samples, organic solvents (in form of free liquid membranes (FLMs)) and acceptor solutions38,39. The µ-EME format also enables use of multiple aqueous and organic phases, which are formed as adjacent plugs in the capillary, and ionic analytes can selectively migrate across multiple phase interfaces into appropriate aqueous solutions40,41. In this contribution, a completely novel principle of EME is demonstrated, which is used for efficient salt removal from µL volumes of saline samples. A five-phase µ-EME set-up is


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formed using an aqueous sample as the central phase, which is sandwiched between two selective FLMs and two aqueous extraction solutions. On application of d.c. electric potential, inorganic cations and anions (i.e. sodium and chloride) migrate in opposite directions from sample solution, cross the neighboring FLM and are finally transferred to the corresponding extraction solution. Simultaneously, composition of the two FLMs eliminates migration of target analytes (i.e. biochemical species) across the organic phases and the analytes are retained in the sample, which is then used for analysis. The µ-EME set-up is also characterized by an excellent variability, which enables rapid optimization of operational parameters for desalting of samples with various salinity. Rapid and efficient salt removal (≥ 99.3% of NaCl) and excellent retention of selected biochemical species (≥ 95% of model protein, peptide and amino acids) was achieved for samples with physiological NaCl concentrations prior to capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) and ESI-MS analyses of the selected biochemical species.

EXPERIMENTAL SECTION Reagents and solutions. All chemicals were of reagent grade and deionized (DI) water with resistivity higher than 18 MΩ·cm was used throughout. Stock solutions of human serum albumin (HSA, 100 µM) and of neurotensin acetate, creatinine, glycine and alanine (1 mM) were prepared in DI water. The chemicals were supplied by Sigma, Steinheim, Germany. Stock solutions (1 M) of NaCl, NaOH, NH4OH (all Lach:Ner, Neratovice, Czech Republic) and formic acid (HFo, Sigma) and stock solution (5 M) of acetic acid (HAc, Fluka, Buchs, Switzerland) were prepared by dissolving the chemicals in DI water. Working solutions of NaCl (10 – 500 mM), NH4OH (10 – 100 mM), HFo and HAc (10 – 100 mM) were prepared by dissolving the stock solutions in DI water. Standard solutions of HSA, neurotensin and amino acids were prepared by dissolving appropriate volumes of stock solutions in working



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solutions of NaCl or in DI water. Background electrolyte (BGE) solutions for CE-C4D analyses were prepared from 5 M stock solution of HAc. Optimum BGE solution for analyses of sodium, HSA, neurotensin, amino acids and chloride consisted of 1 M HAc and 0.05% (v/v) Tween 20 (Sigma) at pH 2.4. Stock solutions of biochemical species were stored at – 20°C and other stock solutions at 4°C for one month. Standard, working and BGE solutions were prepared daily. Organic solvents used in the experiments were 1-butanol (≥ 99.8%, Sigma), 1-pentanol (≥ 99%, Fluka) and 1-hexanol (≥ 98%, Fluka) and were used without further purification.

Capillary electrophoresis. A lab-made CE instrument equipped with a C4D (Admet, Prague, Czech Republic, operated at 50 Vpp/1.84 MHz) was used. The separation voltage was provided by a high voltage power supply unit Spellman CZE1000R (Start Spellman, Pulborough, UK) and was operated at a potential of – 20 kV and + 20 kV applied at the injection side for separations of anions and cations, respectively. Data were collected using Orca-2800 (Ecom, Prague, Czech Republic) data acquisition system. CE-C4D analyses were performed in a fused-silica (FS) capillary (50/375 µm ID/OD, 46 cm Ltot, 13 cm Leff) from Polymicro Technologies (Phoenix, AZ, USA). Prior to the first use, the capillary was preconditioned by flushing with 5 M HAc (15 min), DI water (5 min) and BGE solution (5 min). Between two successive CE analyses, the capillary was rinsed with BGE solution for 1 min. Injections of aqueous solutions were carried out from 1 cm long segments of perfluoroalkoxy tubing (Vici-Jour, Schenkon, Switzerland, 1/1.6 mm ID/OD) by siphoning (7 cm for 10 s) according to a procedure described in reference39. All CE experiments were performed at ambient temperature (25 ± 1°C).


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Mass spectrometry. MS infusion analyses were conducted on the maXis impact mass spectrometer (ESI/TOF/MS, Bruker Daltonics, Bremen, Germany) using a nanospray prepared from a FS capillary (Polymicro Technologies, 10/375 µm ID/OD) with a polished tip. The heated inlet capillary was maintained at 250°C. The MS detection was performed in the positive ion mode with an ESI voltage of 2.5 kV and the scan range of 200 – 2500 m/z. A microinsert was used for sample infusions, which allowed for sampling from volumes down to 5 µL. For infusion of sample after µ-EME salt removal, 2.5 µL of the sample was diluted with 2.5 µL of 200 mM HAc.

Salt removal. PP micropipette tips (Part No. 959, Kartell, Noviglio, Italy) were used as salt removal units. The tips were filled successively with the following five immiscible phases: aqueous extraction solution 1, organic solvent 1 (FLM 1), aqueous sample solution, organic solvent 2 (FLM 2) and aqueous extraction solution 2. Note that up to 100 µL of solutions can be pipetted into the tip, thus experiments with various volumes of all phases were possible. Micropipettes with adjustable volume (Eppendorf AG, Hamburg, Germany and Biohit, Helsinki, Finland) were used for filling the tips with the five solutions. The solutions were pipetted directly into the narrower end of the tip and two tubular platinum electrodes (0.25 mm thick Pt wire, 99.95%, Advent, Oxford, England) were inserted into terminal aqueous solutions41. Cathode was placed into extraction solution 1 and anode into extraction solution 2. µ-EME salt removal was initiated by setting a d.c. power supply ES 0300-0.45 (Delta Elektronika, Zierikzee, Netherlands) to desired operational potential and by switching it on. A photograph of the µ-EME unit filled with five aqueous/organic phases during µ-EME salt removal is depicted in Figure 1A. Electric current values during salt removals were monitored using an M-3800 (Metex, Seoul, Korea) digital multimetre. The µ-EME units were positioned horizontally and solutions in the units were not agitated/stirred. All salt removals were



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performed at ambient temperature (25 ± 1°C) and the units were discarded after each µ-EME. After salt removal, aqueous phases were collected with the micropipettes for analyses and organic phases were discarded. The conical tip of the µ-EME units (left end in Figure 1A) enabled nearly quantitative collection of all phases after µ-EMEs. Moreover, the fact that the right end of the unit has only a slightly conical shape ensured comparable lengths for identical FLM 1 and FLM 2 volumes and thus comparable distribution of electric field across the FLMs. For optimized volumes of operational solutions (extraction solutions, 15 µL; sample solution, 3 µL; FLMs, 5 µL), the differences in lengths of FLM 1 and FLM 2 were less than 10%.

RESULTS AND DISCUSSION FLM composition. Aqueous sample forms the central phase and is sandwiched between two FLMs and two aqueous extraction solutions (see Figure 1A). On application of d.c. voltage, cations and anions from sample solution migrate in opposite directions and can be simultaneously transferred across FLM 1 and FLM 2 into the corresponding extraction solution, respectively. Since each phase, including FLMs, can have unique size and composition, proper selection of the organic phases can significantly affect electrically induced transfers of ionic species from sample to FLMs and to extraction solutions. In the actual procedure, small inorganic cations and anions (i.e. sodium and chloride) are the matrix ions to be removed from sample solution, whereas model biochemical species (i.e. proteins, peptides and amino acids) are the analytes to be retained therein. FLMs, which enable transfers of small inorganic ions and at the same time disable transfers of biochemicals, should thus be preferentially used. The above described fundamental principles of salt removal using µ-EMEs across multiple aqueous and organic phases are schematically depicted in Figure 1B.


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Electrically induced transfers of basic drugs, small inorganic ions, proteins and amino acids were investigated previously38,42. It has been proved that large biochemical species (i.e. HSA) are efficiently retained by organic solvents, such as, aliphatic alcohols and nitrated organic solvents, typically used in EME42. These solvents efficiently hindered EME transfers of small biochemical species (i.e. amino acids) as well42. In addition, low aliphatic alcohols enabled EME transfers of small inorganic ions, such as sodium and chloride, whereas nitrated organic solvents eliminated their transfers efficiently38. Based on these results, low aliphatic alcohols were selected as potentially useful solvents for µ-EME salt removals and 1-butanol, 1pentanol and 1-hexanol were examined. Their suitability for salt removal was examined with 20 mM NaCl sample solution. The µEME units were filled with 10 µL extraction solution 1 (DI water), 4 µL FLM 1 (organic solvent), 3 µL sample solution, 4 µL FLM 2 (organic solvent) and 10 µL extraction solution 2 (DI water). Extraction potentials of 100, 200 and 300 V were applied for µ-EMEs across 1butanol, 1-pentanol and 1-hexanol, respectively. Problems with stability of the organic phases were observed for 1-butanol and mixing of the adjacent solutions occurred at ~ 2 min of µEME. From the three aliphatic alcohols, 1-butanol has highest solubility in water (~ 63 g/L; https://pubchem.ncbi.nlm.nih.gov/compound/1-butanol; 16th May 2017) and instabilities observed at the phase interfaces were ascribed to partial dissolution of 1-butanol in neighbouring aqueous solutions and to concurrent effect of applied electric potential. Compared to 1-butanol, 1-pentanol and 1-hexanol exhibit about three- and ten-fold lower solubility in water, respectively, (https://pubchem.ncbi.nlm.nih.gov/; 16th May 2017) and no stability issues were observed during salt removals across FLMs formed from the two higher alcohols. The contacting surface areas between FLMs and aqueous solutions are rather small (~ 1 mm2) compared to the total FLM plug lengths (~ 2.5 mm) and no measurable decrease in FLM lengths was observed during the experiments with 1-pentanol and 1-hexanol for removal



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times of up to 1 hour (see Section “Electric potential”). Transfer efficiency of sodium and chloride was higher for FLMs composed of 1-pentanol and almost complete salt removal was achieved in about 7 min. On the other hand, more than 30% of salt remained in the sample solution even after 10 min of µ-EME across 1-hexanol and for these reasons, 1-pentanol was selected for further experiments.

Extraction solutions composition. Electrolysis takes place directly in the µ-EME operational solutions, may change their composition and apart from transferring solution components, electrolysis by-products may also be transferred across FLMs during µ-EMEs43. Thus, composition of extraction solutions plays a significant role in µ-EMEs across FLMs. The following extraction solutions were tested for µ-EME salt removal: extraction solution 1 (DI water and various concentrations of HAc or HFo) and extraction solution 2 (DI water and various concentrations of NH4OH). Acidic and alkaline extraction solutions may neutralize electrolytically produced hydroxide (at cathode) and hydronium (at anode) ions, respectively, and help maintain stable pH of sample and extraction solutions. In addition, possible migration of ammonium and acetate (or formate) ions from extraction solutions across/into sample solution constitutes no obstacles since these species are volatile and are fully compatible with ESI-MS. Stabilization of operational solutions pH could be also achieved by application of buffers. However, buffers were not considered as extraction solutions due to possible transfers of buffering substances to sample solutions, which might increase their complexity and limit the subsequent analyses. Experimental conditions were same as in the previous Section except for sample solution (50 mM NaCl) and extraction potential (300 V). Approx. 80% NaCl was removed from sample solution after 8 min for extraction solutions consisting of DI water. After 8 min, electric current dropped to µA level and no further salt removal was observed. Extraction solutions


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retrieved from anodic and cathodic part of the unit were strongly acidic and alkaline, respectively, moreover, sample solution was acidic. Similar electric current behavior and efficiency of salt removal were also observed for combinations of DI water (extraction solution 2) and various concentrations of HAc or HFo (extraction solution 1), although pH stabilization of extraction solution 1 was achieved for acid concentrations ≥ 50 mM. Next, 100 mM HFo/100 mM NH4OH and DI water/100 mM NH4OH were examined as extraction solution 1/extraction solution 2. Extraction solutions remained acidic and alkaline for µ-EMEs using the former conditions and sample solution was neutral, however, salt removal efficiency did not improve compared to previous experiments and approx. 20% NaCl remained in the sample solution after µ-EME. For the latter conditions, extraction solution 1 and 2 were alkaline, sample solution was neutral-slightly alkaline and ≥ 99% NaCl was removed after µ-EME. A comparison of sodium and chloride ions present in sample solutions after salt removal using acid/hydroxide vs. DI water/hydroxide extraction solutions is demonstrated in Figure S1 in Supporting Information. The reasons for the lower efficiency of salt removal in the acid/hydroxide µ-EME system are not fully understood at the moment but might be related to lower transfer of organic anions across 1-pentanol (FLM 1), their accumulation at the phase interface and subsequent reduction of NaCl removal. Less efficient cross-membrane transfers of formate were confirmed by CE-C4D analyses of both extraction solutions after µ-EMEs, however, to fully uncover this phenomenon a comprehensive study is necessary, which was beyond the intended scope of this manuscript. The latter conditions were adopted and were further optimized to obtain stable and efficient µEME salt removal. Extraction solution 1 was DI water in all experiments and concentrations of NH4OH in extraction solution 2 were 10, 25, 50 and 100 mM. Experimental conditions were same as in the previous Section except for sample solution (150 mM NaCl), which corresponds to typical physiological NaCl concentrations. Figure 2 shows salt removal



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efficiency for various NH4OH concentrations. Obviously, low concentrations of NH4OH exhibit lower extraction efficiency and almost 20% of NaCl remained in sample solution for µ-EMEs with 10 mM NH4OH solution. Moreover, sudden increase in electric current was observed at approx. 8.5 and 11 min of µ-EME for 10 and 25 mM NH4OH solutions resulting into compromised stability of the µ-EME system. This current increase was associated with depletion of hydroxide ions in extraction solutions 2 due to electrolysis as evidenced by strongly acidic pH of the solutions after µ-EME. On contrary, electric currents gradually increased, levelled off and rapidly decreased for µ-EMEs with 50 and 100 mM NH4OH solutions and pH of these extraction solutions remained alkaline after µ-EMEs. Electric currents during µ-EME salt removals using various NH4OH concentrations are shown in Figure S2 in Supporting Information. Stable µ-EME process and efficiency of salt removal ≥ 99.7% were achieved for extraction solution 2 consisting of 50 and 100 mM NH4OH and the latter was used for further experiments.

FLM volume. FLM volume determines length of the organic phase, which in turn determines electric resistance of the µ-EME system. At the same time, one has to consider minimum FLM volume, which forms stable phase interface between two aqueous solutions. In the actual experiments, volumes of organic solvent > 5 µL resulted in increased electric resistance and slower removal process, volumes ≤ 3 µL could not form stable phase interfaces between sample and extraction solutions and for these reasons FLM volumes of 3.5, 4.0, 4.5 and 5.0 µL were examined. Efficiency of NaCl removal was not affected by the volume of FLMs and ≥ 99.5% removal was achieved for all FLM volumes. Electric current profiles are depicted in Figure S3 (in Supporting Information) and show the major difference in µ-EME performance at various FLM volumes; longer extraction times and lower electric currents for higher FLM volumes.


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µ-EME salt removals were completed in 10 – 19 min, note, however, that reduction of the extraction time for 3.5 µL FLMs was achieved at the expense of slightly compromised µEME stability and FLM volumes > 3.5 µL were preferably used in subsequent experiments. Variability of FLM volume is an important feature of µ-EMEs, since the volumes (and thus FLM lengths) can be optimized for each particular application with respect to sample composition and total removal time. Lower FLM volumes can be applied to salt removal from samples with lower salinity (i.e. environmental samples) to achieve faster pretreatment times, whereas higher FLM volumes might be advantageous for highly saline samples (such as sea water) to ensure lower electric currents and better µ-EME stability. This unique µ-EME feature is later demonstrated in Section “Samples with various salinity”.

Electric potential. Efficiency and speed of salt removal was examined at five different extraction potentials (100, 150, 200, 250 and 300 V) and the results are depicted in Figure 3. Note that µ-EMEs were stopped at 60 min for extraction potentials 100 and 150 V although the NaCl removal was not completed. NaCl might be removed completely also at these potentials if longer µ-EME times were allowed, however, salt removals were stopped after 1 h to keep µ-EME times reasonable. To confirm the effect of applied electric potential on salt removal, the µ-EME unit was additionally filled with all respective solutions and was kept at diffusive conditions (potential 0 V) for 1 h. A small reduction in NaCl concentration (less than 10%) was observed for 0 V demonstrating that some salting-out process takes place due to the contact of the sample with the organic solvent. A gradual decrease in NaCl concentration in sample solution and reduction of extraction time were achieved for increasing extraction potential. A nearly complete NaCl removal (≥ 99.3%) was achieved at 25, 17 and 12.5 min for 200, 250 and 300 V, respectively. For extraction potential of 100 and 150 V, 40 – 50% and 3 – 10% Na+/Cl- remained in the



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sample even after 1 h of µ-EME, respectively. The removal efficiency showed a non-linear relationship with the applied electric potential and similarly, non-linearity was observed for maximum electric currents during µ-EMEs at different potentials (Figure S4 in Supporting Information). This correlates well with the previously described effects of various extraction voltages on EME efficiencies39,44 and suggests that even faster salt removal might be achieved at higher extraction potentials. It was, however, not possible to confirm this assumption due to maximum voltage limitation of the actually used power supply, moreover, stability of the µEME process might be compromised at higher potentials, which induce higher operational currents. Extraction potential of 300 V was therefore used in all subsequent experiments.

Extraction solutions volume. To examine the effect of extraction solutions volume on NaCl removal, 5, 7.5, 10, 12.5 and 15 µL of DI water and 100 mM NH4OH were used as the terminal aqueous solutions. Sample volume was 3 µL in all experiments. As the geometry of the µ-EME unit is slightly conical over its entire length, volumes of 1-pentanol were adjusted to obtain comparable lengths of FLMs (~ 2.5 mm) for various volumes of extraction solutions. FLM volumes were 3.5, 3.75, 4.0, 4.25 and 4.5 µL for 5, 7.5, 10, 12.5 and 15 µL extraction solutions, respectively. No significant differences in salt removal efficiency and electric current profiles were observed for extraction solution volumes between 7.5 and 15 µL with ≥ 99.5% NaCl removal. Nevertheless, reduced NaCl removal (~ 90 – 95%) was observed for 5 µL extraction solutions. In this particular case, extraction solution 2 was acidified during µ-EME (due to the comparable amount of electrolytically produced protons and lowest volume) and NaCl removal was partially hindered, similarly as in Section “Extraction solutions composition”. Based on the above experiments and with respect to possible effects of electrolysis on µ-EME salt removal, 15 µL was selected as the optimum volume of


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extraction solutions. Further experiments with even larger volumes of extraction solutions (20 – 40 µL) did not offer any obvious advantage over the actually selected 15 µL.

Sample volume. Sample volume directly influences µ-EME time since the number of NaCl moles to be removed is linearly proportional to sample volume and larger sample volumes induce removals of more ions and thus longer removal times. In the actual experiments, minimum sample volume (2.5 µL) was given by the geometry of the salt removal unit, which enabled formation of a stable sample plug between two FLMs. In addition to 2.5 µL, sample volumes of 3.0, 3.5, 4.0 and 5.0 µL were examined. Salt removals ≥ 99.7% were achieved for all examined sample solution volumes with the only difference in µ-EME times, which ranged between 11 and 16.5 min for 2.5 and 5 µL sample volume, respectively. The fact that extraction times did not increase linearly with sample volume was rationalized by calculating total electric charges that passed through the µ-EME systems (according to reference43) and by plotting them against sample volumes. A strictly linear relationship was obtained between the total electric charge and sample volume (r2 = 0.998, see Figure S5B in Supporting Information). Total electric charges calculated for µEMEs of various sample volumes are shown in Figure 4 and corresponding electric currents in Figure S5A. The actual µ-EME system might be suitable for salt removal from even larger sample volumes. However, increase in sample volume will induce increase in extraction time and might possibly require additional optimization of selected operational parameters (i.e. extraction solution volume and/or composition).

Salt removal time. Time profiles for salt removal at different NaCl concentrations (10, 20, 50 and 150 mM) were monitored. The removal rates of sodium and chloride and electric currents for 10 – 50 mM and 150 mM NaCl are depicted in Figure S6 and Figure 5, respectively.



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Maximum electric currents and total removal times were dependent on NaCl concentration in sample solution and were lowest/shortest and highest/longest for 10 and 150 mM NaCl, respectively. Removal rates and electric currents nicely correlate with each other in these Figures. The FLMs consist of pure 1-pentanol and act as efficient resistors in the beginning of µ-EME. Consequently, lowest electric currents and no or only an insignificant NaCl removal are observed in the early stage of the µ-EME process. As the µ-EME proceeds, FLMs are gradually enriched with sodium and chloride ions migrating in opposite directions from sample solution. This is evidenced as a gradual increase in electric current and a gradual decrease in sodium and chloride ion concentrations in sample solution. Finally, electric current reaches a maximum and then rapidly drops to a baseline level. Importantly, the electric current baseline level is obtained at the time when ≥ 99% salt is removed and thus the end of the salt removal can be easily determined by monitoring electric current. This can be seen, for example, by comparing µ-EME times when electric current baseline levels and when ≥ 99% salt removals were achieved (340 and 360 s for 10 mM NaCl; 400 and 420 s for 20 mM NaCl; 580 and 600 s for 50 mM NaCl; 770 and 780 s for 150 mM NaCl).

Samples with various salinity. Until this point, µ-EME operational parameters were optimized for salt removal from samples with physiological salt concentrations. Nevertheless, salt removal might be important for samples from different analytical areas with various salt concentrations. For example, typical samples for environmental studies (i.e. surface waters) contain usually less than 20 mM of inorganic salts, however, environmental samples may also contain up to 500 mM of NaCl (i.e. sea waters). Salt removal from samples with low and high salt content might be performed at different operational conditions, which may considerably reduce total µ-EME time/consumption of organic solvents and improve stability of the µ-


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EME system, respectively. Due to the unique variability of the µ-EME system, operational conditions can be tailored for each particular sample. Two examples are presented, which demonstrate fine-tuning the µ-EME conditions for salt removal from samples with low and high salinity. Salt removal rate is mainly given by the length of the resistive barriers, which encompass sample solution, and too narrow FLMs were shown not sufficiently stable for µ-EME removals of physiological NaCl concentrations (Section “FLM volume”). However, for removal of low NaCl concentrations, the two FLMs can be considerably shorter due to the lower electric currents generated in the µ-EME system. FLM volumes were reduced to 3 µL, volumes of other phases were kept constant (3 and 15 µL for sample and extraction solutions, respectively) and extraction solutions composed of DI water and 20 mM NH4OH. Salt removal ≥ 99% was achieved in less than 180 and 220 s for sample solutions of 10 and 20 mM NaCl, respectively. Higher salt concentrations in sample solution induce higher electric currents and require longer extraction times. In order to ensure stable µ-EME performance for salt removal from highly saline samples, length of the two FLMs was optimized by increasing organic solvent volume to 6 µL. All other parameters were set as in Figure 4. The optimized µ-EME conditions resulted in reduced maximum electric currents and slightly longer extraction times. Nevertheless, salt removal ≥ 99.8% was achieved for 500 mM NaCl sample solution in 20 min of µ-EME.

Biochemical analytes. Suitability of the µ-EME system for simultaneous sample desalting and retention of target analytes was further examined with samples containing 150 mM NaCl and µM concentrations of various biochemical analytes. HSA (5 µM) was selected as model protein, neurotensin (50 µM) as model peptide and creatinine, glycine and alanine (50 µM) as



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model amino acids. µ-EME conditions optimized in the previous Sections were applied and the sample treatment was carried out for 0 – 13 min. Peak areas of the biochemical analytes before and after µ-EME salt removal were compared and drawn as percentage of an analyte remaining in the sample vs. extraction time. Resulting curves demonstrating efficient retention of selected analytes (HSA, neurotensin and creatinine) and removal of sodium and chloride are depicted in Figure 6. Salt removal ≥ 99.3% was achieved and simultaneously more than 95% of all analytes were retained in the sample solution after completion of the extraction process. The curves for glycine and alanine showed same trends and absolute values as those for the other three biochemical analytes but were not included in Figure 6 for better readability. Corresponding electropherograms showing CE-C4D of cationic and anionic species in the sample before and after the µ-EME salt removal are depicted in Figure S7 in Supporting Information. The desalted samples were subsequently subjected to positive ion ESI-MS spectra measurements and further demonstrated usefulness of the proposed salt removal procedure. Neurotensin was selected as the model analyte and resulting ESI-MS spectrum of neurotensin in the sample after µ-EME salt removal is shown in Figure 7A. Two additional spectra were acquired for standard solutions of neurotensin prepared in 100 mM HAc (Figure 7B) and in 100 mM HAc with addition of 10 mM NaCl (Figure 7C) – higher NaCl concentrations were not used in order to eliminate contamination of the MS inlet system. The standard solutions were infused without previous µ-EME pretreatment. The first two spectra demonstrated similar ESI-MS performance with signals typical for triply and doubly charged neurotensin ions at m/z values 558.311 and 836.962, respectively. An additional signal at m/z value 847.956 was observed for the pretreated sample, which corresponds to an adduct formed between sodium and neurotensin due to the presence of residual sodium in the sample after µEME. Nevertheless, as the residual sodium concentration was very low (~ 500 µM), the signal


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was negligible and had no negative effect on ESI-MS of neurotensin. On the other hand, significantly deteriorated ESI-MS performance was observed for untreated standard solution containing 10 mM NaCl, which was characterized by poor ionization, cluster formation and almost complete loss of characteristic neurotensin signals.

CONCLUSIONS A novel µ-EME method for efficient salt removal from saline samples is presented. Disposable and transparent extraction unit is filled with µL volumes of five immiscible solutions, where aqueous sample is the central solution sandwiched between two organic FLMs and two aqueous extraction solutions. On application of d.c. electric potential, inorganic cations and anions in the sample solution migrate in opposite directions towards corresponding FLM, cross the FLM and are quantitatively transferred to terminal extraction solutions. Simultaneously, the two FLMs selectively eliminate migration of target analytes across the organic phases and the analytes are retained in the sample, which is then used for analysis. Each phase in the extraction unit can have unique volume, length and composition and the µ-EME method offers an excellent variability of the salt removal process. Consequently, simple variations in FLM volume (decrease or increase) can be applied for desalting of samples with different salinity, thus achieving faster and more stable salt removal for samples with low and high salinity, respectively. The resulting salt-free aqueous samples are suitable for direct injections to most standard analytical systems, which has been demonstrated by CE-C4D or ESI-MS analyses of selected biochemical species in samples with physiological concentrations of NaCl. Moreover, since retention of analytes in aqueous samples is predominantly ruled by physico-chemical properties of the organic phases, finetuning FLMs composition might be very useful for sample desalting and targeted retention of specific analytes which is currently under investigation.



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AUTHOR INFORMATION Corresponding Author Dr. Pavel Kubáň Tel: +420 532290140 Fax: +420 541212113 E-mail: [email protected]

Author Contributions The manuscript was written by the author. Notes The author declares no conflict of interest.

ACKNOWLEDGMENTS Financial support from the Czech Academy of Sciences (Institute Research Funding RVO:68081715) and the Grant Agency of the Czech Republic (Grant No. 16-09135S) is gratefully acknowledged. The author would also like to thank Jana Křenková (Institute of Analytical Chemistry) for her help with ESI-MS measurements.

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FIGURE CAPTIONS Figure 1. (A) Detailed photograph of a µ-EME unit filled with operational solutions: sample (3 µL), FLMs (5 µL), extraction solutions (15 µL). (B) Fundamental principles of electrically induced salt removal using µ-EME across multiple aqueous and organic phases.

Figure 2. Efficiency of NaCl removal at different concentrations of NH4OH in extraction solution 2. µ-EME conditions: sample, 150 mM NaCl (3 µL); FLM 1 and 2, 1-pentanol (4 µL); extraction solution 1, DI water (10 µL); extraction solution 2, 10 – 100 mM NH4OH (10 µL); extraction voltage, 300 V; extraction time, 13 – 15 min.

Figure 3. Efficiency of NaCl removal at different extraction potentials. µ-EME conditions: sample, 150 mM NaCl (3 µL); FLM 1 and 2, 1-pentanol (4 µL); extraction solution 1, DI water (10 µL); extraction solution 2, 100 mM NH4OH (10 µL); extraction time, 12 – 60 min.



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Figure 4. Total electric charges during NaCl removal at different sample volumes. µ-EME conditions: sample, 150 mM NaCl; FLM 1 and 2, 1-pentanol (5 µL); extraction solution 1, DI water (15 µL); extraction solution 2, 100 mM NH4OH (15 µL); extraction voltage, 300 V; extraction time, 11 – 17 min.

Figure 5. Time dependency of NaCl removal and electric current in the system at optimized conditions. µ-EME conditions: sample, 150 mM NaCl (3 µL); other conditions as for Figure 4.

Figure 6. Time dependency of NaCl removal and retention of biochemical species at optimized conditions. µ-EME conditions: sample, 150 mM NaCl, 5 µM HSA and 50 µM neurotensin, creatinine, glycine and alanine (3 µL); extraction time, 12 min; other conditions as for Figure 4.

Figure 7. Positive ion ESI-MS spectra of neurotensin in the sample after µ-EME salt removal (A), 25 µM neurotensin in 100 mM HAc (B) and 25 µM neurotensin in 100 mM HAc with addition of 10 mM NaCl (C). µ-EME conditions as for Figure 6, ESI-MS conditions as in Experimental Section.


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Salt Removal from Microliter Sample Volumes by Multiple Phase

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