Suppression of Apparent Fluid Flow in Capillary Isotachophoresis

Article pubs.acs.org/ac

Suppression of Apparent Fluid Flow in Capillary Isotachophoresis without Recourse to Capillary Coating Farid Oukacine†,‡ and Myriam Taverna*,†,‡ †

Université Paris-Sud, Faculté de Pharmacie, Laboratoire des Protéines et Nanotechnologies en Sciences Séparatives, 92296 Châtenay-Malabry, France ‡ CNRS UMR 8612, Institut Galien de Paris-Sud, 92296 Chatenay-Malabry, France ABSTRACT: This manuscript describes a new analytical methodology that allows one to stop the apparent fluid flow in uncoated fused silica capillaries. This method, which requires neither capillary coating nor buffer additive, is based on the use of a 22% polyethylene oxide gel which is placed at the anodic end of the capillary. Its high viscosity precludes its penetration into the capillary and the apparent electro-osmotic pumping effect is stopped. To avoid sample depletion in the gel, the concentration of the background electrolyte in the gel has to be at least 10-fold higher than that of the background electrolyte. This method was applied to isotachophoretic experiments performed in unmodified silica capillaries and at high alkaline pHs. The sharpness of the zone boundaries was perfectly conserved. The separation voltages recorded during the isotachophoretic processes demonstrate a very good stability of the system preventing fluctuations of the analyte migration times between runs. This approach can be virtually applied at any pH value of the background electrolyte, in the presence of organic solvents or surfactants. Using this methodology, it has been possible to detect underivatized Amyloid-β peptide 1-40, one of the potential Alzheimer’s disease biomarkers, at lower concentrations than ever previously achieved using UV detection.

A

Furthermore, Liu et al.11 have recently demonstrated that the EOF affects the dynamics of ITP. Indeed, in absence of EOF, the leading electrolyte (LE) zone and terminating electrolyte (TE) zone migrate at the same velocity and therefore adapt themselves to generate low and high electric fields (E), respectively. Current literature commonly assumes that the adaptation of E is fast. However, in EOF-driven ITP processes, the TE zone is split into two zones: a high-E zone and a low-E zone. The formation of the highly adapted TE zone requires a large-scale mass transfer and is therefore a rate-limiting step in EOF-driven ITP.11 Different ways are described in the literature to modulate or to suppress the EOF. These include derivatizing the inner surface of the capillary,12,13 the use of acidic buffers,14 and the addition of surface active species or organic modifiers to the buffer.15 Capillaries dynamically coated with polymers through hydrogen bonding, hydrophobic effects, and/or Van der Walls interaction have been used in CE for many years.16−20 Viscous additives like ethylene glycol or glycerol can be also added in the electrolyte in order to decrease the EOF magnitude;21 however, they modify the electrolyte composition and the separation process. Moreover, covalent or noncovalent coatings are often easily removed at extreme pHs or in the presence of organic solvents. The aim of this work is to propose a new methodology allowing one to stop the apparent FF in uFSC, in cITP, without

n important parameter in capillary electrophoresis (CE) is the electro-osmotic flow (EOF). The EOF occurs when an ionic current passes through the capillary column that contains surface charges.1−4 Then, when the uncoated fused silica capillary (uFSC) is in contact with an electrolyte, the inner capillary surface acquires a negative charge at pHs above 3. The positive counterions in the liquid phase compensate the negative charge of the wall so that an electrical double layer is created at the interface between the solid wall and the liquid phase. In the presence of an external electric field, the fluid in this charged double layer acquires a momentum which is then transmitted to adjacent layers of fluid through the effect of viscosity. This creates a fluid flow (FF) inside the capillary called electro-osmosis.5 The EOF is uniform throughout the capillary cross-section except for a very thin electric double layer near the capillary wall where the flow velocity rapidly decreases from its free stream value to zero at the substrate/fluid interface.6 This results in very narrow uniform bands and high separation efficiencies with very short run times. In electrokinetic preconcentration methods, the EOF can be detrimental. For example, in stacking methods, the local electroosmotic velocity (Veo) in the sample plug is generally greater than the bulk Veo of the background electrolyte (BGE). The pressure difference caused by this mismatch may generate a laminar flow inside the capillary which broaden the sharp zone generated by the stacking process.7,8 Capillary isotachophoresis (cITP) is also generally performed under suppressed EOF conditions9 and with constant current10 to minimize zone broadening due to any EOF mismatch arising from variations in the composition of sample zones. © 2014 American Chemical Society

Received: October 15, 2013 Accepted: February 27, 2014 Published: February 27, 2014 3317

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recourse to any capillary coating or any buffer additive. This method is based on the use of a 22% polyethylene oxide gel, which is placed at the anodic end of the capillary during the run. Its high viscosity precludes its penetration into the capillary and the electro-osmotic pumping effect is stopped. This system can be described as a closed separation system with a flow near the wall still existing because EOF cannot be suppressed without any surface modification of the fused silica capillary. Thus, the electrolyte flow near the wall goes back down the center core of the cross-section area to ensure zero net flow.22,23 This generates a back pressure gradient that creates peak dispersion in capillary zone electrophoresis mode (CZE) and particularly at high pHs. However, in cITP, the peak dispersion is expected to be counteracted by the ITP mechanism itself. In this method, the apparent FF is stopped by a physical constraint applied onto the system. Thus, it can be virtually applied, in cITP, whatever the pH value of the BGE, in the presence of organic solvents or surfactants. In the first part of this manuscript, the proof of concept is demonstrated. In the second part, using small molecules as model compounds, the limitations and the advantages of the method have been evaluated. Finally, the method was applied to the preconcentration of an Alzheimer’s disease biomarker (amyloid-β peptide 140) reaching limits of detection (LOD) never previously attained for this biomarker using UV detection.

For the experiments performed in uFSC, a new capillary was conditioned by performing the following washes: 1 M NaOH for 15 min, 0.1 M NaOH for 10 min, and water for 5 min. Between two analyses, the capillary was successively flushed at 50 psi with 1 M NaOH for 3 min, water for 1 min, and BGE, LE, or TE for 5 min. After the analyses performed with polymer gel (PG), the capillary was flushed for 5 min under simultaneous applied voltage (+30 kV, Imax = 50 μA) and pressure (50 psi), with a solution of 0.1 M NaOH containing 35 mM SDS and placed at both ends of the capillary. This step was used to dissolve the small plug of the gel that had eventually penetrated inside the capillary and which could clog it. During this washing step, the SDS enters in the capillary with continuous electrokinetic injection and interacts with the small plug of PEO. The complex PEO/SDS24 becomes negatively charged and is removed from the capillary by the electric field effect. In order to improve the timeliness of this washing step, a pressure of 50 psi was simultaneously applied. For the experiments performed in PEG-coated capillary, a new capillary was conditioned by flushing with water for 10 min. Between two analyses, the capillary was successively flushed at 50 psi with water for 2 min and BGE for 5 min. CZE to Study the Effect of PEO Concentration, in PG, on the FF Velocity. After the conditioning of the capillary, DMSO was first injected, at the inlet end of the capillary, by hydrodynamic injection (HDI) at 0.1 psi for 5 s. Then, positive mobilization pressure (4 psi) was applied for 72 s. After that, benzoic acid (BA) was injected, at the outlet end of the capillary, by electrokinetic injection (EKI) at −10 kV for 3 s. Finally, negative mobilization pressure (−2 psi) was applied for 21 s. Then, separations were performed at 30 kV. A new capillary was used for each new experiment. cITP Experiments with Polymer Gel (cITP-uFSC-PG) for BA and HEPES Analysis. The capillary was first fully filled with LE (97.8 mM NH4OH + 21.6 mM CH3COOH, pH 9.8). Then, the sample, diluted in TE (392.0 mM NH4OH, pH 11.4), was injected in the capillary by HDI of 0.3 psi. The injection of the samples was followed by HDI of the short plug of TE (0.5 psi, for 5 s). The separation was performed at constant current (4 μA) with TE and PG (containing 22% wt of PEO) at the inlet and at the outlet side of the capillary, respectively. The concentration of the LE in the gel was 16 times higher than the concentration of the LE in the capillary while the inlet reservoir contained nonconcentrated TE. For the control of the cITP experiment in the presence of EOF (without PG), the same experimental conditions were used with a slight modification. Because of the presence of EOF, the capillary was first fully filled with the TE and the separation was performed with LE and TE at the inlet end and at the outlet end of the capillary, respectively. cITP-uFSC-PG for Aβ 1-40 Analysis. After the washing process, the capillary was fully filled with the LE (194.4 mM NH4OH + 43.4 mM CH3COOH, pH 9.9). Before the electrokinetic injection (EKI) of the Aβ 1-40, a small plug of TE was injected at the inlet side of the capillary (1 psi for 6 s). Then, a voltage of −15 kV was applied during 0.2 min with TE (392.0 mM NH4OH, pH 11.4) and PG (containing 22% wt of PEO) at the inlet and at the outlet end of the capillary, respectively. Indeed, it was observed that when a PG is placed at the outlet end of the capillary, the apparent FF does not stop immediately after the voltage application but it rather decreases rapidly until it stops. Even if this decrease is very fast (few seconds), this step ensures that the apparent FF is stopped at

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EXPERIMENTAL SECTION Chemicals. Benzoic acid, phthalic acid, dimethyl sulfoxide, polyethylene oxide (Mw 200 000 g mol−1), acetic acid ≤ 99.99%, HEPES, sodium dodecyl sulfate, and ammonium hydroxide (containing 28% of NH3) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Sodium hydroxide was from VWR (Fontenay-sous-bois, France). All other reagents were of analytical or HPLC grade. Amyloid-β peptide 1-40 was purchased from Eurogentec (Angers, France). Ultrapure water (18.2 MΩ cm−1) was prepared from Direct-Q3 Water Purification System (Millipore, Milford). The 0.2 mL PCR tubes were from VWR (Leuven, Belgium). Gel Preparation. A volume of 7.1 mL of 20-fold concentrated BGE or LE was added to 2 g of polyethylene oxide (PEO). The solution was gently mixed, at room temperature, for 4 h with a magnetic stirrer. The gel was stored at 4 °C overnight in order to eliminate any air bubble. The final concentration of the PEO in the gel was ∼22% wt. Prior to the CE experiments, ∼200 μL of the gel were transferred into a PCR tube. The vial was covered with parafilm and was sonicated (model USC900D, VWR, Leuven, Belgium) for 5 min at 25 °C. Immediately after the sonication the gel was used for the experiment. To ensure reproducibility, sonication time of the gel must be the same for each experiment (5 min). The temperature of the ultrasonic bath must be approximately constant and ≤25 °C, otherwise the viscosity of the gel may vary between runs. New electrolytes were prepared every day due to their high volatility. For each experiment, new electrolytes and new gel were used. Capillary Electrophoresis. Uncoated fused-silica capillaries (50 μm i.d., 365 μm o.d.) were from Phymep (Paris, France). Polyethylene glycol (PEG) coated capillaries (50 μm i.d, 0.10 μm film) were from Interchim (Montluçon, France). CE experiments were carried out with a PA800+ ProteomeLab instrument (Beckman Coulter Inc., Brea, CA). The temperature of the capillary cartridge was set at 25 °C. 3318

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Figure 1. (A) Scheme depicting the methodology used for the suppression of FF in CE. (B) Effect of the PEO concentration, in the PG, on the migration times of DMSO and BA. Prior to the analysis, BA was placed at ∼5 cm and DMSO at ∼7.5 cm from the UV detector using the protocol described in the Experimental Section. (C) Effect of the PEO concentration, in the PG, on the FF velocity (VFF). Experimental conditions: uFSC 48.5 cm (38.2 cm to the detector) × 50 μm i.d. BGE: NH4OH 94.8 mM + CH3COOH 20.5 mM, pH 9.9. Samples: DMSO at 1% v/v, BA at 0.4 g L−1. Samples are diluted in BGE. During the runs, PG, containing PEO and BGE, was placed at the anodic end of the capillary. The concentrations of the PEO in the PG are depicted in the figures. Other conditions as described in the Experimental Section.

Figure 2. Comparison of electrophoretic profiles of two small molecules, analyzed using three different BGE pHs, in neutral CZE-PEG (bottom trace) and in a CZE-uFSC-PG (top trace). Experimental conditions: uFSC and PEG coated capillary (neutral capillary), 48.7 cm (38.3 cm to the detector) × 50 μm i.d.. Sample: BA at 1.0 g L−1 + PA at 0.9 g L−1 in BGE. Sample injection: Sample was first injected at 0.3 psi for 5 s, then a small plug of BGE was injected at 0.5 psi for 5 s. BGE: (A) CH3COOH 26.1 mM + NaOH 10 mM, (B) H3PO4 20.3 mM + NaOH 30 mM, (C) NH4OH 97.1 mM + CH3COOH 21.6 mM. Applied voltage: −20 kV. For the experiments performed in CZE-uFSC-PG, PG, containing 22% wt of PEO and 16-fold concentration of BGE, was placed at the anodic end of the capillary during the runs, while the cathodic reservoir contained nonconcentrated BGE. Other conditions as described in the Experimental Section.

analysis of two small molecules. Prior to the analysis, one neutral (DMSO) and one negatively charged (BA) molecules were positioned on either side of the detection window of an unmodified uFSC (Figure 1A). BA was placed at ∼5 cm and DMSO at ∼7.5 cm from the UV detector. Then, a vial containing the PG (PEO + BGE) was placed at the anodic end of the capillary, and a separation was performed using an ammonium acetate buffer (pH ∼ 9.9). As described in Figure 1B, using a PG with 3.0% wt of PEO, only the neutral molecules were detected. The DMSO was detected at ∼0.6 min and the PEO, migrating from the anodic compartment, at ∼2.7 min. This shows that the cathodic FF is too high to allow the migration of the BA toward the cathode. When the concentration of the PEO in the gel was increased to 8.5% and 11.5% wt, respectively, longer migration times for the DMSO and PEO were observed. Furthermore, because of the decreased FF, the BA was also detected. With 17.0% wt of PEO, the FF was strongly stopped. DMSO took ∼6.2 min to reach the detector while PEO could not be detected under 10

the beginning of the EKI. After this step, Aβ 1-40 was electrokinetically injected, at the inlet side of the capillary, by an applied voltage of −5 kV with PG at the outlet compartment. Finally, the separation was performed with a constant current of 8 μA with TE and PG at the inlet and at the outlet side of the capillary, respectively. The concentration of the LE in the gel was 16 times higher than the concentration of the LE in the capillary while the inlet reservoir contained nonconcentrated TE.

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RESULTS AND DISCUSSION Apparent Fluid Flow Suppression in Capillary Electrophoresis. An innovative way to stop the fluid flow (FF) in CE using a high viscosity polymer gel (PG) is proposed. During the electrophoretic separation, a BGE containing the PG is placed at the anodic end of the capillary. The high viscosity of the gel precludes its penetration into the capillary and stops the apparent electro-osmotic pumping effect. The technical feasibility of this method was demonstrated by the CZE 3319

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Figure 3. (A) Isotachophoretic profiles of BA and HEPES obtained in cITP-uFSC-PG system. (B) Separation voltages recorded during the experiments. Experimental conditions: uFSC 36.5 cm (26.1 cm to the detector) × 50 μm i.d. Samples: (1) BA at 2.0 g L−1, (2, 3) BA at 4.1 g L−1 + HEPES at 5.3 g L−1. Injection time of the sample: (1, 2) 12 s, (3) 24 s. The insert of part A is a cITP experiment control performed in the presence of EOF (without PG) and obtained in the same conditions as those used for trace (3). Other conditions as described in the Experimental Section.

section area to ensure zero net flow.22,23 This generates a backpressure gradient that creates important peak dispersion particularly at high pH.27 During the electrophoretic process, the mass transfer from the gel electrolyte vial to the tip of the capillary was hindered. This resulted in ion depletion in this zone and in current leakages during the electrophoretic process. To avoid this phenomenon, the concentration of the BGE in the gel had to be, at least 10 times higher than that of the BGE present in the capillary. When the concentration of the BGE in the gel was 16-times higher, the electrophoretic process was found to be stable for more than 60 min. This stability is explained by the fact that the amount of electrolyte that enters the capillary is governed by the Kohlrausch Regulating Function (KRF),28 which stipulates that its numerical value w is locally invariant in time and is defined as

min. Indeed, because of the high viscosity of the gel, the FF was greatly stopped. For each concentration of the PEO, the FF velocities (VFF) were estimated using the migration time of DMSO. Figure 1C shows that VFF linearly decreased until the percentage of PEO reached ∼8.5%. Beyond this concentration, the decrease of VFF followed a logarithmic trend. Finally, it appears from this figure that a concentration of PEO above 20% wt is required to completely suppress the FF. In an attempt to ensure that this observation was equivalent to what is observed when a neutral capillary is used, two model molecules, phthalic acid (PA) and benzoic acid (BA), were analyzed by CZE using either a PEG-coated capillary, which is neutral, or a uFSC combined to the PG (CZE-uFSC-PG), using strictly the same electrophoretic conditions. Samples were injected at the cathodic end of the capillaries, and a negative voltage was applied in both cases. For the experiments performed by CZE-uFSC-PG, the BGE containing 22% wt of PEO was positioned at the anodic compartment during the separation. The experiments were performed at three different pHs: 4.5, 7.0, and 9.8. As shown in Figure 2, the CZE-uFSC-PG system allows stopping the apparent FF, whatever the pH value of the BGE, since the migration times of the ions are approximately the same in both capillaries. It should be noted that at pH 4.5, the migration times of the two ions were slightly higher with the neutral coated capillary. In these electrophoretic conditions, the theoretical migration times, estimated by the freeware program PeakMaster,25,26 were 5.72 min for PA and 7.43 for BA. These theoretical values are much closer to those obtained experimentally in the CZE-uFSC-PG system (5.91 min for PA and 8.11 min for BA). This indicates the presence of residual cathodic EOF, in the neutral capillary, that slows the movement of the two anions. However, as shown in Figure 2, with the CZE-uFSC-PG system, significantly more peak broadening is observed at pH above 7. Higher is the pH of the BGE and lower is the peak efficiency. This feature is mainly due to the fact that the use of PG induces a hydrodynamic dispersion contribution. Indeed, the CZE-uFSC-PG system used in this work can be described as a closed separation system. Because the net charge density in the electrical double layer (EDL) is not zero, the ions in the EDL region still move toward the cathode. The electrolyte flow near the wall goes back down the center core of the cross-

w(x) =

∑ i

Ci(x , t ) μĩ

(1)

where Ci and μ̃ i refer to the concentrations and actual mobilities of all ionic species. In other terms, the concentration of electrolyte that enters the capillary cannot exceed the concentration of electrolyte present initially in the capillary. So the incorporation of high concentrations of BGE in the gel allowed the electrolyte to be continuously delivered from the gel to the capillary. Capillary Isotachophoresis in Uncoated Fused Silica Capillary under Alkaline Conditions. In order to demonstrate the relevance of the use of a PG for the FF suppression, cITP was performed in uFSC under a high alkaline pH (cITP-uFSC-PG). Figure 3A displays three successive isotachophoregrams of two model molecules at different concentrations and injection times. The pH values of the LE and TE are 9.8 and 11.4, respectively. After the injection of the sample into the capillary, gel containing 22% wt of PEO was positioned at the anodic compartment for the FF suppression during the cITP. The concentration of the LE in the gel was 16times higher than that of the LE in the capillary. The results clearly show the laws that govern cITP still control the process. Thus, the zone heights are independent of the solute concentration while the zone widths increase with the solute concentration. In addition, sharp and stable boundaries are formed between the two discrete zones of BA and HEPES. The 3320

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Figure 4. (A) Effect of injection time from 15 to 120 s, in EKI, on isotachophoretic peak shape of Aβ 1-40 in cITP-uFSC-PG system. (B) Calibration curve obtained for Aβ 1-40. Experimental conditions: uFSC 36.5 cm (26.1 cm to the detector) × 50 μm i.d. Sample: Aβ 1-40 (A) at 11.5 μM and (B) other concentrations indicated in the figure. Injection time: (A) as indicated in the figure and (B) 120 s. Other conditions as described in the Experimental Section.

function of the ionic mobility, concentration, and charge of the injected ion relative to all other competing ions).33 Thus, the optimal injection of the Aβ 1-40 was anticipated to be at pH around 11. However, covalently or noncovalently coatings are often easily removed at extreme pHs. We therefore employed the cITP-uFSC-PG system to stop the apparent FF. Figure 4A displays the effect of the injection time on the isotachophoretic profiles of the Aβ 1-40 diluted in NH4OH (pH 11.4) and injected by EKI mode. After the injection, cITP was used in order to restack the sample zone. A linear plot was observed for the measured peak area as a function of injection time. The slope of the calibration curve was 2.06 × 102 mAU s μM−1 with a correlation coefficient of R2 = 0.990. A linear relationship was observed between the peak intensity (I) of the Aβ 1-40 and the injection time (tinj) (at 5 kV, I = 5.61 × tinj). This linearity was only observed over a small range of injection times (≤45 s) with a correlation coefficient of R2 = 0.997. However, it is interesting to note that despite the high alkaline pHs of the electrolytes, the dispersion of the sample observed in CZE was totally avoided. Sharp peaks with high efficiencies were observed especially for low injection times. This is mainly due to the fact that the sample diffusion is counteracted by the ITP mechanism. At high injection times, the peak is less sharp and symmetric than expected in peak mode isotachophoresis.34,35 This suggests some interaction of the Aβ 1-40 with the inner capillary wall. During the EKI and the ITP process, gel containing 22% wt of PEO was positioned at the anodic compartment. In this way, it was possible to inject Aβ 1-40 for 120 s under an electric field of 145 V cm−1. Figure 4B shows the calibration curve obtained for different concentrations of the Aβ 1-40 ranging from 0.7 to 11.5 μM. The very good linearity of the response (measured by the peak areas) with a correlation coefficient of 0.990 proved that the method is quantitative. Under these conditions, it is possible to detect concentrations of Aβ 1-40 as low as ∼50 nM using only UV detection.

bands remain in contact and migrate as broad zones according to their μep. Moreover, the zone widths were found to be related to the injected sample volume, as expected by the KRF. Figure 3B displays the separation voltages recorded during the three respective experiments. Due to the fact that the cITP is performed under constant current intensity, the separation voltages increase linearly (in absolute value) as the leading ion is replaced progressively by the lower mobility terminating ion inside the capillary. The slopes of the voltages measured over time are perfectly parallel. This reflects a great stability of the system that prevents fluctuation of migration times of the analytes that could arise from variations of the EOF. A control of cITP experiment in presence of EOF was also performed in the same experimental conditions as those producing trace 3 in Figure 3A with slight modifications. The capillary was first fully filled with the TE. The separation was performed with LE and TE at the inlet and outlet ends of the capillary, respectively. The result is shown in the insert of the Figure 3A. Despite the fact that only two molecules were analyzed (BA and HEPES), three different zones were present in the isotachophoregram. This can be explained if one considers that the detection length is too short for the focusing ITP process to be completed. Thus, in the presence of EOF, the two molecules are detected before being completely focused. This result clearly shows that the suppression of the FF is really mandatory to achieve an efficient ITP of the molecules under these conditions. Electrokinetic Injection for Isotachophoretic Analysis of Amyloid-β Peptide in Uncoated Fused Silica Capillary. In this section, the PG was exploited to stop the apparent FF during EKI-cITP analysis of an amyloid-β peptide which is considered as a potential Alzheimer’s disease biomarker. At low pHs, Aβ 1-42 is not stable and tends to form oligomers and aggregates,29 whereas this aggregation can be prevented at alkaline pHs.30,31 As a proof of concept, we analyzed the Aβ 140 which is less prone to aggregation and is very close structurally to the Aβ 1-42. The experimental values of the μep of the Aβ 1-40 measured in our laboratory, at pH 7.8 and pH 11.0, were estimated to be −10.8 × 10−9 m2 V−1 s−1 and −21.1 × 10−9 m2 V−1 s−1, respectively. Due to the fact that, in EKI, if the EOF is in the opposite direction to the μep of the analytes, the analyte injection is less effective,32 it becomes interesting to suppress the EOF during the EKI of the Aβ 1-40. Moreover, under suppressed EOF conditions, the injected sample amount depends on the transference number of the analyte (which is a

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CONCLUSION In this manuscript, a new approach has been developed to stop the apparent FF in uFSC. This system is described as a closed separation system. During the experiments, a polymer gel, with high viscosity, is placed at the anodic end of the capillary. The high viscosity of the gel precludes its penetration into the capillary and the electro-osmotic pumping effect is stopped. Due to the fact that the EOF is not suppressed, the electrolyte 3321

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flow near the wall goes back down the center core of the crosssection area to ensure zero net flow. In CZE, this generates a back pressure gradient that creates peak dispersion particularly at pH above 7. However, in cITP, the peak dispersion is counteracted by the ITP mechanism itself. The advantage of this method is that it is very easy to implement, is both fast and inexpensive, and requires neither capillary coating nor buffer additive. This method can be virtually applied, in cITP, at any pH value of the BGE, in the presence of organic solvents or surfactants. Due to the fact that the FF is stopped by a physical constraint, the small variations of the FF between the experiments are avoided. This prevents fluctuations in the migration times of the analytes and confers a better reproducibility for the ITP experiments. By using PG to stop the FF, cITP can now be performed in uFSC and whatever the pH or the composition of the BGE. Further studies are currently being performed to exploit this strategy to other preconcentration methods. Finally, the interest of this new strategy lies probably beyond the area of the preconcentration techniques as for instance to stop residual FF in coated capillaries for CZE experiments.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33-1-4683-5462. Fax: +33-1-4683-5944. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Fundings for this research and the postdoctoral grant of F.O. were provided by European Union’s Seventh Framework Program FP7/2007-2013 under the Grant CP-IP 246513Nadine and also by the French National Agency (ANRDIGIDIAG, Grant No. ANR-10-NANO-02-07). We are grateful to Stella Ghouti-Baxter for proofreading the English of this manuscript.

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dx.doi.org/10.1021/ac403337j | Anal. Chem. 2014, 86, 3317−3322