Journal of Chromatography A, 1364 (2014) 59–63

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Study of behavior of carboxylic magnetite core shell nanoparticles on a pH boundary ˇ cík a , Radek Zboˇril b , Jan Petr a,∗ Carmen Cacho a , Zdenka Marková b , Juraj Sevˇ a Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science, Palack´ y University in Olomouc, 17. listopadu 12, 77146 Olomouc, Czech Republic b Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palack´ y University in Olomouc, Sˇ lechtitel˚ u 11, 78371 Olomouc, Czech Republic

a r t i c l e

i n f o

Article history: Received 19 March 2014 Received in revised form 16 August 2014 Accepted 27 August 2014 Available online 2 September 2014 Keywords: Nanoparticles Stacking Boundary effects Dynamic pH junction pH boundary

a b s t r a c t During the last years, several authors have focused on the characterization of the size and charge of the nanoparticles by capillary electrophoresis. However, considering that nanoparticles are generally suspended in a solvent different from those commonly used as background electrolytes (BGE), an appropriate characterization of the behavior of the nanoparticles in the sample-BGE interface is required, as this might affect the overall electrophoretic behavior of the nanoparticles. In the present work, we address the evaluation of the behavior of COOH-coated maghemite nanoparticles in the vicinity of a pH boundary. To do so, different suspensions of nanoparticles prepared in acid media were injected into a borate/NaOH pH 9.5 BGE. The formation and evolution of boundaries in the sample-BGE interface in such systems was modeled by computer simulation. A systematic evaluation of the effect that parameters such as the coion, the sample pH or the injection time have on the electrophoretic behavior of the nanoparticles was carried out. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Since 1989 when VanOrman et al. [1] first reported the separation of sulphated polystyrene nanospheres in a CTAB-coated capillary, capillary electrophoresis has been regarded as a useful technique for the characterization of the size and charge of the nanoparticles, providing similar results to those obtained by transmission electron microscope (TEM) and dynamic light scattering (DLS) [2]. In this sense, qualitative studies carried out aiming at the electrophoretic separation of e.g. gold nanoparticles [3–5], silica sols [6] or polystyrene latex microspheres [7–10] demonstrated that separation of the particles was mainly controlled electrophoretically, that is, in a size- and charge-dependent manner. In order to better understand the theoretical mechanism underlying the electrophoretic separation of nanoparticles according to their size and charge density, Radko et al. [9,10] compared the performance of negatively charged polystyrene latex nanoparticles of sizes ranging from 300 to 800 nm using Tris-borate background electrolytes of varying ion strength. Two main conclusions arose

∗ Corresponding author. Tel.: +420 585 63 4416. E-mail address: [email protected] (J. Petr). http://dx.doi.org/10.1016/j.chroma.2014.08.090 0021-9673/© 2014 Elsevier B.V. All rights reserved.

from such study: (i) the size-dependent electrophoretic separation of the nanoparticles is strictly related with R, where  stands for the reciprocal value of the thickness of the electric double layer around the nanoparticle and R for its radius, and (ii) if the capillary is adequately thermostated, the electrophoretic heterogeneity of the nanoparticles is the major contributor to peak broadening. They also observed that the presence of a charged layer on the surface of polystyrene nanoparticles can affect the size-dependent separation of the particles, most specially when using buffers of high ionic strength. Considering, however, that suspension of the nanoparticles is prepared in different solvents than those commonly used as background electrolytes, in order to characterize their electrophoretic properties it is necessary to evaluate their behavior in the boundary between the background electrolyte and the sample solvent. Such boundaries have extensively been applied for the on-line preconcentration of several analytes [11–16], due to a dramatic change in the electrophoretic mobility of the analytes in the vicinity of the boundary. Briefly, four main different strategies have been described for the on-line preconcentration of analytes within the capillary, either separately or combined: stacking, isotachophoresis, sweeping, and dynamic pH junction [13]. The injection of the sample at a pH value different from that of the background electrolyte can lead

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into the on-line enrichment of the analytes by two different mechanisms: In pH-mediated stacking, a low conductivity zone is created by the titration of the sample buffer with the penetrating background electrolyte. In such low conductivity zone, the velocity of analytes is increased, thus resulting in their focusing on the boundary with high conductivity zone [16]. In dynamic pH junction, on the other hand, the electrophoretic mobility of weakly ionic analytes is dramatically changed due to a change in their ionization state in the vicinity of their pKa value [17]. While pH-mediated stacking can be applied for the on-line enrichment of any kind of analytes (strong ions [18], weak ions [19] or neutral compounds [20]), enrichment due to dynamic pH junction is limited to weak ions [17], whose electrophoretic mobility depends on the pH of the background electrolyte. To the best of our knowledge, only large volume stacking of gold and silver nanoparticles has been described up to the moment [21–25]. The nanoparticles were stabilized with SDS to prevent their agglomeration. In the optimum conditions, enrichment efficiencies up to 260-fold were obtained. Recent experiments carried out in our laboratory indicated that carboxylic maghemite core–shell nanoparticles can be stacked by field-amplified sample stacking using borate/NaOH pH 9.5 background electrolyte [26]. The main aim of the present work is to describe the behavior of carboxylic maghemite core–shell nanoparticles in the presence of a pH boundary. 2. Materials and methods 2.1. Chemicals Boric acid, citric acid, acetic acid, sodium hydroxide, 45% (w/v) aqueous solution of poly(acrylic acid) sodium salt (PAA), iron(II) sulphate heptahydrate, and hydrochloric acid were purchased from Sigma Aldrich (St. Louis, MO, USA). The different buffer solutions were prepared by dissolving the corresponding acid and adjusting to the desired pH by titration with NaOH. 2.2. CE apparatus and conditions Electrophoretic measurements were performed with a HP 3D CE system equipped with a DAD detector (Agilent Technologies, Waldbronn, Germany) using 50 ␮m i.d. bare fused-silica capillaries (Polymicro Technologies, Phoenix, USA) with 33.5 cm total length and 25.0 cm effective length. Prior to the first use, capillaries were subsequently rinsed with 1 M and 0.1 M NaOH solutions for 15 min, and water for 10 min, under a pressure of 930 mbar (93 kPa). Samples of nanoparticles prepared in either citrate- or acetate-based buffer solutions were hydrodynamically injected (50 mbar = 5 kPa, 5–120 s) into a background electrolyte consisting of 50 mM borate/NaOH pH 9.5. Unless stated otherwise, all CE measurements were carried out at a voltage of 20 kV, and the nanoparticles were monitored at 280 nm. 2.3. Synthesis of the nanoparticles Magnetite NPs stabilized by PAA were prepared according modified protocol described earlier by Bakandritsos et al. [27]. Briefly, PAA (660 mg) was dissolved in double distilled H2 O (30 mL). 10 M NaOH solution (1 mL) was added to mechanically stirred (350 rpm) PAA solution and heated up 40 ◦ C. FeSO4 ·7H2 O (720 mg) was dissolved in double distilled H2 O (20 mL), pre-acidified by 37% HCl (60 ␮L) and added to PAA solution under mechanical stirring. The mixture was heated up to 60 ◦ C and stirring was continued for 90 min. The formed nanoparticles were left to cool down to room temperature (RT) and were magnetically separated. The magnetic pellet was re-suspended in double distilled H2 O (40 mL) and

then was centrifuged (10,000 g, RT) for 60 min. The pellet was resuspended in double distilled H2 O and sonicated for 15 min. A second centrifugation (10,000 g, RT, 60 min) and sonication were performed and pellet was re-suspended in double distilled H2 O. The final colloid was centrifuged (2000 g, RT) for 20 min to remove any aggregates. The supernatant was collected and stored at 4 ◦ C. NPs were then dispersed in the injection buffer. 2.4. Computer simulations SIMUL (version 5) [28,29] was used for computer simulation of the different buffer zones formed during the injection of acetic and citric acids into a borate background electrolyte. The capillary length used for the simulation was 33.5 mm with the detector placed at 25 mm from the injection point, and the sample plug was varied from 0.1 to 10 mm. Simulations were continued up to 600 s of migration time applying a constant voltage of 200 V (electric field strength of 8 kV/m). Unless stated otherwise, simulations were done using 2000 grid points. Different solutions of acetic acid/NaOH and citric acid/NaOH with an ionic strength of 33.5 ± 1.5 mM and pH values ranging from 3.5 to 6.5 were injected into a 50 mM borate/NaOH pH 9.5 background electrolyte to simulate the experimental conditions used in the real CE experiments carried out with nanoparticles. Mobilities and pKa values of acetic acid, citric acid and sodium were used from the program database based on Hirokawa’s tables [30]. Boric acid (pKa = 9.23 and  = −36.2) was added according to Mala et al. [31]. 3. Results and discussion The main objective of the present work is to study the behavior of the nanoparticles in the presence of a pH boundary. In order to maximize the difference in the behavior of the nanoparticles at different pH values, COOH-coated maghemite nanoparticles were selected as model compounds, as the COOH moieties present in the particle surface are expected to behave as weak acids due to the deprotonation of the carboxylic groups. Zeta-potential and the mean hydrodynamic diameter of the nanoparticles in water, measured by dynamic light scattering and zetametry, were of −59 mV and 80 nm, respectively. 3.1. Behavior of NPs in electrolytes at different pHs The changes in the electrophoretic mobility of the nanoparticles under different pH values were initially evaluated. In this sense, the mean electrophoretic mobility of the nanoparticles in background electrolytes with pH values ranging from 3.5 to 9.5 using borate, citrate and acetate with sodium as counter-ion was estimated by means of the Williams–Vigh method [32] using 0.1% (v/v) dimethylsulfoxide as a neutral marker. Different sets of experiments were done at voltage values ranging from 5 to 20 kV and the time during which the electrophoretic separation was carried out ranging from 5 to 30 s. The ion strength of all the background electrolytes was fixed at 33.5 ± 1.5 mM. Fig. 1 presents the mean electrophoretic mobility of the nanoparticles in each of the evaluated background electrolytes. As can be observed; there is a slight decrease of the electrophoretic mobility of the nanoparticles at pH values below 5.5. This fact can be explained considering that at such pH, the carboxylic moieties created during the coating of the nanoparticles can get protonated, thus diminishing the net charge of the nanoparticle and its electrophoretic mobility. Interestingly, the decrease of the electrophoretic mobility of the nanoparticles depends not only on the pH of the sample, but also on the anion, as it is considerably higher in the case of acetate-based background electrolytes in comparison

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Fig. 1. Mean electrophoretic mobility of the nanoparticles using different BGEs (N = 6).

with those of citrate. Here, the anion affects their zeta potential which seems to be a key fact for this effect. 3.2. Behavior of NPs at pH boundary A further experiment was designed in which suspensions of the nanoparticles in the different samples were injected into a background electrolyte consisting on 50 mM borate/NaOH pH 9.5, as such background electrolyte had shown excellent results in terms of the reproducibility of the electrophoretic behavior of COOHcoated maghemite nanoparticles [26]. In such system, two different mechanisms for the online preconcentration of the nanoparticles can be expected: (i) pH mediated stacking due to the titration of borate with either citric or acetic acids, and (ii) dynamic pH junction of the nanoparticles, if the sample is loaded at pH below 5.5, considering that we observed a significant reduction of their electrophoretic mobility at such pH value. Results from these experiments are depicted in Fig. 2, where Fig. 2A represents the electropherograms obtained for the injection of the nanoparticles in the different solvents (acetic acid pH 4–5 or citric acid pH 3.5–6.5). As can be observed, Gaussian peaks are obtained for all the samples injected using citrate as solvent. As

expected, a slight decrease in peak width (Fig. 2B) is observed at pH values below 5.5, corresponding to the already observed reduction of the electrophoretic mobility of the nanoparticles at such pH value. This can be attributed to the on-line preconcentration by a mechanism similar to that described for dynamic pH junction. Results obtained for samples injected in acetic acid are quite surprising, as a very efficient on-line enrichment takes place at pH values of 4.5 and 5.0, which consequently resulted in much sharper peaks than that obtained at pH 4.0. In this sense, peak heights were increased by approximately 12-fold, while peak width was approximately one third in comparison with that obtained for the injection of the samples under CZE conditions. The difference between acetate- and citrate-based electrolytes is probably due to the different effect of those ions on the zeta potential of NPs. Also an opposite pH combination system using injection of NPs in alkaline solutions to acetate-based separation buffer was examined. However, no preconcentration was observed. Further experiments were performed by varying the amount of injected sample into the capillary using acetic acid as sample solvent at pH of 4.5 and 5.0 and 50 mM borate/NaOH pH 9.5 as background electrolyte. Injection times ranged from 5 to 100 s, accounting for up to 40% of the capillary length. As can be observed

Fig. 2. Electropherograms of behavior of NPs at the pH boundary (a) electropherograms obtained for the injection of samples of nanoparticles in acetic acid (pH 4.0–5.0) and citric acid (pH 3.5–6.5) into a 50 mM borate/NaOH pH 9.5 background electrolyte. Hydrodynamic injection of the nanoparticles for 20 s. (b) Influence of pH of citric acid and acetic acid used on the obtained peak widths for nanoparticles (N = 6).

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Fig. 3. Effect of the injection time on the electrophoretic behavior of nanoparticles (a) sample solvent: 87 mM acetate/NaOH pH 4.5, (b) sample solvent: 50 mM acetate/NaOH pH 5.0, background electrolyte for both: 50 mM borate/NaOH pH 9.5.

Fig. 4. Evolution of the pH profile in the capillary during electrophoresis, calculated by Simul software (a) sample solvent: 87 mM acetate/NaOH pH 4.5, (b) sample solvent: 50 mM acetate/NaOH pH 5.0, background electrolyte for both: 50 mM borate/NaOH pH 9.5.

from Fig. 3, a different effect was observed for the injection of the nanoparticles in acetic acid at pH 4.5 (Fig. 3A) and 5.0 (Fig. 3B). In this sense, the nanoparticles seemed to be efficiently stacked when injected at pH 5.0, while some “de-stacking” effect was observed at pH 4.5 for injection times over 20 s. The observed on-line preconcentration of the nanoparticles under such conditions can be attributed to the modification of the surface of the nanoparticles (change of co-ions from acetate to borate), the decrease of thickness of the electric double layer on the nanoparticles, the variation in the dispersion of the nanoparticles and their agglomeration on the pH boundary. Finally, simulation experiments were carried out using the Simul 5.0 software in order to have as much information as possible about the formation of any boundary and their migration through the capillary. Fig. 4 shows the temporal evolution of the pH boundary formed by the injection of 87 mM acetate/NaOH pH 4.5 (Fig. 4A) and 50 mM acetate/NaOH pH 5.0 (Fig. 4B) into a 50 mM borate/NaOH pH 9.5 background electrolyte. All other profiles (conductivity, sodium, boric acid and acetic acid concentrations) are presented in the supporting information. As can be observed, a sharp boundary is formed in the front interphase between acetic acid and borate (right-hand side of the sample zone in Fig. 4). Such boundary is moving through the capillary, being its mobility higher at pH 5.0 in comparison with that of pH 4.5. From the concentration profiles, two moving boundaries are formed when injecting acetate/NaOH to borate/NaOH electrolyte,

one sharp where the on-line preconcentration can occur and one which is not sharp. The sharp boundary (acetate/borate) migrates through the zone of NPs (acetate has a higher mobility than borate) and it stacks the nanoparticles. Interestingly, it seems that the stacking due to the differences in pH and the nature of ionic atmosphere of NPs (zeta potential of NPs varied from −30 mV in acetate buffer pH 4.5 to −45 mV in borate buffer pH 9.5) is reversible. After the migration of the acetate/borate boundary, NPs are de-stacked and form again the stable dispersion. In this view, a sharp peak of preconcentrated NPs can be observed at the end of the peak of NPs, in the middle of the NPs’ peak, and at the beginning of the peak of NPs, see Fig. 3A: 10 s, 20 s, and 30 s. 4. Conclusions In the present work, we have addressed the evaluation of the behavior of nanoparticles at the pH boundary formed between the sample solvent (acetic or citric acids) and the background electrolyte (borate). We have found a considerable influence of parameters such as the co-ion (acetate, citrate) and the sample pH on the retention time and peak shape of the nanoparticles, thus confirming that it is important to study the behavior of nanoparticles on a pH boundary, since it affects the overall electrophoretic behavior of the nanoparticles and the characterization of their charge and size by capillary electrophoresis. When such effects are appropriately taken into account, capillary electrophoresis provides a fast

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and easy tool for the characterization of nanoparticles. Finally, we have found that the large volume stacking of the nanoparticles can be used for their preconcentration on a borate/acetate pH boundary. Thus, the observed increase in peak height (up to 12-fold under the optimum conditions) is of interest, and could be exploited in the determination of nanoparticles, e.g. in the environment (see the supporting information). Acknowledgements

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Authors thank the financial support by the Operational Program Research and Development for Innovations – European Regional Development Fund (project CZ.1.05/2.1.00/03.0058), and the Operational Program Education for Competitiveness – European Social Fund (projects CZ.1.07/2.3.00/30.0004 and CZ.1.07/2.3.00/20.0018). Authors thank Dr. Martina Riesová (Charles University, Prague) for providing assistance with the Simul software.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.08.090.

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Study of behavior of carboxylic magnetite core shell nanoparticles on a pH boundary.

During the last years, several authors have focused on the characterization of the size and charge of the nanoparticles by capillary electrophoresis. ...
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