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Nanoparticle Characterization by Cyclical Electrical Field-Flow Fractionation Julien Gigault,† Bruce K. Gale,‡ Isabelle Le Hecho,† and Ga€etane Lespes*,† †
Universite de Pau et des Pays de l’Adour (UPPA)/CNRS Laboratoire de Chimie analytique Bio-Inorganique et Environnement, UMR IPREM 5254—Technop^ole Helioparc, Av. du President Angot, 64053 Pau Cedex, France ‡ Department of Mechanical Engineering, University of Utah, 50 S. Central Campus Drive Room 2110, Salt Lake City, Utah 84112-9202, United States ABSTRACT: In this work, the analytical potential of cyclical electrical field flow fractionation (CyElFFF) for nanomaterial and colloidal particle characterization has been experimentally demonstrated. Different operating parameters were investigated in order to evaluate their effect on the mechanisms of retention and fractionation power of CyElFFF. The voltage and frequency of the oscillating electrical field appeared to be the most influential parameters controlling the separation mode. Mobile phase flow rate was also found to be a key parameter controlling the fractionation efficiency. This work allowed the definition of operating conditions such that a reliable CyElFFF analysis could be performed on different nanoparticles on the basis of the direct comparison of their theoretical and experimental behavior. The results show that this technique in optimized conditions is a powerful tool for electrophoretic mobility based separation and characterization of various nanoparticles.
N
anotechnology and nanomaterials offer new opportunities in several fields of application. The principal physicochemical properties of nanomaterials are linked to their specific size, shape, and mobility (electrophoretic, sedimentation, and so forth). Indeed, these parameters control their industrial performance as well as their potential impact on the environment.1,2 Nevertheless, while these new nanomaterials are starting to be mass produced and used everywhere, there is currently not enough understanding of their physicochemical properties or their future in the environment. This lack of knowledge may be explained by the lack of appropriate analytical methods for the physicochemical characterization of nanoparticles and nanomaterials. A variety of techniques exist to separate and characterize nanomaterials and colloidal particles. For size characterization, flow field-flow fractionation appears to be a versatile technique for diverse kinds of nanoparticles.35 Concerning both size and charge (electrophoretic mobility) characterization, capillary electrophoresis (CE) has been used for a few years.69 Indeed, several kinds of nanomaterials and colloidal particles have been studied using CE such as polymers (polystyrene particles), inorganic nanoparticles, microorganisms (viruses, bacteria, cells), and humic substances.1015 Nevertheless, CE has several drawbacks associated with the challenging experimental conditions required such as electrical fields in the range of kilovolts, carefully crafted mobile phases (pH, ionic strength, and so forth), or a small size capillary. These operating conditions can damage or change the analytes, leading to aggregation, shear degradation, capillary r 2011 American Chemical Society
adsorption, and aqueous particle instability. In addition, the pH of the buffer in CE can modify the particle charge.3 Electrical field-flow fractionation (ElFFF) is a technique which can separate analytes on the basis of their electrophoretic mobility and size without the drawbacks cited for CE.3,1618 Indeed, the much smaller electrical field used in ElFFF (voltage range from 0 to 2 V) as well as ElFFF channel dimensions in the range of several micrometers significantly reduce the risk of particle damage.19 Moreover, with ElFFF the mobile phase is generally water, which has less interaction with analytes than the mobile phases used in other techniques such as CE. Nevertheless, there are some drawbacks in ElFFF such as low effective electrical field efficiency.3 However, the simplicity in design and the ease of use, as well as the comparability in time and resolution with commonly used CE, make ElFFF a very promising technique for separation of nano- and microscale particles. Another subtechnique of FFF, which is not well-known, termed cyclical FFF, separates particles based on their migration rates which depend on transport coefficients.20 Migration rates have been shown to depend on the generalized mobility of particles. In cyclical FFF the direction of the field is reversed according to a periodic or cyclic pattern (e.g., square waveform) during the course of the separation. With each inversion, the sample population seeks to re-establish a new equilibrium distribution. Received: April 7, 2011 Accepted: July 20, 2011 Published: July 20, 2011 6565
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Analytical Chemistry This subtechnique of FFF has been little used and is not wellknown. A cyclical sedimentation field was first investigated for the separation of particles based on their size.21 Recently, a cyclical electrical FFF (CyElFFF) has been developed.22,23 In CyElFFF the electrical field that is applied can be more effective than in traditional ElFFF because the ac field short circuits the large capacitance associated with the parallel plate channel electrodes as the frequency increases. In addition, a too high voltage (typically over ∼1.7 V) induces water electrolysis and bubble formation in normal ElFFF, but the higher frequencies in CyElFFF generate less disturbance from bubbles than in normal ElFFF, so voltages up to ∼8 VPP can be applied. Thus, the higher electrical field used in CyElFFF is potentially more powerful and can be used to retain charged molecules according to differences in electrophoretic mobility (μ). Nevertheless, there is no comprehensive study on the analytical performances of this technique across a range of nanomaterial and colloidal particles, and there have been no demonstrations of particle retention below 80 nm. With the recent emphasis in the literature on particles in this smaller size range, and especially for engineered nanoparticles in this size range, demonstration of particle characterization capabilities in this range would be valuable and potentially allow characterization of nanoparticles that is not currently possible. Moreover, it was admitted that considerable experimental and theoretical work will be necessary to more fully evaluate this new form of FFF.22 Thus, there are multiple reasons for investigating the capabilities of CyElFFF with these nanomaterials and the development of analytical techniques in these application areas. Accordingly, the objective of this work was to investigate the different operating parameters in CyElFFF in order to evaluate their effect on the retention mechanisms and fractionation power for different nanoparticles in a wide range of size and electrophoretic mobility using CyElFFF. The key parameters of CyElFFF investigated were the voltage applied, the field frequency, and carrier flow rates. The goal was to develop a method sufficiently robust that it could be adapted for heterogeneous colloidal particle samples. Finally, the first time demonstration of some applications of this technique are presented using both manufactured and natural particles. The knowledge acquired on the basis of this work is essential to reach the capability and potential of this technique in the field of nanomaterial characterization and environmental monitoring.
’ THEORY A CyElFFF system is composed of two electrodes separated by a thin spacer defining a flow channel. The flow in the channel is laminar with a parabolic velocity profile. As particles are introduced into the channel, they interact with the applied oscillating electric field and move back and forth between the electrodes. In CyElFFF, depending on their electrophoretic mobility, the particles will spend more or less time in the fast flow lines compared to other particles. If they spend more time in the fast flow lines, they will elute from the far end of the channel sooner than particles that are confined to slower flow lines. Three different modes have been proposed to describe particles motion in the channel during elution as is illustrated by Figure 1.20 In mode I, nanoparticles only move near the accumulation wall in an oscillatory pattern. In mode II, nanoparticles cross the main flow and reach the opposite wall, then move back to the original accumulation wall (bottom electrode).
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Figure 1. Principal modes of elution in CyElFFF.
In mode III, particles move across the channel and slip along the opposite wall before the field is reversed and the particles reverse their motion.20,21 In CyElFFF a nondimensionnal retention parameter λ0 is used to describe the motion of a particle and is defined by20 λ0 ¼
μEeff 2f ω
ð1Þ
where f is the frequency of the applied electric field, μ is the electrophoretic mobility of the particle, ω is the channel spacer thickness, and Eeff is the magnitude of the effective field experienced by the particles inside the channel. Moreover, in CyElFFF, Eeff is equal to VB/ω, where VB is the effective voltage across the bulk phase. The nondimensional retention parameter can be then rewritten as23,20 λ0 ¼
μVB 2f ω2
ð2Þ
Moreover, the retention ratio R is defined by3 R ¼
t0 tR
ð3Þ
where t0 is the experimental void time (elution time of unretained particles) and tR is elution time of retained particles. R can be related to λ0 depending on the motion of the particles and on the mode according to which this motion can be described. Thus, R in mode I is given by20 2 R ¼ 3λ0 1 λ0 , for λ0 e 1 ð4Þ 3 whereas R in mode III is given by R ¼
1 , for λ0 g 1 λ0
ð5Þ
At λ0 = 1, eqs 4 and 5 connect to create a continuous function with tR = t0 which corresponds to mode II.
’ EXPERIMENTAL SECTION Chemicals. Ammonium carbonate ((NH4)2CO3, 99.5%) and sodium dodecyl sulfate (SDS, 98.5%) were purchased from SigmaAldrich. Latex nanospheres came from Jasco. The water used was 18 MΩ deionized (DI) water. Single walled carbon nanotubes were purchased from Nanocyl (SWCNT/NC) and Sigma Aldrich (SWCNT/SA) (numbered lot from Sigma Aldrich, St. Quentin Fallaire, France) with size characteristics previously determined.5 Fe3O4 and Ag nanoparticles were purchased from Sigma Aldrich and NLC (Salt Lake City), respectively. 6566
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Table 1. Electrophoretic Mobility of Nanosphere Standards size (nm), manufacturer data 20
40
100
size measured (nm)
22.6 ( 0.4
40 ( 0.8
electrophoretic mobility (104 V2 cm1 s1)
1.3 ( 0.3
1.9 ( 0.1
To evaluate the applicability of the optimized conditions for natural colloids, two soil leachates (A and B) were used. They were obtained from two agricultural soils with a silty nature sampled at the surface. The natural pH of these soils are 8 and 6 for soils A and B, respectively. Sample Preparation. Latex nanospheres were diluted in Milli-Q water in order to obtain size-standard solutions with a detectable UV signal. Their characteristics were calibrated (size, electrophoretic mobility, zeta potential, etc.) and are presented in Table 1. Aqueous dispersions of CNT in the presence of SDS were realized. A suspension was prepared by adding SWCNT powder to SDS aqueous solution, with the suspension obtained then being sonicated. The final CNT concentration was 0.01 g L1. A dispersion of 0.1 g L1 Fe3O4 nanoparticles in oleic acid aqueous solution and a dispersion of Ag nanoparticles in D2O water at 22 mg L1 were used. The two soil leachates (A and B) were prepared according to a standardized protocol from the French Agency for Normalization (NF X31-210, 1992). Briefly, the preparation included three steps: First, 100 g of soil was stirred with 1 L of DI water in a bottle shaken for 16 h. Then, the mixture was centrifuged at 3500g for 30 min. Finally, supernatant was collected and filtered at 0.45 μm in order to have a continuum of size under 450 nm. Instruments. The CyElFFF channel used for all the experiments was 64 cm long, 2 cm wide, and 178 μm deep. It is illustrated in Figure 2 and is identical to that used in a previous publication.22,24 The channel was kept in a horizontal position as it was assumed that the very small particles used in this work would not be subject to significant sedimentation. Flow rates were controlled with a HPLC pump (model BioInstrumenta). A UV detector was a variable wavelength UVvis spectrophotometer model 520 (ESA, Inc., MA) tuned at 254 nm and connected online to the ElFFF channel. All injections were performed with a 100 μL Hamilton microliter syringe. Data from the UV detector were collected and processed using LabVIEW 8.6 software (National Instruments). The size and electrophoretic mobility of the polystyrene nanospheres were measured using a zeta potential analyzer (Zetasizer 3000, Malvern). Method. The electrophoretic mobility is governed by both the charge on the particle at the plane of shear and the apparent particle size (i.e., the physical diameter increased by about twice the thickness of the double layer), rather than the charge at the solidliquid interface.16 In order to separate all the particles in the same mode with a convenient efficiency, our objective was to have λ0 g 1, i.e., to operate according to mode III.20 Indeed, with this condition R = 1/λ0 and a linear relation between tR and λ0 appears: ð6Þ tR ¼ t0 λ0 Then, eq 2 can be inserted in eq 6, and the results rearranged to obtain VB ð7Þ tR ¼ t0 2 μ ¼ Aμ 2ω f where A is a constant at given operating conditions.
200
400
107 ( 4
211 ( 4
434 ( 14
3.3 ( 0.2
4.7 ( 0.2
5.7 ( 0.3
In mode I this relationship between retention time and electrophoretic mobility is more complicated and nonlinear and becomes tR ¼
2f ω2 t0 A ¼ VB2 2 Bμ Cμ2 3VB μ 2 μ fω
ð8Þ
where A, B, and C are constants at given operating conditions. A linear relation between the analyte’s retention time and electrophoretic mobility allows determination of the selectivity of separation (Sμ):20 dðlog t Þ R ð9Þ Sμ ¼ dðlog μÞ Selectivity defines the difference in retention time with electrophoretic mobility (for CyElFFF). This parameter is particularly useful in order to evaluate the ability of an FFF technique to separate particles.3 Finally, a more specific parameter of FFF techniques that also describes the ability to separate components is the fractionating power (Fμ) and is defined by3 Fμ ¼
Sμ N 1=2 4
ð10Þ
with N the number of theoretical plates and Sμ the selectivity. In order to have an optimal nanoparticle fractionation, all these parameters were considered and studied together.
’ RESULTS AND DISCUSSION Optimization of CyElFFF. The aim of this optimization effort is to explore the range of operating conditions and key parameters to find those giving the highest separation efficiency with regard to future analytical use with nanoparticles. Preferably these conditions would provide a linear relationship between electrophoretic mobility and retention time. Conditions such as voltage, frequency, flow rates, and stop flow influence were explored. Several measures have to be considered in order to evaluate the CyElFFF results: retention time, selectivity, and fractionating power. For this work different nanospheres calibrated in size and electrophoretic mobility were used in order to provide a reliable evaluation of the CyElFFF separation effectiveness. All experiments were realized with an ultrapure water mobile phase. Indeed, a change of mobile phase from DI water to a higher ionic strength carrier causes changes to channel capacity and efficiency through double layer polarization effects.25 Voltage. The main parameter that controls particle motion through the channel is the applied voltage field.22 For square cyclical waveforms, the voltage is usually expressed in volts peakto-peak (VPP). Across the range of applied voltage, the three different modes of separation previously described can occur while only one of these modes (mode III) is desired since it generates a linear separation of analytes following their 6567
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Figure 2. Representation of the CyElFFF channel.
electrophoretic mobilities. In this work, the applied voltage was tested over the range 08 VPP. Over 8 VPP, bubbles appear in the channel, which can be explained by electrolysis of the water carrier. The cyclical frequency was fixed at 1 Hz and flow rate at 1 mL min1, and only DI water was used as the mobile phase. Figures 3 and 4 show the retention time of the various nanospheres according to the applied voltage and according to their electrophoretic mobility for a selected voltage, respectively. All these results illustrate that for highest voltage we are typically in mode III and for low voltages in mode I with a transition point at voltages ranging from ≈1.5 to 3 VPP (frequency 1 Hz). In general, the retention curves follow the pattern predicted by theory with a few important exceptions, which will be discussed shortly. When these results are examined by considering a given particle (electrophoretic mobility), it can be seen that the retention time generally increases with voltage in mode III (Figure 3). As noted, our preference was to use CyElFFF in mode III to allow for a linear retention with electrophoretic mobility. Nevertheless for high voltages deep in mode III, no linear variation was observed due to what is most likely diffusion effects for the smallest particles (which also have the smallest mobility) causing the retention times to be less than anticipated (see especially Figure 4c, left part). In Figure 3, the retention curves for the 20 and 40 nm particles should cross over the curves for the 100, 200, and 400 nm particles, but do not due to decreased retention due to significant diffusion. Note that the basic theory for CyFFF does not currently include diffusion. Also deep in mode III (at 8 VPP) the 400 nm particles also elute earlier than predicted, possibly because of steric effects at this size and voltage, i.e., by limiting the approach of particles to the accumulation wall.26 This deviation from the linearity expected in mode III caused us to conclude that this mode of separation is not convenient in order to separate the largest nanoparticles with the highest voltages (from 4 to 8 VPP). Nor did it appear to work well for the smallest particles. As mode III did not appear to be a good choice for linear retention of particles, we explored mode I. Interestingly, retention of particles in mode I between 0.5 to 1.5 VPP allowed linear
Figure 3. Representation of the retention time for different nanospheres following the different voltages applied (obtained with a frequency of 1 Hz and evaluated from 10 replicated analysis, mean RSD = 7%).
retention of nanoparticles (Figure 4), which is not necessarily consistent with theory, but is consistent with some previously published work.23 It corresponds to the fact that the transition point between mode I and mode III is not reached at the same voltage value according to the considered particles (typically ≈1.5 VPP for 20 and 40 nm, and ≈3 VPP for g100 nm). In Figure 4a it appears a linear relation between tR and μ exists and that the relationship is optimized by keeping low mobility particles in the low retention region where diffusion will also be minimized. For voltages under 0.5 VPP, there is no measurable retention, which indicates that, for very low voltage (below 0.25 V above or below 0 V), there is insufficient field to produce an effect and the particles elute with the void peak. In order to find the optimal voltage from these three values that produced linear retention mostly in mode I (0.5, 1.0, and 1.5 VPP), all metrics of separation were investigated. Table 2 6568
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Figure 5. Representation of the retention time for different nanospheres following the frequency applied (obtained with a voltage of 0.5 VPP and evaluated from 8 replicated analysis, mean RSD = 6%).
Figure 4. Representation of the retention time for different nanospheres following their electrophoretic mobility for various voltage ranges from 0.5, 2, and 8 VPP (obtained with a frequency of 1 Hz and evaluated from 10 replicated analysis, mean RSD = 6%).
Table 2. Fractionating Power (Fμ) Determined from Different Nanosphere Standards for Voltage of 0.5, 1.0, and 1.5 VPP fractionation power (Fμ) nanospheres standard
0.5 VPP
1.0 VPP
1.5 VPP
20 nm
0.41 ( 0.03
0.16 ( 0.02
0.12 ( 0.01
40 nm
0.48 ( 0.02
0.27 ( 0.08
0.30 ( 0.02
100 nm
0.72 ( 0.02
0.64 ( 0.03
0.31 ( 0.02
200 nm
0.93 ( 0.01
0.71 ( 0.02
0.33 ( 0.01
400 nm
1.19 ( 0.01
0.73 ( 0.01
0.30 ( 0.03
summarizes the different fractionation power (Fμ) determined from the particle retention for these three voltages. The results indicated that 0.5 and 1.0 VPP give the highest F values. Moreover, except for the 400 nm nanosphere standard, no significant difference in effectiveness appears between these two voltages. Complementary selectivity which is the slope of the linear relation between retention time and electrophoretic mobility was determined for these three voltage. It appears that a voltage of 0.5 VPP allows having the highest selectivity value, which is 0.291 ( 0.003, while eq 8 predicts a maximum selectivity value of 1. Although this Sμ value could seem relatively low, the voltage of 0.5 VPP
corresponds to the optimal voltage that can be obtained in order to have a satisfactory nanoparticle separation and was chosen and used in later experiments. Frequency. If the voltage applied has a major influence on the separation efficiency, another parameter which controls particle retention in the channel is the frequency. Indeed, as was indicated in eq 2, retention time depends on both voltage and frequency. So in order to determine the influence of frequency, voltage was kept constant at 0.5 VPP and frequency was varied in the range from 0.1 to 100 Hz. Figure 5 represents the retention time of nanosphere standards following the frequency applied. On this figure it appears clearly that frequency has a significant influence on the nanoparticles retention only between 1 and 10 Hz. The very large and very small frequencies appear to be too fast or too slow to have a meaningful impact at this voltage, which is relatively small. It was previously seen that results obtained with 1 Hz give an efficient separation. Figure 6 illustrates the retention time of nanosphere standards versus their electrophoretic mobility for these two conditions cited above. This figure shows that a polynomial-like relation links tR and μ for 10 Hz which is characteristic of a transition from mode III to mode I according to eq 4. Near the transition point between the modes, the resolution and selectivity are at a minimum, as well as retention times, so the retention times of all the nanospheres obtained for 10 Hz were globally under those obtained with a frequency of 1 Hz. For 100 Hz, most of the particles would be caught in an early peak and not retained (no offset voltage is used in these experiments), and the peaks are known to become quite flat at high frequencies making retention difficult to detect.22 Additionally, the sample can drift away from the wall after many cycles, which induces at once shorter retention times and lower selectivity. Thus, in order to have a reasonable elution, the optimal frequency appears to be 1 Hz. Flow Rates. Particle velocity in the channel is associated with the mobile phase velocity at that point above the wall at which the particle is located. Mobile phase velocity increases with increasing distance from the wall according to the parabolic profile associated with laminar flow. It is therefore generally known in cyclical FFF that the elution flow rate affects the effective field and the retention time20 and in some cases has been shown to improve resolution and plate heights.22 In this study, the flow rate was tested in the range from 0.8 to 1.6 mL min1 in order to find if the 6569
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Table 3. Fractionating Power (Fμ) and Selectivity (Sμ) Determined from Different Nanosphere Standard Signals for Mobile Phase Flow Rates of 0.8, 1.0, 1.3, and 1.6 mL min1 flow rate (mL min1) 0.8
1.0
1.3
1.6
0.28 ( 0.02
0.28 ( 0.01
Selectivity (Sμ) 0.080 ( 0.006
0.29 ( 0.01
Fractionating Power (Fμ) 20 nm 40 nm
Figure 6. Representation of the retention time for different nanospheres following their electrophoretic mobility for two characteristic frequencies: 1 Hz and 10 Hz (obtained with a voltage of 0.5 VPP and evaluated from 8 replicated analysis, mean RSD = 6%).
change of the velocity profile has an influence on retention time. In order to find the optimal flow rate, Table 3 summarizes the variation of the fractionating power of the different standard nanospheres and selectivity determined for the different flow rates tested. It appears clearly that flow rates of 1.0, 1.3, and 1.6 mL min1 give the highest values of selectivity. The low Fμ values for 0.8 flow rate may be attributed to some interactions (e.g., wall sticking effect as in Fl-FFF) between analytes and the channel wall or more likely diffusion effects, which are known to lead to band spreading and poor retention in CyElFFF.22 It is also observed that for the three higher flow rates tested the selectivity remains constant while the fractionation power decreases with an increasing flow rate. This decrease is more important for the smallest particles, which have the highest diffusion coefficient and the lowest electrophoretic mobility. The most probable hypothesis is that it is more difficult to reach an equilibrium when the flow rate increases and especially for these small particles. This induces a broadening phenomenon leading to a lower fractionation power. By also considering fractionating power, the flow rate of 1.0 mL min1 gives globally the best results. As expected the retention time decreases with increasing flow rate. Stop Flow. In FFF techniques, this step is important in order to have a sample in equilibrium in the FFF channel before elution. Indeed, after injection into any FFF channel, the sample becomes subject to the influence of the applied field and begins its migration toward the accumulation wall. It was previously shown that the relaxation time in ElFFF depends on the channel thickness and has little influence when it exceeds 30 s.3 So here the time was kept constant at 30 s and several dc (direct current) voltages were tested in order to see if this step has an influence of the selectivity by CyElFFF for nanoparticle separation. The selectivity was evaluated for different relaxation time. No significant variation of selectivity was found if a voltage is applied during the relaxation step in mode III, as the sample essentially relaxes during every half-cycle of the analysis when in mode III. Nevertheless, analysis realized without voltage during relaxation step was less repeatable than analysis with voltage applied. This result can be explained by the fact that, during this step, even if the time is relatively short and if no voltage is applied, the diffusion, which is a function of size, leads to a distribution of the particles across the height of the ElFFF channel and a different starting point in the experiments. So for further experiments, a voltage of 1 V was
0.040 ( 0.007 0.055 ( 0.002
0.41 ( 0.03 0.48 ( 0.02
0.14 ( 0.02 0.24 ( 0.01
0.14 ( 0.01 0.26 ( 0.03
100 nm
0.164 ( 0.006
0.72 ( 0.02
0.56 ( 0.04
0.51 ( 0.01
200 nm
0.241 ( 0.004
0.93 ( 0.01
0.80 ( 0.03
0.88 ( 0.05
400 nm
0.253 ( 0.003
1.20 ( 0.01
1.00 ( 0.02
1.19 ( 0.01
Table 4. Optimal Conditions of CyElFFF for Nanoparticle Characterization parameter
optimized condition
mobile phase
DI water
injection volume (μL)
100
relaxation time (s)
30
relaxation voltage (VPP)
1
relaxation frequency (Hz) elution flow rate (mL min1)
1 1
elution voltage (VPP)
0.5
elution frequency (Hz)
1
analysis duration (s)
≈400
kept in order to be sure that all nanoparticles traveling in the channel only follow their electrophoretic mobility. Analytical Performance. The optimized conditions are summarized in Table 4. From these conditions, the repeatability of the fractionation was evaluated on the nanosphere standards by six replicated analyses. There was no difference in the peak position (tR) nor in the standard deviation (σt). In addition, to evaluate the quality of the fractionation by CyElFFF, calibration using nanosphere standards allows estimating the electrophoretic mobility variation according to the retention time and by checking the linearity of the relationship (eq 7). As shown in Figure 7 which represents typical fractograms of nanospheres, the signals are well resolved, and the linear curve is precise (R2 = 0.991) and significant. Applications. After calibrating the instrument and determining optimal experimental parameters, different particles were characterized by CyElFFF using the conditions previously described (Table 4). There are a wide variety of nanoparticles and nanoparticle mixtures based on how they are produced (manufactured or natural), shape (spherical, rod), and size (monodisperse or polydisperse). So the capabilities of CyElFFF were evaluated for particles which have various physicochemical characteristics and natures. In order to see only the influence of electrophoretic mobility in CyElFFF separation, different manufactured nanoparticles were studied: silver nanoparticles, iron oxide nanoparticles, and carbon nanotubes in the optimal conditions cited above. Figure 8 represents distributions obtained for samples of these various 6570
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Figure 7. Representation of typical fractograms of nanosphere standards realized in optimal conditions with the variation of corresponding electrophoretic mobility according to the retention time (evaluated from 6 replicated analysis, mean RSD = 6%).
nanoparticles with electrophoretic mobility corresponding to the retention time calculated from calibration. Case of Manufactured Nanoparticles. First Ag and Fe3O4 nanoparticles were analyzed (Figure 8a). Fe3O4 which has a diameter larger than Ag nanoparticles (50 and 10 nm, respectively) was eluted first with an electrophoretic mobility close to zero. This result is in accordance with the literature. Indeed, it has been shown that Fe3O4 nanoparticles have no charge and an electrophoretic mobility close to 0, electrophoretic mobility being directly proportional to the number of charges on the surface.8 Moreover, Ag nanoparticles, which have a smaller size compared to Fe3O4 nanoparticles have a higher electrophoretic mobility. This figure confirms the fact that CyElFFF separates only on the basis of the transport rate which is the electrophoretic mobility in the case of CyElFFF. Note that this is the first demonstration of retention of both 10 nm particles and Ag nanoparticles in CyElFFF. Carbon nanotubes were also fractionated by CyElFFF. These new nanomaterials have a large size range and elongated shape. It was previously theoretically seen that particle shape can have an influence of their retention time.19 Figure 8b illustrates the distribution obtained for single walled carbon nanotubes from different manufacturers but with approximately the same length range (1002000 nm).5 It appears from these results that these two kinds of carbon nanotubes have different electrophoretic mobility ranges. A possible explanation for this difference could be that, on the basis of the structural configuration of the graphene sheet which composes them, they can be conducting (as SWCNT/SA) or semiconducting (typically SWCNT/NC) materials. Moreover, it is important to note that the electrophoretic mobility in the case of polydisperse conducting analytes is spread over a large range of values compared to the mobility found for Ag nanoparticles, which are monodisperse nanoparticles. All these results using different manufactured nanomaterials, and especially those with variations in size and shape, show the potential of CyElFFF to obtain electrophoretic mobility information about a variety of nanoparticles. Case of Natural Nanoparticles. Finally, natural colloidal particles were also fractionated and the two different soil leachates previously described analyzed. A typical distribution as a function of electrophoretic mobility is presented in Figure 8c. The non-Gaussian signal suggests a variety of particle
Figure 8. Distributions obtained for various nanoparticle samples according to electrophoretic mobilities (evaluated from 6 replicated analysis, mean RSD = 6%).
populations make up the leachate and form a complex matrix, which leads to a polydisperse sample with different concentrations of particles with different mobilities. Indeed, this bulk solution, which contains numerous more or less soluble ionic or nonionic chemical species, can influence the colloidal particle displacement into the electrical channel. Nevertheless, on the basis of the CyElFFF fractionation, these two soils appear to have different ranges in electrophoretic mobility. It is well-known that the electrophoretic mobility and the charge of colloids have an influence on the speciation and mobility of different chemical compounds present in the environment,6 so these results show the relevance of CyElFFF for obtaining information of environmental interest. 6571
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’ CONCLUSION In this work, CyElFFF for particle characterization was optimized. The voltage and frequency of the electrical field appeared to act as major influential parameters controlling the mode of separation in CyElFFF. Elution flow rate remains one of the key parameters controlling the fractionation efficiency also. One of the main advantages of CyElFFF relies in its ability to obtain a distribution based on electrophoretic mobility, which was experimentally confirmed here. Additionally, this work shows how defining the operating conditions can provide a reliable CyElFFF analysis which can be performed on different colloidal particles and manufactured nanomaterials. Analysis of Ag, Fe3O4, carbon nanotubes, and soils was performed for the first time in CyElFFF. Finally, the results show that this technique is a powerful tool for the separation according to the electrophoretic mobility and the characterization of various manufactured and natural particles in a wide range of submicrometer sizes. More generally the cyclical FFF (CylFFF) approach has the advantage that any field having a strong interaction with the sample particles could be used, so additional applications can easily be envisioned. Moreover, CyElFFF should be able to be coupled with multiple detectors permitting complementary information to be obtained. Work is continuing to confirm the potential of this fractionation technique and actually reveal it as a promising tool of characterization in the field of nanomaterials and nanotechnology.
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’ AUTHOR INFORMATION Corresponding Author
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dx.doi.org/10.1021/ac2008948 |Anal. Chem. 2011, 83, 6565–6572
