Inductively-Coupled Plasma-Mass

Oct 29, 2014 - Capillary electrophoresis coupled with inductively coupled mass spectrometry as an alternative to cloud point extraction based methods ...
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Capillary Electrophoresis/Inductively-Coupled Plasma-Mass Spectrometry: Development and Optimization of a High Resolution Analytical Tool for the Size-Based Characterization of Nanomaterials in Dietary Supplements Haiou Qu, Thilak K. Mudalige,* and Sean W. Linder* U.S. Food and Drug Administration, Office of Regulatory Affairs, Arkansas Regional Laboratory, 3900 NCTR Road, Jefferson, Arkansas 72079, United States S Supporting Information *

ABSTRACT: We report the development and optimization of a system consisting of capillary electrophoresis (CE) interfaced with inductively coupled plasma mass spectrometry (ICPMS) for rapid and high resolution speciation and characterization of metallic (e.g., gold, platinum, and palladium) nanoparticles in a dietary supplement. Multiple factors, including surfactant type and concentration, pH of running buffer, and applied voltage, were investigated to optimize the separation conditions. It was found that by using the anionic surfactant sodium dodecyl benzenesulfonate (SDBS) in the running buffer the separation resolution was significantly improved, allowing for easy distinction of adjacent size fractions in a gold nanoparticle mixture with very small size differences (e.g., 5, 15, 20, and 30 nm). The type and concentration of the surfactant was found to be critical in obtaining sufficient separation while applied voltage and pH values of the running buffers largely affected the elution times by varying the electroosmotic flow. Quantum dots were used as mobility markers to eliminate the run-to-run variation. The diameters of the nanoparticles followed a linear relationship with their relative electrophoretic mobility, and size information on unknown samples could be extrapolated from a standard curve. The accuracy and precision of this method was confirmed using 10 and 30 nm gold nanoparticle standard reference materials. Furthermore, the method was successfully applied to the analysis of commercially available metallic nanoparticle-based dietary supplements, as evidenced by good agreement between the particle sizes calculated by CE/ICPMS and transmission electron microscopy (TEM).

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sample preparation, and the potential for artificial particle aggregation due to the drying effect are also common limitations associated with TEM.10 Other popular sizing techniques such as differential centrifugal sedimentation and dynamic light scattering (DLS) either are time-consuming or produce erroneous results in polydisperse samples as a result of the strong biases caused by the presence of larger particles.11,12 Coupling advanced separation techniques with various online detection systems, such as light scattering and mass spectrometry, enables the acquisition of additional information on nanomaterials.13 Asymmetric flow field flow fractionation (AFFFF) has been widely accepted as a powerful tool in the characterization of nanomaterials over a diverse range of sizes. When coupled to multiple online detectors, mixtures of polystyrene or gold nanoparticles have been separated leading to the determination of their sizes and mass concentration.14 A common problem for AFFFF is the potential for decreased

ngineered nanomaterials have been considered for application in a variety of fields due to their distinctive and novel properties compared to their bulk counterparts.1−4 Lately, nanomaterials are being incorporated in the manufacturing process by several companies in order to improve the performance of their products, such as antimicrobial silver nanoparticles in athletic socks, ultraviolet radiation blocking titanium dioxide particles in sunscreens, and essential element nanoparticles in dietary supplements.5,6 Since a range of physical, chemical, and biological characteristics of nanomaterials are closely associated with their size, accurate characterization of this parameter is essential to acquire an in-depth understanding of their properties. Transmission electron microscopy (TEM) is widely used for the determination of nanoparticle morphology and size distribution.7 The statistical significance of TEM results from studies may be relatively low when considering the amount of nanoparticles examined only constitutes a very small fraction of the entire sample.8,9 The number of particles that need to be measured to obtain meaningful results depends largely on sample polydispersity. Additionally, the high cost of the instrument, complicated © 2014 American Chemical Society

Received: July 11, 2014 Accepted: October 29, 2014 Published: October 29, 2014 11620

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recoveries of the analytes of interest, due to particle adhesion to the separation membrane, which limits its application in accurate quantitative analysis.14,15 Liquid chromatography (LC) coupled with inductively coupled plasma mass spectrometry (ICPMS) has been used to characterize nanomaterials such as gold nanoclusters and silver nanoparticles.16,17 In contrast to an AFFFF approach, which is more suitable for macromolecule or nanometer-sized particles, a LC-ICPMS method is capable of detecting particles and dissolved metal ions simultaneously.18−20 However, previous reports suggest that some LC techniques (e.g., hydrodynamic chromatography) are not very effective in separating multicomponent nanoparticle mixtures. Additional studies are needed to improve the instrumentation and optimize the operational conditions for higher resolution.16 Capillary electrophoresis (CE) is another well-developed separation technique that has a diverse range of applications. The relatively simple open-tubular separation environment, which does not have a stationary phase in most cases, and small sample volume offer advantages over chromatographic techniques. The use of a high electric field and the planar flow velocity profile of electroosmotic flow (EOF) allows for efficient high resolution analysis to be obtained in short measurement times.21 CE has been utilized in the studies of a variety of samples including DNA, proteins, peptides, metabolites, pharmaceuticals, etc.22−24 In recent years, there has been a growing interest in the use of CE as a tool in the characterization of different types of nanomaterials.25−29 Liu and Wei successfully used CE to study 5.3 and 19 nm gold colloids.25 Similar methodologies were also applied in the characterization of CdSe quantum dots in aqueous medium, as well as in the detection and quantification of surface functionalized fullerene-based nanomaterials in serum.26,27 Common detection systems utilized with CE are diode array UV−visible absorption detection or laser-induced fluorescence detection.30 Neither technique provides information on the elemental composition of the sample, which is a vital parameter when studying materials. ICPMS is a powerful analytical technique for elemental analysis that provides excellent sensitivity and selectivity. It has been accepted as a valuable tool in the study of nanoscale engineered materials.31,32 CE coupled with ICPMS was developed to combine the efficient separation capacity with the ultrasensitive detection ability for high resolution studies. It has been demonstrated to be an important technique for a variety of applications, including the speciation of environmental samples and metalloproteomics studies.33,34 A recent publication by Engelhard and co-workers has reported the utilization of CE coupled with ICPMS for size base separation and size determination of gold and silver nanoparticles.29 The objective of this work is to establish a high resolution analytical system consisting of CE interfaced with ICPMS to separate and characterize multimode gold nanoparticle mixtures containing a size fraction with small differences (e.g., 5, 15, 20, and 30 nm). Multiple factors, including surfactant type and concentration, pH of running buffer, and applied voltage, were investigated to optimize the separation conditions. The developed method was tested by using standard reference materials to demonstrate the accuracy and reliability. The practical application and performance were further evaluated by utilizing it in the analysis of commercial dietary supplements containing metallic nanoparticles.

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EXPERIMENTAL SECTION

Materials and Chemicals. N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS, 99%), sodium dodecyl sulfate (SDS, 99%) and decyl sodium sulfate (S10, HPLC grade) were purchased from Acros Organics (Waltham, MA, USA). Sodium dodecylbenzenesulfonate (SDBS, 97.5%) and cyclohexylaminoethanesulfonic acid (CHES, 99.9%) were purchased from MP Biomedical (Carlsbad, CA, USA). Tris[hydroxymethyl]aminomethane (TRIS, 99.8%) was purchased from BioRad (Hercules, CA, USA), and N-cyclohexyl-3aminopropanesulfonic acid (CAPS, 98%) was acquired from Fisher Scientific (Waltham, MA, USA). 1,2-Diaminocyclohexanetetraacetic acid monohydrate (CDTA, 98.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and gold ICP standard (1000 μg mL−1) was purchased from Ultra Scientific (North Kingstown, RI, USA). All chemicals were used as received without further purification. Gold nanoparticle suspensions (5, 10, 15, 20, 30, 40, and 50 nm, stabilized by citrate with a net negative surface charge) were acquired from Ted Pella (Redding, CA, USA) and stored at 4 °C. A total of four commercially available dietary supplements (labeled as DS 1−4) which claim the inclusion of nanomaterials or the utilization of nanotechnology were purchased from Internet resources. Each of the dietary supplements was an aqueous suspension and was used as received for the analysis without further purification. Deionized water (>18MΩ·cm) was used throughout the experiments. Polymer coated CdSe/ZnS quantum dots (QDs) (Emax = 665 nm) with carboxylic acid surface groups were purchased from Ocean NanoTech (Springdale, AR, USA). According to the manufacturer, QDs used in this study have an average inorganic core diameter of 8 nm measured by TEM and a hydrodynamic diameter of 17 nm measured by DLS. These values were experimentally confirmed for this study by TEM and DLS measurements. Before use, QDs (4 nM) were washed with deionized water and spinfiltered for three times to eliminate leached metal ions from QDs and other chemical residues. The purified QDs were then redispersed in water, and the suspension was diluted 15-fold. Gold nanoparticle standard reference materials of 10 and 30 nm (RM 8011 and RM 8012) were purchased from National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA). All gold nanoparticles were directly used for analysis without any dilution or other treatments. Instrumentation. Gold nanoparticles standards and dietary supplements were studied on a JEOL 1400 TEM (Peabody, MA, USA) operated at 80 kV. The images were acquired by a TVIPS TemCam F416 camera, and ImageJ software was used to process the images and to obtain size statistics of gold nanoparticles. A few drops of samples were placed directly on a 300 mesh copper grid and air-dried overnight. Capillary electrophoresis separation was performed on a 7100 Capillary Electrophoresis system (Agilent Technology, Santa Clara, CA, USA). Coated fused-silica capillaries (i.d. 50 μm; o.d. 360 μm; length 67 cm) were obtained from Molex (Phoenix, AZ, USA). A new capillary was initialized by flushing with 1 N NaOH for 30 min and running electrolyte for 30 min, followed by rinsing with water for 5 min. The capillary was conditioned each day before use with 1 N NaOH for 15 min, water for 3 min, and running buffer (e.g., TRIS 10 mM, SDBS 70 mM, pH = 9) for 15 min. Between each run, the capillary was rinsed with 1% of HNO3 for 1 min and with water for 30 s and then equilibrated with 1 N of NaOH, water, and running 11621

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buffer for 3 min, respectively. All conditioning and rinsing steps above were performed under pressure of 970 mbar to eliminate the possibility of carryover effect. Samples were hydrodynamically injected at 30 mbar for 5 s, followed with an injection of running buffer at 50 mbar for 5 s. The temperature of the cartridge was set at 23 °C (ambient temperature), and the applied voltage for the separation was 30 kV. The hyphenation of CE with ICPMS (Agilent 7700x, Agilent Technology, Santa Clara, CA, USA) was realized by directly connecting the outlet of the capillary to a Mira Mist CE nebulizer (Burgener Research Inc. Mississauga, Ontario, Canada). A solution containing 1% HNO3 (v/v) and 10% methanol was used as the makeup solution, and it was introduced to the nebulizer as sheath liquid through a platinum tubular electrode. An insulated copper wire with a crocodile clip head was used to connect the cathode in CE to the platinum electrode. The pH value of running buffer was adjusted by adding 1 N of NaOH or 0.1 N of HCl solutions. ICPMS was operated under no gas mode, and the mass isotopes of 197Au, 195Pt, and 105Pd were monitored for metallic nanoparticles and 112Cd for QDs. The peristaltic pump speed was set at 0.06 rpm. The details of the operation conditions are listed in Table 1. The pH value of the buffer solution was measured with an Orion Star A214 pH meter (Thermo Scientific. Waltham, MA, USA).

Scheme 1. Illustration of the Separation of a Gold Nanoparticle Mixture Using Capillary Electrophoresis

where q is the ionic charge of the particle, η is the solution viscosity, and r is the particle radius.21 Because the net negative charge of the particle, which is derived from the ionized surfactants, depends on the number of surfactant molecules absorbed on the particle surface, the charge of the particle can be considered to be proportional to the surface area of the analyzed particles:25 q ∝ 4πr 2

Then, the relationship between μem and particle size can be expressed by

Table 1. CE/ICPMS Operating Parameters μem ∝

CE Parameters capillary running electrolyte make up solution voltage temperature sample injection

polymer coated fused silica capillary, I.D. 50 μm, O.D. 360 μm, length 67 cm TRIS 10 mM, SDBS 70 mM, pH = 9.0

4πr 2 2 = r 6πηr 3η

(3)

Since η is considered to be a constant, the μem of a particle is then directly proportional to its size. The relationship suggests that negatively charged particles with larger sizes travel at a faster rate toward the anode. Previous studies have also suggested that there is a proportional relationship between μem and particle size.38 Using this theory, it is reasonable to expect that particles with different radii would exhibit different μem leading to separation effects between each size fraction. Since the EOF flows at a much greater magnitude and is in the opposite direction of the μem under basic conditions, all nanoparticles are flushed toward the cathode at different rates depending on their sizes. All of the samples eluted at the cathode were delivered directly to the ICPMS for elemental analysis. In order to optimize the separation efficiency and resolution of the methodology, multiple experimental parameters were evaluated. First, three anionic surfactants (SDS, SDBS, and S10) were tested as potential components of the running buffer at identical concentrations (70 mM). It was determined that the highest resolution was obtained with buffer solutions containing SDBS (Figure 1a). Although previous studies have suggested that adding SDS to the running buffer enhances the separation of gold nanoparticles,25,29 when the size difference of the adjacent nanoparticle fraction was less than 10 nm, no baseline separation was achieved in any buffers containing SDS. When S10 was used, a slight improvement in nanoparticle separation was observed, accompanied by a greater degree of peak broadening as compared to the use of SDBS, especially for 5 nm gold nanoparticles. A hypothesis to explain the variation in separation efficiency is that the interaction between the surfactants and the surface of the gold particles is a contributing factor. Among the three surfactants under investigation, there are differences in the hydrophobicity of the molecules. For example, SDS has a longer carbon chain than S10, and compared to SDS, SDBS contains an additional phenyl group

1% HNO3 and 10% methanol in water 30 kV 23 °C hydrodynamic, 30 mbar, 5 s ICPMS Parameters

RF power sample depth plasma gas nebulizer gas flow dwell time monitored isotope nebulizer

(2)

1500 W 8.0 mm 15.0 L min−1 0.90 L min−1 mobility marker (1 s), analyte (3 s) 112 Cd+, 197Au+, 195Pt+, 105Pd+ Mira Mist CE

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RESULTS AND DISCUSSION Optimization of the Separation Conditions. As shown in Scheme 1, the sample suspension containing a mixture of gold nanoparticles with variable sizes was injected into the capillary at the anode side and a voltage was then applied. The capillary was prefilled with electrolyte containing a high concentration of surfactants. It has been reported that anionic surfactant molecules, such as SDS, are able to self-assemble on a gold surface through hydrophobic interaction and form a monolayer.35,36 Under basic conditions, the surfactant molecules are deprotonated and the particle carries a net negative charge.37 The electrophoretic mobility (μem) of an analyte can be expressed by q μem = 6πηr (1) 11622

dx.doi.org/10.1021/ac5025655 | Anal. Chem. 2014, 86, 11620−11627

Analytical Chemistry

Article

nanoparticle becomes very small (i.e.,