Preparation of Narrow Dispersity Gold Nanorods by Asymmetrical

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Preparation of Narrow Dispersity Gold Nanorods by Asymmetrical Flow Field-Flow Fractionation and Investigation of Surface Plasmon Resonance J. Ray Runyon,† Adam Goering,† Ken-Tye Yong,‡ and S. Kim Ratanathanawongs Williams*,† †

Laboratory for Advanced Separations Technologies, Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, Colorado 80401, United States ‡ School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798, Singapore ABSTRACT: The development of an asymmetrical field-flow fractionation (AsFlFFF) method for separating gold nanorods (GNR) is reported. Collected fractions containing GNR subpopulations with aspect ratios, sizes, and shapes which are more narrowly dispersed than the original population were further characterized by UV−vis spectroscopy and transmission electron microscopy. This ability to obtain different sizes and shapes of nanoparticles enabled the evaluation of a new approach to estimating the retention time and hydrodynamic size of nanorods and the investigation of GNR optical properties at a previously unattainable level of detail. Experimental results demonstrate that the longitudinal surface plasmon absorption maximum of GNRs is correlated with the effective particle radius in addition to the aspect ratio. This may account for some of the variabilities reported in published empirical data from different research groups and supports reports of simulated absorption spectra of GNRs of different physical dimensions. The use of AsFlFFF with dual UV−vis detection to rapidly assess relative changes in GNR subpopulations was demonstrated for irregularly shaped gold nanoparticles formed at different synthesis temperatures.

G

which occupies the same volume as that of the nonspherical particle) of the GNR increased, but the aspect ratio remained constant.18,19 Brioude used computer simulations to demonstrate that the slope of the plot of SPl versus GNR length would decrease as the diameter of the rod increased. The implication of these simulation results is that a single linear regression between SPl and the aspect ratio does not completely capture all contributions to the position of SPl, especially for particles of different diameters but similar aspect ratios.20 Experimentally delving into this question requires the availability of GNRs that are uniform in aspect ratio but have different effective sizes. Commonly utilized solution-based synthesis approaches yield GNRs which vary in size by 10−25% and often contain subpopulations of particles of different shapes and sizes.2,5,6,8,21−31 Variations in reaction parameters such as time, temperature, type of surfactants, and reactant ratios can affect the size and shape distributions.21,23 The synthesis challenge is to produce high yield, high quality, narrowly disperse GNRs in both shape and size with desired dimensions and aspect ratios. Alternatively, more monodisperse gold nanoparticle samples may be obtained using separation methods. Size exclusion chromatography (SEC),32,33 capillary electrophoresis (CE),34 gel electrophoresis (GE),23,31 diafiltration,35 and centrifuga-

old nanorods (GNR) have two distinct surface plasmon resonance absorption bands, a longitudinal band (SPl) and a transverse band (SPt), corresponding to electron motion along the long axis and the short axis of the particle, respectively.1 The absorption maximum for SPl can be tuned across the visible to near-infrared spectrum by varying the aspect ratio (length/diameter), surface coating, and refractive index of the GNR.2−9 Their tunability, bioinertness, facile synthesis, and ease of surface modification make GNRs attractive candidates for bioimaging, biosensing, self-assembly, and molecular characterization applications.2,10−16 Monodisperse GNRs with respect to size and shape are needed to attain the desired optical properties for these applications and for fundamental investigations of the individual contribution of size and shape to SPl. Linear relationships between the GNR aspect ratio and the SPl have been experimentally determined,2,5,6 and theoretical studies have been conducted to explain this observed absorption behavior.17−21 Link et al. applied Gans theory to model the absorption spectra of GNRs (approximated as elongated ellipsoids) of different aspect ratios and demonstrated that SPl was linearly dependent on the GNR aspect ratio and that the dielectric constant of the surrounding medium at the surface of the particle also played a critical role.17 They also raised the question as to whether the SPl would shift for different sized particles of the same aspect ratio. Jain and Prescott used discrete dipole approximations to predict a red shift in SPl when the effective radius (reff) (radius of a sphere © XXXX American Chemical Society

Received: September 5, 2012 Accepted: December 10, 2012

A

dx.doi.org/10.1021/ac302571g | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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tion36,37 have been used to separate gold or silver nanoparticles by size, shape, charge, and/or density. The SEC of 5.3 to 19.3 nm GNRs and gold nanospheres yielded enrichment of different shapes at different retention times, but a mixed surfactant mobile phase was needed to mitigate complete adsorptive sample loss.33 Gold nanocrystals from 1 to 3 nm in diameter and which differ in size by as little as 0.6 nm were resolved by recycling-SEC. However, the analysis times were long, sample dilution and adsorptive loss were a concern, and the technique was not demonstrated for larger nanoparticles or nonspherical shapes.32 Enrichment of gold nanospheres and GNRs from a mixture was achieved via centrifugation, but the potential for remixing is of concern because the separation occurs in a single tube and the centrifuge is stopped during sample collection.37 The isolation of different aspect ratio nanorods may prove difficult. Silver nanoparticles of different shapes were separated via GE, and the bands were analyzed by UV spectroscopy; however, sample preparation and recovery for further applications are tedious.31 There is a clear need for the development of separation methods for nanomaterials which can overcome these limitations. Field-flow fractionation (FFF) is a family of separation techniques that has been successfully used to separate and quantitatively recover nanosize analytes such as proteins,38−40 polymers,41,42 carbon nanotubes,43−45 and nanoparticles.46−53 These studies demonstrated FFF’s ability to separate different sized nanomaterials and the ease with which more monodisperse fractions can be collected for further studies. Moreover, when FFF is coupled with other detectors and orthogonal sizing techniques, the absolute number concentration of analytes such as gold nanoclusters can be quantified.51 This work employs the asymmetrical flow FFF (AsFlFFF) technique to fractionate GNRs into more discrete subpopulations. Our focus on nanorods distinguishes this work from others that have dealt predominantly with spherical nanoparticles. AsFlFFF utilizes two perpendicular fluid flows to effect sample retention and separation.54 An AsFlFFF fractionation is performed in an open ribbonlike channel devoid of packing material in which one wall is impermeable and the other is comprised of an ultrafiltration membrane supported by a microporous frit. This hardware design provides AsFlFFF unique advantages for nanoparticle separations compared to those of the previously mentioned techniques. AsFlFFF can accommodate a wide size range of particles (2 nm to 50 μm) and a high particle number load (∼106 to 108 particles per injection). Sample loss due to irreversible adsorption is minimized, and undesirable components and contaminates can be eliminated through the ultrafiltration membrane during the fractionation. Very importantly, AsFlFFF is an elution-based separation method, and thus fractions are easily collected for further off-line studies. The AFlFFF separation is based on differences in analyte diffusion coefficient (D), which in turn, can be related to the hydrodynamic radius (rh) of a spherical particle through the Stokes−Einstein equation (D = kT/6πηrh, where k is Boltzmann’s constant, T is temperature in Kelvin, and η is the viscosity of the suspending liquid). In eq 1, the retention time (tr) of the analyte is related to D (and subsequently rh) through experimental parameters such as the channel thickness (w), the cross-flow rate (Vċ ), and the flow rate exiting through the channel outlet (V̇ ).54

tr =

V̇ ⎞ w 2πηrh ⎛ V̇ ⎞ w2 ⎛ ln⎜1 + c ⎟ = ln⎜1 + c ⎟ 6D ⎝ V̇ ⎠ kT V̇ ⎠ ⎝

(1)

The right-hand side of eq 1 invokes the Stokes−Einstein equation and is commonly employed in AsFlFFF calculations of rh of a spherical (equivalent) particle from the measured tr. However, the diffusion of rod-shaped particles in solution is more complex and is affected by the particles' length and aspect ratio.37,44,45,55 Aragon and Flamik used the boundary element method to compute numerical solutions for the hydrodynamic transport properties of cylinders with axial ratios between 1 and 100.55 The solution for cylinders with rectangular ends is shown in eqs 2 and 3, which relate the translational diffusion (Dτ) of the particle to its length (L) and aspect ratio (p). Dτ =

kT [ln(p) + Xτ (p)] 3πηL

Xτ (p) = 0.374304 − +

(2)

1.11097 1.71453 0.149474 + − p p p2

0.4914531ln(p) 0.091666ln(p) − p p2

(3)

In practice, Dτ can be calculated from TEM measurements of GNR length and width. This Dτ value can then be used in place of D in eq 1 either to predict retention time or to calculate the rh for a cylinder-shaped particle that elutes at the retention time at peak maximum. This work seeks to assess the importance of using a shapespecific diffusion treatment for AsFlFFF of nanorods and to demonstrate the importance of this technique in the study and characterization of nanomaterials. AsFlFFF is an ideal technique to facilitate the investigation of the aspect ratio and reff on GNR optical properties because fractions of different hydrodynamic (or effective) sizes and aspect ratios can be separated and readily collected for further analyses. The specific goals of this study are to develop AsFlFFF for the separation of GNRs, evaluate the use of the Aragon−Flamik diffusion treatment for predicting AsFlFFF retention times and sizes of nanorods, experimentally determine the relative contribution of the GNR aspect ratio and effective radius to the position of the surface plasmon absorption maximum, and demonstrate the ease with which AsFlFFF can be used to monitor relative changes in GNR size and shape subpopulations, as a function of synthesis temperature.

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EXPERIMENTAL SECTION Five different GNR samples (labeled as G1−G5) were used for the AsFlFFF studies. G1, G2, and G3 were synthesized at 296 K, 301 K, and 363 K, respectively. G4 is the supernatant of GNRs synthesized according to the procedure described in reference 4. Sample G5 is a 1:1 mixture (v/v) of G1 and G4. GNR Synthesis. GNR samples were synthesized following a seed-mediated method.2,5,10 Gold(III) chloride trihydrate (HAuCl4·3H2O), sodium borohydride (NaBH4), silver nitrate (AgNO3), and L-ascorbic acid were purchased from Sigma Aldrich and used as received. Cetyltrimethylammonium bromide (CTAB) and deionized ultrafiltrated water (DIUF) were purchased from Fluka and used as received. Aqueous solutions of HAuCl4, CTAB, AgNO3, NaBH4, and L-ascorbic acid were prepared in DIUF water. The seed-mediated GNR synthesis procedure involved introducing a seed solution of B



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TEM was performed on a Philips CM200 with a LaB6 filament and at an accelerating voltage of 200 kV. Images were collected using Keen View Soft Imaging System with iTEM Universal TEM Imaging Platform Software. TEM samples were prepared by placing 2 μL of the sample onto a 400 mesh Formvar/carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA) followed by air drying. The lengths and diameters of at least 100 particles in each sample were measured. The standard deviations of the TEM measurements are consistent with previously published data.23 Safety Considerations. Caution should be practiced when working with gold(III) chloride trihydrate (HAuCl4·3H2O), sodium borohydride (NaBH4), silver nitrate (AgNO3), cetyltrimethylammonium bromide (CTAB), and sodium azide (NaN3), as all can pose serious health and/or environmental effects (refer to the chemical Material Safety Data Sheet).

gold nanoparticles into a growth solution of gold salt. The seed solution was prepared by mixing in the following order: 5.0 mL of 0.2 M CTAB, 5.0 mL of 0.96 mM HAuCl4, and 1.0 mL of 0.01 M iced NaBH4. A translucent brown solution was formed after 30 min of vigorous stirring. The growth solution was prepared by mixing in the following order: 5.0 mL of 0.2 M CTAB, 5.0 mL of DIUF water, 200 μL of 0.025 M HAuCl4, 100 μL of 0.08 M L-ascorbic acid, and 300 μL of 0.004 M AgNO3. The growth solution changed from orange to a clear and colorless solution upon adding the L-ascorbic acid solution. Twelve microliters of the seed solution was then added to the growth solution, and the mixture was allowed to sit undisturbed for a few hours. The GNRs were collected from solution by centrifugation. Subsequently, the GNR samples were redispersed in ∼1.0 mL of DIUF water. AsFlFFF Instrumentation and Conditions. The AsFlFFF instrument (constructed in house) was comprised of a Waters 590 (Milford, MA) pump to drive the carrier liquid through the channel, a Shimadzu LC-6A (Columbia, MD) pump for sample injection, an AsFlFFF channel, and a Waters 486 (Milford, MA) UV−vis detector to monitor sample elution. The experimental procedure consists of sample introduction, focusing, and separation steps. These various stages are implemented using a sample injection valve and 4-way switching valves to direct carrier fluid flow. The retention of GNRs was evaluated in different aqueous carrier liquids (e.g., deionized water, 20 mM and 100 mM sodium chloride, and 0.03% (w/v) CTAB), under different flow rates and sample injection and focusing times. The conditions that optimally balanced GNR retention time and resolution were carrier liquid composed of 0.03% (w/v) CTAB and 0.02% w/v NaN3, V̇ = 0.6 mL/min, Vċ = 0.7 mL/min, Vċ /V̇ was ∼1.1, sample injection volume = 20 μL, sample injection time = 30 s, and total focusing time = 2.0 min. The channel thickness was calculated to be 185 ± 6 μm, using the retention times of 22, 32, and 100 nm polystyrene latex bead standards. A 5 kDa molecular weight cutoff (MWCO) regenerated cellulose membrane provided permeability for the cross-flow while retaining the GNR sample in the AsFlFFF channel. Chrom and Spec 1.5 chromatography data collection software (Ampersand International, Inc., Beachwood, OH) was used to collect FFF UV−vis data. Fraction Collection and Preparation. Twenty microliters of a GNR suspension was injected into the AsFlFFF channel. Fractions collected from multiple injections were pooled and then reduced to ∼1.0 mL by liquid evaporation. Each concentrated fraction was centrifuged at 11600 rpm for 5 min using a Beckman Microfuge B microcentrifuge, followed by washing with 0.5 mL of DIUF water and additional centrifugation for 3 min. The final washed GNR fractions were redispersed in 0.25 mL of DIUF water. The two centrifugation−washing cycles were essential to obtaining fractions with sufficiently high GNR and low CTAB concentrations for TEM analysis. UV−vis and TEM Sample Preparation. UV−vis spectra were obtained using a Thermo Electron Corporation Nicolet Evolution 300 BB spectrophotometer (Madison, WI). UV−vis samples of unfractionated GNR samples were prepared by transferring 6−8 drops of sample into a quartz UV cell (1 cm path length) and then adding DIUF water. UV−vis spectra of collected GNR fractions were obtained after concentration by centrifugation. The UV−vis spectrum of CTAB showed no absorbance from 400 to 1100 nm.

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RESULTS AND DISCUSSION Careful selection of the carrier liquid, the semipermeable membrane, and the flow conditions are essential to the success of the AsFlFFF separation. CTAB surfactant was chosen as a carrier liquid modifier because it is used during GNR synthesis and provides a similar environment in the AsFlFFF channel. Furthermore, the formation of positively charged bilayers on the GNR surfaces helped prevent the formation of aggregates2,5,10,28,56 and minimized sample adsorption to the FFF channel walls. A CTAB concentration of 0.03% (w/v) in the carrier liquid (which is below the critical micelle concentration) yielded retention and separation of GNRs, as shown in Figure 1. By comparison, no GNR elution was observed when NaCl was used as a carrier liquid modifier and purple spots resulting from irreversible GNR adsorption on the

Figure 1. Comparison of GNR fractograms obtained when (a) 0.03% (w/v) CTAB was added (solid line) or not added (dashed line) into the carrier liquid and (b) when Vċ /V̇ was set at 1.1 (gray line) or 5.8 (black line), using a 0.03% (w/v) CTAB carrier liquid. C



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intervals across the elution peak. The five fractions indicated by colored areas (G1−5, G1−9, G1−13, G1−17, and G1−21) were further characterized by TEM, UV−vis spectroscopy, and reinjection into the AsFlFFF channel. The TEM images in Figure 2b show that the original sample consists mainly of rods with a broad distribution of sizes and aspect ratios while the fractionated samples were more narrowly distributed in these features. A useful parameter for comparing size distributions among fractions of nonspherical particles is reff, which is defined as the radius of a sphere having a volume equal to that of the nonspherical particle. The average reff of each fraction was determined by calculating the cylindrical volume each GNR occupied as measured from TEM images, and then translating this to a spherical volume. Table 1 summarizes TEM measurements of the GNR length and width, calculated aspect ratio, and reff for each as-synthesized sample and collected fraction. The original G1 sample had an reff distribution of 10%. Fractions G1-5, G1-13, and G1-21 had narrower distributions ranging from 3 to 5%, demonstrating the effectiveness of AsFlFFF to produce more uniform fractions. This data suggests that even narrower dispersity fractions can be prepared by collecting over shorter time intervals. Fractions collected at longer retention times generally contain larger reff particles, as expected. This trend was observed for all GNR samples and their fractions. The conclusion that each fraction of the G1 sample has a distinct size population was further confirmed through reinjection of each fraction into the AsFlFFF channel, as shown in Figure 2c. The absorbance for each fraction is normalized according to height to more clearly demonstrate their differences in retention time. The retention time at the peak maximum of each reinjected fraction matches the time at which the fraction was collected from the original GNR sample injection. Figure 3 compares the UV−vis spectra of unfractionated G1 with collected fractions. The SPl for unfractionated G1 has a peak maximum at 706 nm, which represents the average absorbance of all GNRs in the original suspension. The more

membrane surface were observed near the AsFlFFF sample inlet. Increased GNR retention times were observed at a higher cross-flow rate to channel flow-rate ratios, Vċ /V̇ , as described in eq 1 and shown in Figure 1b. The use of CTAB in the carrier liquid had the added benefit of increasing the lifetime of the regenerated cellulose membrane to 50 sample injections without any signs of compromised system performance. An AsFlFFF separation of GNRs is demonstrated by the fractogram and TEMs of sample G1 and collected fractions shown in Figure 2. Fractions were collected at one minute

Figure 2. (a) AsFlFFF fractogram of GNR sample G1; conditions: Vċ /V̇ = 5.8, to = 3.0 min, λ = 530 nm. (b) TEM images of unfractionated G1 and selected fractions. (c) Fractograms of fractions reinjected into AsFlFFF.

Table 1. GNR Dimensions and SPl sample

SPl (nm)

shape

G1a G1-5 G1-13 G1-21 G1′-1b G1′-2b G1′-3b G2a G3a G3-1 G3-2 G4a

706 757 692 673 740 700 682 740 674 677 679 804

rod rod rod rod rod rod rod rod rod rod rod sphere short rod long rod rod rod/sphere rod rod

G5-1 G5-2 G5-3 G5-4

713 762 796 1160

length (nm) 47.1 45.0 55.9 62.3 46.4 57.7 64.7 55.7 42.3 40.8 44.8 56.9 123.8 329.5 45.7 86.0 120.0 356.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.7 1.7 1.4 2.8 1.4 1.4 2.1 2.0 1.3 1.1 2.0 1.0 4.4 12.1 1.5 5.8 3.0 11.9

width (nm) 18.6 15.9 24.5 34.1 16.2 21.4 25.4 18.7 17.1 16.5 20.1 56.9 41.5 28.5 20.3 32.7 41.4 28.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.2 0.9 1.2 1.8 0.8 0.8 1.2 1.0 0.7 0.5 1.1 1.0 0.9 0.7 0.7 1.6 0.9 0.5

aspect ratio 2.7 3.2 2.7 2.2 3.0 2.8 2.6 3.1 2.6 2.5 2.3 1.0 3.0 11.6 2.3 2.6 2.9 12.7

± ± ± ± ± ± ± ± ± ± ±

0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1

± ± ± ± ± ±

0.1 0.4 0.1 0.1 0.1 0.4

reff (nm)

AF rhc (nm)

SE rhd (nm)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

24.0 22.4 28.1 33.4 22.8 27.1 30.1 25.5 22.5 21.9 24.2 41.6 47.6 69.8 24.8 37.1 46.9 73.0

35.6 30.6 39.6 49.7 − − − 38.3 34.8 27.8 36.4 − − − 36.4 52.8 66.0 97.2

14.4 12.8 18.3 23.6 13.2 17.0 19.8 15.3 13.2 12.7 15.0 28.5 33.8 36.7 15.1 25.7 33.7 37.5

1.4 0.6 0.6 1.0 0.8 0.8 1.2 0.6 0.4 0.3 0.7 0.5 0.9 0.9 0.4 1.4 0.6 0.7

a

Unfractionated sample. bRepeat synthesis of G1 at 296 K. cCalculated from eqs 2 and 3, assuming a 6 nm CTAB bilayer thickness. dCalculated using eq 1. D



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Figure 3. Comparison of SPl of unfractionated G1 with collected fractions G1-5, G1-9, G1-13, G1-17, and G1-21. Aspect ratios are shown in parentheses. The absorbance below 600 nm has been omitted for clarity.

narrowly dispersed fractions yielded SPl positioned on either side of 706 nm. This again confirms that each collected fraction consists of GNRs with distinctively different physical dimensions. The aspect ratios noted in parentheses were determined from TEM measurements. As expected, the SPl increased with an increasing aspect ratio. SPl data for all original and fractionated GNR samples are also summarized in Table 1. Figure 4a shows a fractogram of sample G5 (a 1:1 by volume mixture of G2 and G4) and demonstrates the effectiveness of AsFlFFF to separate polydisperse multimodal GNRs. The fractogram shows a void peak (to) at ∼3 min that is attributed to an unretained sample matrix or pressure pulses from switching valves. The elution profile of the retained sample suggests the presence of four subpopulations. The peak observed at ∼13 min matches that obtained when only G2 is injected into the system (see Figure 5), while the three peaks at ∼17, 21, and 30 min match those observed for G4 (data not shown). The cross-hatched regions designate the time intervals at which fractions were collected, as they eluted from the AsFlFFF channel. A photograph, TEM image, and UV spectrum corresponding to each collected fraction are shown in Figures 4 (panels b−e). Since retention time is proportional to hydrodynamic size, the GNRs in the early eluting fraction are smaller than those collected in later eluting fractions. For nonspherical nanoparticles, the hydrodynamic size-based separation can also translate to a concurrent shape or length fractionation. Fractions G5-2, G5-3, and G5-4 consist of spheres, short rods, and long rods, respectively. Measurements from TEM micrographs confirm that G5-1, G5-3, and G5-4 fractions contain GNRs with increasing lengths, and that G5-1 and G5-3 have similar aspect ratios, albeit different lengths and widths. UV−vis data shows different SPl values for each fraction and the presence of a peak near 1100 nm for G5-4. Conversely, G5-2 shows a high intensity peak at 530 nm, indicating an enrichment of spherical particles within the fraction as observed in TEM images. The results from the TEM and SP l measurements for each G5 fraction are summarized in Table 1. The AsFlFFF fractionation of G5 resulted in enrichment of different species such as spheres or GNRs of different lengths at different retention times. However, TEM images show the presence of a small number of spherical (low aspect ratio) particles among predominantly rod-shaped particles. This may be due to the flow rate ratio that was used to perform the AsFlFFF separations. A constant Vċ /V̇ ratio of 1.1 was chosen so that the separation is completed within ∼35 min. A higher

Figure 4. (a) AsFlFFF fractogram of G5; Vċ /V̇ = 1.1, λ = 530 nm. The shaded regions labeled G5-1 to G5-4 were collected for further characterization. (b−e) Photographs, TEM images, and UV−vis spectra (solid traces) of fractions G5-1 to G5-4. The dashed UV−vis traces in (b−e) correspond to the unfractionated G5 sample and are included for comparison. The TEM scale bars for (b−d) are 200 nm and (e) is 500 nm.

Figure 5. AsFlFFF fractogram comparison between GNRs synthesized at 301 K (G2) and 363 K (G3), Vċ /V̇ = 1.1, λ = 530 nm.

Vċ /V̇ would yield a higher resolution separation but at the cost of increased analysis time. The AsFlFFF fractogram is a direct reflection of the hydrodynamic size and size distribution and can thus be used to monitor subtle changes in particle size and populations. To illustrate this point, a comparison between fractograms of GNRs synthesized at 301 K (G2) and at 363 K (G3) is shown E



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Figure 6. (a) AsFlFFF fractogram of GNRs synthesized at 363 K (G3); conditions: Vċ /V̇ = 1.1, λ = 530 nm. (b) TEM of G3. (c) UV−vis spectra of unfractionated (G3) and fractionated (G3-3). (d) TEM of G3-3.

in Figure 5. Higher reaction temperatures have been shown to produce shorter rods.57,58 As expected, shorter GNRs were produced at 363 K, as evidenced by the shorter retention time of G3 compared to that of G2. A decrease in size for G3 compared to that for G2 is also corroborated in the reff values shown in Table 1. Furthermore, the shift of G3 to shorter SPl compared to G2 points to smaller aspect ratio GNRs. The fractogram of GNRs synthesized at 363 K (G3) exhibits a shoulder at ∼15 min which suggests the presence of a subpopulation of larger nanoparticles. The fractogram shown in Figure 6a shows time intervals when fractions were collected. Large irregular-shaped particles are observed in the TEM image of the as-synthesized G3 sample shown in Figure 6b. UV−vis (Figure 6c) and TEM (Figure 6d) characterization of fraction G3-3 confirm that a subpopulation of larger, irregular-shaped particles with low aspect ratios predominate in the shoulder at 15 min. The results shown in Figures 2−6 demonstrate that AsFlFFF can indeed separate nanoparticles into fractions of different effective sizes and aspect ratios. These narrow dispersity fractions enable us to experimentally investigate the relative impact of GNR physical dimensions on the SPl absorption maximum. Figure 7 plots SPl versus the aspect ratio for GNR fractions collected from AsFlFFF of G1, G1′, and G3. G1′ is a repeat synthesis of G1 (296 K) with fractions collected across 4 min intervals instead of 1 min intervals. The higher aspect ratio samples exhibited SPl at longer wavelengths and are in agreement with published experimental and theoretical data. The minimum least-squares fit has a slope of 86 which falls within the previously reported range of 71 to 90.17−20 The bars extending from each data point in Figure 7 are derived from TEM measurements and represent the distribution of aspect ratios within each collected fraction. It is interesting to note

Figure 7. Plot of SPl vs aspect ratio for G1, G1′, and G3 fractions.

that despite the overlap in aspect ratios, each collected fraction has a visibly different color and clearly exhibits different SPl. In other words, the GNRs are indeed separated and the subpopulation is distinctly different in each fraction. Figure 8 presents experimental evidence to support theoretical predictions that the reff of the GNR has an effect on the position of SPl. Figure 8a is a plot of the SPl versus reff for fractions G5-1, G5-2, and G5-3 and clearly shows that increasing the size of the nanoparticles results in a red shift of SPl. Applying a linear fit to the data of Figure 8a yielded a slope of 4.5, which agrees with the slope of 4.2 obtained by approximating a linear fit to Jain’s data.18 The smaller value of the slope in Figure 8a compared to that observed in Figure 7 for aspect ratio (4.5 vs 86) indicates that reff has a smaller, albeit significant, effect. If the aspect ratio and reff were the only contributors to the SPl position, the variability in the slope of the SPl versus aspect ratio plot for unfractionated GNRs would be 86 ± 5 or 81−91. This accounts for variability in the upper end of the published range of 71−90.17−20 Figure 8b shows a plot of SPl versus reff for G1 and G1′ fractions. At first glance, F



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Table 2. Comparison of Experimental (AsFlFFF) and Calculated (Aragon and Flamik, AF) Retention Times for GNR Fractions with Different Nanorod Lengths and Aspect Ratios

a

sample

AsFlFFF tra (min)

AF tr (min)

G1-5 G1-13 G1-21 G3-1 G3-2 G5-1 G5-2 G5-3 G5-4

21.3 29.7 37.4 10.0 12.5 12.6 17.2 20.8 30.0

19.2 23.4 27.3 8.8 9.5 9.6 13.2 16.0 23.5

Measured at midpoint of fraction collection.

trs values and suggest that the Aragon and Flamik approach holds promise for correlating tr with nanorod dimensions. The AsFlFFF separation, when combined with dual detection UV−vis, can give an estimate of the relative amounts of different shape populations present in a GNR sample. Since spherical gold nanoparticle absorption and transverse surface plasmon resonance both occur at 530 nm, all gold nanoparticles (irrespective of shape) are detected. However, a UV−vis detector setting at 720 nm will selectively register the elution of gold nanorods. A comparison of the absorbance of eluting gold nanoparticles at these two wavelengths can give insight to the particle shape. The 530 and 720 nm fractograms of the G5 sample are superimposed in Figure 9a. At ∼19 min, the 530 nm fractogram shows a distinct peak that is not present in the 720 nm fractogram. This peak is attributed to the elution of spherical-like nanoparticles. The 720 nm fractogram shows two peaks at ∼12 and 21 min, which suggests the elution of nanorods at these retention times. When the areas under the peak were calculated for the intervals marked in Figure 9a, the

Figure 8. Plot of SPl vs effective radius (reff) for (a) G5 GNR fractions and (b) G1 and G1′ fractions. The aspect ratios are shown in parentheses.

the negative slope appears to disagree with that observed in Figure 8a. However, on closer inspection, this negative slope follows theoretical predictions and further supports the data presented in this study. The TEM measurements confirm that larger fraction numbers (i.e., longer retention times) correspond to larger reff. More importantly, the TEM measurements show that the increase in GNR size is accompanied by a decrease in aspect ratio. Since a shift to shorter wavelengths for the SPl is expected with a decreasing aspect ratio, and the aspect ratio has a larger impact than reff, it follows that the slope in Figure 8b has a negative value. These findings help explain reported differences between experimental and theoretical values of SPl. In addition to separating nanoparticles, AsFlFFF can also provide information about their size. The right-hand side of eq 1 is commonly used to calculate the average hydrodynamic radii of spherical nanoparticles at the retention time at peak maximum. This treatment is based on the Stokes−Einstein equation for diffusion of a sphere and yields the SE rh values listed in Table 1. The SE rh values are 2−2.5 times larger than the TEM-derived reff values. This is not surprising since the TEM measurements are for “dry” particles. An alternate treatment employs Aragon and Flamik’s translational diffusion of a cylinder Dτ in place of D in eq 1 and yields the AF rhs that are also listed in Table 1. The calculated Dτs incorporated an ∼6 nm CTAB bilayer shell around the particle, which is more representative of GNRs in solution.2 The AF rh values are 0.67−0.77 times those of the SE rh values. These substantial differences in rh demonstrate the importance of utilizing a diffusion treatment that is more appropriate for rods and which incorporates the rod lengths and aspect ratios. The availability of narrow dispersity GNR fractions enabled us to further experimentally assess Aragon and Flamik’s diffusion treatment for cylinders and to compare calculated and experimental retention times for nanorods of known lengths and aspect ratios. Table 2 summarizes the calculated AF tr and the retention times at which fractions were collected. The AF tr values are 0.73−0.90 times those of experimental AsFlFFF

Figure 9. Dual UV−vis detection of (a) G5 at 530 nm (solid trace) and 720 nm (dashed trace) and (b) G3 at 675 nm (solid trace) and 600 nm (dashed trace). Vċ /V̇ = 1.1. The vertical dashed lines indicate the time intervals used in the peak area calculations. G



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relative amounts of spherical nanoparticles, short rods (AR 2.2 to 3.3), and long rods (AR 12.3 to 13.1) are 31%, 64%, and 5%, respectively. The fractograms shown in Figures 4a and 9a are both for G5 but vary slightly in their appearance and retentions times. These variations result from differences in void volumes that arise when the membrane is replaced and the channel is disassembled and reassembled. Also, small variations in membrane performance, even within the same batch of membranes, have been observed in flow FFF.59 Dual-wavelength detection was also utilized to characterize the relative amounts of rods and irregular-shaped particles in sample G3 as shown in Figure 9b. UV−vis of unfractionated G3 showed two peak maxima at ∼600 nm and at 674 nm. The fractogram obtained at 675 nm is representative of the rods present in the sample, while the 600 nm trace represents the large, irregular-shaped components of the sample (see UV and TEM data in Figure 5, panels c−d). The combination of the UV traces taken at 600 and 675 nm were used to guide the selection of peak areas from which to calculate the relative amount of each subpopulation. Sample G3 was found to consist of 75% rods and 25% irregular-shaped particles. This is in good agreement with the 78% and 22% determined from TEM images (206 particles measured). The advantage to using AsFlFFF with dual-wavelength UV− vis detection to estimate the relative amounts of subpopulations in a GNR sample is that between 106 and 108 particles are characterized compared to between 102 and 103 by TEM. It should also be noted that if an array of wavelengths was monitored, nanorods with different aspect ratios eluting from the AsFlFFF channel will cause a change in peak intensity at the wavelength that corresponds to their maximum absorbance. This would allow the determination of relative amounts of GNRs with various aspect ratios. Unfortunately, we did not have access to a photodiode detector to illustrate this point. Finally, the GNR sample can be directly injected into the AsFlFFF channel, thereby eliminating the multiple washing steps needed to remove excess organic reactants that can adversely affect the quality of the TEM image.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 303-273-3245. Fax: 303-2733629. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the NSF (CHE-1013029) for funding and Professor Stephen Boyes from CSM for guidance in the synthesis of GNRs.

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REFERENCES

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CONCLUSION Asymmetrical flow FFF was successfully used to generate more uniform size (and effectively shape) fractions of gold nanorods. The availability of these narrow dispersity GNR samples of known lengths and widths facilitated studies pertaining to both FFF and surface plasmon resonance absorption of GNRs. More specifically, the Aragon and Flamik diffusion equation for cylinders was shown to provide a good estimate of hydrodynamic sizes and retention times of nanorods of different lengths and aspect ratios. The effects of GNR aspect ratio and reff were decoupled and revealed not only that the position of the SPl is dependent mainly on the aspect ratio but also that reff has a significant role. These findings provide experimental confirmation of recently published theories, suggesting the importance of reff. In addition, we provide an explanation for some of the variabilities in the SPl−size relationships that have been reported in the literature. Finally, we demonstrated the use of the developed AsFlFFF method for rapid analysis of changes in GNR subpopulations that result from variations in experimental conditions. Quantitative estimations of these subpopulations in terms of relative amounts are possible using dual-wavelength UV−vis detection. H



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Preparation of Narrow Dispersity Gold Nanorods by Asymmetrical

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