Synthesis and Rapid Characterization of Amine-Functionalized Silica

Mar 20, 2012 - Sculpting the internal architecture of fluorescent silica particles via a template-free approach. Cornelia Rosu , Andrew J. Gorman , Ra...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Langmuir

Synthesis and Rapid Characterization of Amine-Functionalized Silica Erick Soto-Cantu, Rafael Cueto, Jerome Koch, and Paul S. Russo* Department of Chemistry and Macromolecular Studies Group, Louisiana State University, Baton Rouge, Louisiana 70803, United States S Supporting Information *

ABSTRACT: Amine-functionalized colloidal silica finds use in a variety of applications and fundamental investigations. To explore convenient methods of synthesis and characterization of research-grade materials in relatively large quantities, nearly monodisperse colloidal silica particles were prepared by base-catalyzed hydrolysis of reagent-grade tetraethyl orthosilicate (TEOS) without the traditional time- and energy-consuming distillation step. Radius was varied reliably from 30 to 125 nm by changing the water/TEOS ratio. Asymmetric flow field flow fractionation (AF4) methods with online light scattering detection proved effective in assessing the uniformity of the various preparations. Even highly uniform commercial standards were resolved by AF4. The surface of the colloidal silica was decorated with amino groups using (3-aminopropyl) trimethoxysilane and spacer methyl groups from methyl-trimethoxysilane. The surface density of amino groups was quantified spectrophotometrically after reaction with ninhydrin; the nature of this analysis avoids interference from sample turbidity. As an alternative to the ninhydrin test, an empirical relationship between surface density of amino groups and zeta potential at low pH was found. The size of the colloidal silica was predictably decreased by etching with HF; this method will be effective for some preparations, despite a modest reduction in size uniformity.

■

INTRODUCTION Silica has several advantages over other inorganic materials for the synthesis of functionalized colloids. The difference of refractive index between silica and nonpolar liquids is small, which minimizes multiple scattering. The chemistry of silica and silicon compounds is well known. The 1968 work by Stöber et al.1 explored the formation of colloidal silica by the hydrolysis of alkyl silicates using linear alcohols as solvents and ammonia as catalyst in the presence or absence of water. A readily available and widely used alkyl silicate is tetraethyl orthosilicate (TEOS). The effects of the concentration of components,2,3 temperature,4 solvent,5 addition of surfactants,6,7 addition of electrolytes,8 use of seeds,9 etc. on the characteristics of the silica resulting from TEOS hydrolysis have been examined. Indeed, most of these factors were explored in the original Stöber work, but the mechanistic details have received much subsequent attention.10,11 In general, an increase in either the water or ammonia concentration results in larger particles. An increase in temperature has the opposite effect. Alcohols of higher molecular weight yield larger particles but with an increase in size polydispersity. In most studies, colloidal silica is obtained by the direct addition of TEOS to an ammonia/alcohol solution. The original Stöber method tends to produce spherical colloidal silica of reasonably low polydispersity. To achieve these narrow distributions, it has been a virtual requirement that the TEOS used in the process be of high purity. Either reagent grade material was distilled prior to use, or chemicals of even higher purity were selected. Distillation adds a slow and energy-intensive step to a process that is otherwise fast and economical, but purchasing lab-scale quantities of TEOS with a degree of purity higher than reagent grade raises the price by a factor of 2−20. In 2003, © 2012 American Chemical Society

Zhang et al. reported the synthesis of colloidal silica of low polydispersity using undistilled, reagent grade TEOS.12 Their method is a modification of Stöber’s. Like Bogush et al.2 before them, one of the key differences is that the TEOS is predissolved in ethanol. The authors claim that dilution of the TEOS suppresses the formation of new nuclei, rendering the distillation unnecessary for the production of monodisperse silica spheres. Colloidal silica synthesized using this very simple method seems to have a polydispersity comparable to certified silica standards. Coating the silica particles with amino groups leads to a large number of new applications, such as gene delivery,13 plasmid DNA transport,14 capture and release of bacteriophage viruses,15 and synthesis of silica-polypeptide composite particles.16,17 In this last application, the amino groups on the colloidal silica acted as initiators for the polymerization of N-carboxyanhydrides (NCAs). The surface functionalization was accomplished using (3-aminopropyl) trimethoxysilane (APS). Partial passivation of the surface with a nonreactive spacer such as methyl-trimethoxysilane (MTMS) seemed to reduce steric crowding during polymerization of the monomer.18,19 The concept of using a mixture of passive and active moieties was previously explored on flat substrates20as well as on colloidal particles.21,22 Quantitation of the surface concentration of amino groups is crucial in many applications, especially when the particles are used as initiators. For example, in the polymerization of NCAs the monomer-to-initiator ratio can be used to predict the Received: December 17, 2011 Revised: February 8, 2012 Published: March 20, 2012 5562

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

purification system. Anhydrous tetrahydrofuran (THF) and anhydrous hexanes were obtained from a Pure-Solv system. The following chemicals were purchased from Sigma-Aldrich and used without further purification, unless otherwise specified: APS (97%), MTMS (98%). Standard certified colloidal silica was obtained from Duke Scientific (catalog no. 8050, lot no. 30156, claimed mean diameter 0.49 μm ± 0.01 μm). Characterization Methods. Batch-mode dynamic light scattering (DLS) measurements were conducted in a custom-built apparatus using a He−Ne laser as the light source (632.8 nm) and ALV-5000 digital autocorrelator. Measurements were made in homodyne mode34 at five scattering angles from 30° to 90° in 15° increments. The apparent diffusion coefficient is defined as Dapp = Γ/q2 where Γ is the decay rate of the electric field autocorrelation function, g(1)(t), and q is the scattering vector magnitude, q = 4πn sin(θ/2)/λ0, where n is the solvent refractive index, θ is the scattering angle and λo is the wavelength of the incident light. Dapp becomes D0 in the limit of zero concentration and zero scattering angle; only then can it be used to compute the true hydrodynamic radius (Rh) by applying the Stokes− Einstein equation, Rh = kT/6πη0D0 where k is Boltzmann’s constant, T is the absolute temperature and η0 is the viscosity of the solvent. In the present experiments, concentrations were sufficiently low that the c = 0 extrapolation could be ignored. Multiangle DLS measurements were performed in order to determine whether the q = 0 extrapolation could be similarly ignored so that single-angle measurements would suffice. Once this was confirmed, a Malvern Zetasizer Nano-ZS was used for rapid measurements. This instrument performs backscatter DLS measurements at θ = 173° external to the measurement cell (internal angle 174.7°) using a 4 mW He−Ne laser of λ0 = 632.8 nm. The hydrodynamic radii obtained from multiangle measurements and from the Malvern Zetasizer Nano-ZS were the same within uncertainty. The Malvern Zetasizer Nano-ZS was also used for zeta potential measurements but the scattering angle for zeta potential measurements is θ = 17°. Clear polycarbonate disposable capillary zeta potential cells from Malvern were chosen. Silica particles were dispersed in 0.1 M HCl and 1 mM NaNO3 at concentrations ∼0.1% (w/v). Asymmetric Flow Field Flow Fractionation (AF4) experiments were performed using an Eclipse 2 (Wyatt Technology Corp., Santa Barbara, CA, henceforth referred to as Wyatt). An Agilent 1100 HPLC system (isocratic pump, autosampler, and degasser, Agilent Technologies, Palo Alto, CA) was used to inject the samples and deliver the mobile phase (water with 200 ppm sodium azide). The AF4 channel was assembled with a 490 μm-thick Mylar spacer. The membrane used was made of regenerated cellulose with a 10 kDa molecular weight cutoff (Wyatt). AF4 parameters for all but the Duke certified silica standard: channel length, 24 cm; channel width at inlet, 2.15 cm; channel width at outlet, 0.6 cm; channel flow, 1 mL/min; focus flow, 1.5 mL/min, 2 min. All samples were injected at an injection flow rate of 0.2 mL/min with a focus flow rate of 3 mL/min for 3 min, and then focus at 3 mL/min for 5 min. The channel flow rate during separation was always 1.0 mL/min. A three-stage cross-flow sequence was used for particles prepared for this work: 1) 0.2 mL/min, hold constant for 30 min; 2) ramp 10 min to 0.01 mL/min; 3) 0.0 mL/min, hold constant for 20 min. For the Duke certified silica standard particles, which are larger, the cross-flow sequence was: 1) 0.2 mL/min, constant for 40 min; 2) ramp 15 min to 0.01 mL/min; 3) 0.0 mL/min for 20 min. The detector set (all from Wyatt) included: Optilab rEX Differential Refractive Index (DRI) detector operating at 658 nm; Heleos Multi Angle Light Scattering (MALS); QELS (single-angle quasielastic light scatteringi.e., DLS at a single angle, 100.29 degrees adjusted for refractive index). The source for scattering measurements was a GaAs 50 mW laser operating at λo = 658 nm. Calibration of the MALS detector to obtain Rayleigh factors was done using filtered toluene. Normalization of the individual detectors was performed using an AF4 run of bovine serum albumin (BSA, Pierce Biotechnology). Under the conditions used, BSA elutes as several peaks, representing various aggregates. The peak for monomeric BSA was used to compute the normalization factors and also the interdetector delay volumes. The finite size of BSA is taken into consideration

molecular weight of the resulting polymer. Most methods for surface characterization, such as X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle, and atomic force microscopy (AFM) work best (or only) on flat surfaces. Extensive characterization of silane films has been demonstrated using the above-mentioned methods.23−26 Attempts at titration were not successful.16 Later Vadala et al. used a backtitration approach to quantify the concentration of amino groups on silica-coated cobalt particles but the lowest concentration measured was 2.4 mmol/g.27 Solid-phase synthesis of peptides requires methods to verify the presence of amino groups. Kaiser et al. reported a simple method for testing the completion of the reaction in the Merrifield solid-phase synthesis. Their method, with reported a sensitivity of 5 μmol/g,28 involves the reaction of ninhydrin with a terminal primary amine, which produces a bright purple complex. The colored product formed has the “sequestered” nitrogen atom of the amine that was tested. In other words, the nitrogen atom “immobilized” on the solid substrate, where its absorption characteristics could be altered, is extracted by the ninhydrin molecule and becomes part of the colored species in solution. Fluorescamine29 and 2,4,6-trinitrobenzenesulphonic acid30 have been used in qualitative approaches for the detection of completeness in solid-phase coupling reactions. Both methods are more sensitive than the ninhydrin test, but in both cases scattering from the particles can interfere with spectroscopic quantitation of the fluorescent or colored product attached to the solid surface. The present work at the lab scale is devoted to the eventual production of “large” quantities (made from more TEOS than one would typically care to distill in a lab setting) of uniformly sized particles with amino groups on the surface, as well as passivating alkyl groups. Such passivated/functionalized particles offer greater flexibility than fully passivated or fully functionalized materials. To characterize the particles, traditional sizing methods such as dynamic light scattering (DLS) and transmission electron microscopy (TEM) are joined by asymmetric flow field flow fractionation with online scattering detection (AF4/MALS).31−33 The lattermost method promises unprecedented ability to assess the size uniformity of samples by separating them prior to sizing them, while at the same time sampling many more particles than are typically seen in one or even several electron microscopy images; however, the method has not been vetted for this purpose until now. A particular goal of the research is to develop an easy and rapid method to determine the surface amino functionality. Thermogravimetric analysis (TGA) cannot accurately quantify the amino groups on the surface due to the preponderance of mass that is silica and the low molecular weight of the decomposable, amine-bearing moiety (58 g/mol). Instead, we explore the use of a ninhydrin-based spectroscopic method for the quantification of surface amino groups, whose surface density was varied by adjusting the molar ratio of amine-bearing APS and amine-lacking MTMS used in the functionalization reaction. A relationship between the measured surface density of amino groups and the zeta potential at low pH is investigated to determine whether amino group functionality can be measured by the simple and fast zeta potential method.

■

EXPERIMENTAL SECTION

Materials. TEOS reagent grade 98% was purchased from SigmaAldrich. Absolute ethanol 200 proof was purchased from PharmcoAAPER and used as received. Concentrated ammonia (29%) and, hydrofluoric acid (48%) in water were purchased from EMD Chemicals. Water was obtained from a Barnstead Nanopure 5563

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

cule35,36 is 4 molecules per nm2. An amount of APS/MTMS solution typically exceeding that calculated to functionalize a smooth surface with a single layer by 10−20% was added rapidly to the colloidal silica cores dispersion while stirring. The functionalization reaction was allowed to proceed at room temperature for ∼12 h. The resulting passivated/functionalized (P/F) colloidal silica particles were centrifuged at 3000 g for 45 min. The supernatant was tested for amines using ninhydrin. The P/F particles were redispersed in absolute ethanol. This washing procedure was repeated until the supernatant tested negative for amines (absence of blue coloration after ∼10 mg of ninhydrin were added and placed in a water bath at ∼65 °C for ∼30 min). Typically more than 5 washes were required to remove all the unreacted APS.

during the former calculation. Data acquisition and processing were performed using Astra V (5.3.4.13) software (Wyatt). Sizes were obtained by fitting the angular dependence of the scattered light intensities to a spherical form factor; details are supplied in Results and Discussion. For most transmission electron microscopy (TEM) measurements, approximately 2 μL of sample were placed on top of a 400mesh, carbon-coated, copper grid from Electron Microscopy Sciences and allowed to air-dry. The dry samples were then observed using a JEOL 100-CX TEM working at an accelerating voltage of 80 kV unless otherwise indicated. The resulting images were analyzed using ImageJ 1.38x from the National Institutes of Health, U.S.A. Between 80 and 170 particles were counted to obtain particle sizes. For the quantification of amino groups, a 0.35% (w/v) ninhydrin solution in absolute ethanol was freshly prepared. Standard hexylamine solutions were freshly prepared in order to build the calibration plot. The concentration range was 0.12 to 0.87 mM. Functionalized silica samples were dried at 120 °C for 4−6 h, and then 0.2 g of sample was placed in a capped vial along with 4 mL of absolute ethanol. The mixture was placed in a bath ultrasonicator for 30 min. Next, 1 mL of ninhydrin solution was added to the vial containing the sample and it was sonicated for 10 more minutes. The ninhydrin-sample dispersion was then placed in a water bath at 65 °C for 30 min. The samples were allowed to cool down for 10− 15 min. Once the samples were at room temperature they were centrifuged at 7600g for 30 min. Approximately 1 mL of supernatant was pipetted out and the absorbance at 588 nm was measured in an Agilent 8453 spectrophotometer. Measurements were repeated three times. Synthesis of Colloidal Silica Without Predilution of TEOS. Absolute ethanol (350 mL), concentrated ammonia (10−25 mL) and water (0−9 mL) were combined in a 500-mL round-bottom flask. They were vigorously stirred using a magnetic bar for 10−20 min in order to reach homogeneity. While maintaining stirring, a 25 mL aliquot of undistilled TEOS was added rapidly (∼3−5 s). The reaction was allowed to proceed at room temperature for 24 h. Then the colloidal dispersion was centrifuged at 7600g for 60 min. The silica was subsequently redispersed in absolute ethanol. This centrifugation-dispersion, or washing, procedure was repeated at least 5 times. Typical yield: 5 g solid silica (0.2 g of solid silica/1 g of TEOS). Synthesis of Colloidal Silica with Predilution of TEOS. The two-solution procedure of Zhang et al.,12 which uses diluted-but-notdistilled TEOS, was scaled up and modified slightly. Solution I was prepared by adding 230−260 mL of absolute ethanol and 20−50 mL of concentrated ammonia to a 500 mL round-bottom flask. This solution was agitated vigorously for 10−20 min using a magnetic stirplate. Solution II was prepared in a 50 mL beaker by dissolving 5 mL of TEOS in 20 mL of absolute ethanol. Solution II was loaded into a 50 mL polypropylene syringe and added rapidly (∼ 0.8 s) into solution I while vigorous stirring was maintained. The reaction was allowed to proceed at room temperature for 2 h. The resulting colloidal silica was centrifuged at 3000g for 30 min. The silica pellet was subsequently redispersed in absolute ethanol. This washing procedure was repeated at least 5 times. The seeded initiation procedure outlined by Zhang et al. was not used.12 Typical yield: 1.3 g solid silica (0.24 g of solid silica/1.0 g of TEOS). Functionalization/Passivation of Colloidal Silica with Amino Groups. The procedures of an earlier work19 were generally followed, but the amino surface group functionalization was explored over a wider range (xAPS = 0.082−1.00) compared to 0.10 and 0.25 previously). Dispersions of colloidal silica (∼10 wt %) in absolute ethanol were placed in 200 mL Erlenmeyer flasks. Solutions of APS and MTMS were freshly prepared in Eppendorf tubes. The solutions had predetermined compositions and the total volume of solution was typically 100 μL. The amounts of the two silanes (APS and MTMS) to be added were calculated considering the estimated total surface area of the silica cores (assuming spherical shape and using the radius from DLS measurements and taking density of colloidal silica16 as 1.96 g/cm3). It was also assumed that the parking area of a silane mole-

■

RESULTS AND DISCUSION Control of Particle Size by Growth. The size and size distribution of colloidal silica are influenced by a number of factors, including temperature, the nature and concentration of the components (alkoxy silane, alcohol, ammonia, and water), surfactants or electrolytes added, rate of mixing, and predilution of components. Temperature has a great effect.4 From the point of view of lab-scale chemistry it is convenient to vary initial concentrations of the reactants or catalyst in order to obtain the desired result. Water sensitively affects the final size of the colloidal silica obtained by the Stöber method, so the water/TEOS ratio was chosen as a convenient independent variable. The concentration of TEOS was held approximately constant at 0.35 M, and water was added to the ethanol/ammonia solution. In addition, the concentrated ammonia solution was a source of water itself. Figure 1

Figure 1. Hydrodynamic radius of colloidal silica prepared with predilution of TEOS as a function of water/TEOS ratio in the formulation.

shows a graph of hydrodynamic radius (Rh) vs water/TEOS w/w. The observed hydrodynamic radii data were fitted by a power law (eq 1) R h = P(H2O/TEOS)Q

(1)

with P = 110 ± 2.6 and Q = 1.73 ± 0.10 and a third-order polynomial (eq 2). R h = A(H2O/TEOS)3 + B(H2O/TEOS)2 + C(H2O/TEOS) + D

(2)

.with A = −237 ± 100, B = 555 ± 210, C = −250 ± 136, and D = 45 ± 27. The purpose of fitting the data is to obtain a reliable size predictor, not to explain the growth phenomenon. The polynomial provides a better fit, shown in Figure 1, which is the expected effect of more adjustable parameters. 5564

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

Bogush et al.2 explored the production of colloidal silica by hydrolysis of distilled TEOS in the ranges 0.1−0.5 M TEOS, 0.5−17.0 M H2O, and 0.5−3.0 NH3, all using ethanol as solvent. An empirical expression was developed Rh =

1/2 A [H2O]2 e−B[H2O] 2

Another difference from the traditional Stöber synthesis is the total concentration of TEOS. While Zhang et al. held it at 15.4 mg/mL, in the present study the TEOS concentration ranges from ∼55−60 mg/mL in order to increase the yield. The concentrations of the other components vary accordingly depending on the targeted size. Figure 2 shows images of

(3)

where A = [TEOS]1/2 (82 + 151[NH3] + 1200[NH3]2 − 366[NH3]3 )

(4)

and B = 1.05 + 0.523[NH3] − 0.128[NH3]2

(5)

where the reagent concentrations are given in mol/L. Equation 3 includes the correction proposed by Razink and Schlotter.37 Neither the expression published first by Bogush nor the correction proposed by Razink and Schlotter are consistent with our results, as shown in Table 1. Table 1. Composition of the Formulations Predicted by eqs 3−5 and Measured Hydrodynamic Radii of Colloidal Silica Prepared in This Study preparation code 1 2 3 4 5 6 7 8 a

[NH3] [TEOS] 0.44 0.66 0.86 0.99 1.07 1.06 1.05 1.04

0.29 0.28 0.28 0.28 0.28 0.27 0.27 0.27

[H2O]

H2O/ TEOS w/w

Rh/nma

Rh/nm observed

1.02 1.52 2.00 2.28 2.47 2.86 3.25 3.63

0.30 0.45 0.60 0.69 0.76 0.88 1.01 1.14

27.6 70.2 125.7 164.0 189.3 207.5 225.8 240.7

12.5 27.9 39.3 52.9 63.8 87.7 110.6 117.5

Using eqs 3−5. Figure 2. TEM images of silica particles. (a) Prepared using the traditional Stöber procedure (no predilution of reagent grade TEOS); (b) prepared by predissolving reagent grade TEOS in absolute ethanol, as described by Zhang;12 (c) certified standard silica.

The disagreement is not surprising, as Bogush followed the practice of most workers and used high-purity, freshly distilled TEOS. These authors also prepared alcohol solutions by direct addition of ammonia. The results in the present work were obtained using “as received” TEOS reagent grade (98%) without the distillation step. It is of interest to determine whether a particle preparation could be altered after synthesis. In particular, is it possible to reduce the size of particles in a large, readily available stock solution? This question was pursued briefly by HF etching (danger: HF is highly toxic). The result is that a size decrease can be accomplished, albeit with some broadening of the particle size distribution. Details appear in the Supporting Information. Uniformity of Size. Although the polydispersity of particles made without predilution of TEOS is low enough for many applications, the resulting particles are not strictly monodisperse, owing partly to impurities in the starting materials. As discussed already, Zhang et al.12 circumvented the need for high-purity TEOS by prediluting reagent grade material in ethanol. The authors claim that, because of the dilution step, the formation of new nuclei and the aggregation or adhesion of particles is suppressed.

colloidal silica prepared without predilution (size polydispersity from TEM > 12% measured as the quotient of the standard deviation of size and the average size) and the predilution, twostep method (size polydispersity from TEM = 4.9%). Also appearing is an image of a standard reference silica from Duke Scientific (size polydispersity from TEM = 4.0%,). The material made with predilution of “as received”, undistilled, reagent grade appears to be nearly as uniform as the commercially available silica standard; this is assessed further in the next section. AF4/MALS Measurements. Figure 3a−c shows the AF4/ MALS light scattering intensity and radius traces for the colloidal silica obtained with and without the predilution of the TEOS recommended by Zhang et al. Before comparing these figures to the corresponding electron micrographs, Figures 2a−c, it is appropriate to consider how the radius is determined, and how reliably. The particle module in the Wyatt Astra software38 5565

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

Figure 4. AF4 slices collected for the commercial silica standard, shown together with fits to eq 6 for uniform silica spheres. Even for a narrow sample, the order of elution follows the AF4 norm (large particles last). The logarithmic scale exaggerates errors of fit at high values of q.

As described in the Supporting Information, the core−shell model does indeed improve the fit in the high-angle regime, but two features were noted: (1) the fits are not unique, with multiple parameters (such as core vs shell thickness, and the refractive index of each) resulting in fits of similar quality and (2) sometimes better fits in the high-angle regime could be had at the price of modest worsening in the low-angle regime. In Figure 3 the peak for the sample prepared without predilution of TEOS is broader than that for the sample prepared using predilution. The scattered intensity at any single angle can be misleading; as shown in Figure 3a, the large particles are significantly under-represented if the 90-degree data are displayed. The effect is less when the 35-degree data are shown (using results extrapolated to zero angle would offer a modest additional improvement). The slope of the radius vs elution volume plot, taken in the region where most of the sample is eluted (∼18−45 min for Figure 3a and ∼28−37 min for Figure 3b), yields qualitative information about the polydispersity of the sample. In the case of the sample obtained using predilution of TEOS the slope is almost flat; this is a good indication of low polydispersity. In the case of the sample made without predilution, the slope is clearly positive. The average radius for both samples is ∼100 nm, which is relatively close to the value from TEM of 130 nm ±10 nm, but the very large number of particles simultaneously sampled in the AF4/MALS device reveals information that might only be suspected in TEM images showing, as they typically do, mere hundreds of particles. The images for the particles prepared without predilution (Figure 2a) suggest the existence of a population of large particles. This suspicion is convincingly confirmed by the corresponding AF4/MALS trace (Figure 3a). The particles made by predissolving TEOS in ethanol (Figure 2b, 3b) are comparable in terms of uniformity to a commercial silica standard (Figures 2c and 3c). One of the most significant observations is that AF4 can improve upon the uniformity of even a reference size standard; the particles eluting near the peak in Figure 3c differ significantly in size from those eluting near the end.

Figure 3. AF4 intensity (left scale; black) and radius (right scale; blue) traces of colloidal silica. (a) Prepared without predilution of reagent grade TEOS; intensities from two angles are shown to emphasize that large particles are under-represented at high scattering angles. (b) Prepared with predilution of reagent grade TEOS in ethanol. (c) Commercial silica sphere standards. Left axis (black curve), relative intensity of the light scattering detector at 90°.

was set to determine size. For a uniform, solid sphere, the form factor P(qR) = I(qR)/I(q = 0) is39 P(qR ) =

9 6

(qR )

[sin(qR ) − qR cos(qR )]2 (6)

The quality of the data and the fits to the sphere form factor were good at low angles, as shown in Figure 4. This permits an accurate radius to be determined, but the fit is worse at higher angles for the largest particles (the problem is much less obvious when a linear vertical scale is used). It was thought that perhaps the instrument’s nonlinear least-squares algorithm got trapped in a local, not global, minimum, but tests using a brute-force (grid search40) algorithm confirmed the results of the vendor’s method. One possible explanation for the small (on a linear scale) misfitting at high angles is internal particle structure; for example, there could be refractive index gradients within the particles. A core− shell model was developed41 and applied to test this notion. 5566

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

Not all of the information from AF4/MALS could be used reliably. In particular, we do not report the particle radius obtained by the single DLS detector in the online measurements. Two problems are associated with this parameter in the case of the present particles. First, unless the sample in the detected volume at any given time is absolutely uniform the DLS and SLS experiments will respond differently to the distribution. The DLS radius is the inverse of the z-average of the inverse of the radius,42 while the SLS signal is the square root of the z-average of the square of the radius.39 Possibly even more significant is the short (10 s) acquisition time relative to the diffusion time; this problem is imposed by the flow conditions. The correlation function in DLS is defined in the limit of infinite acquisition time. As a practical matter, the acquisition time ought to exceed the correlation time (q−2D−1) by at least a factor of 104, with factors of 106 being easily achieved for polymers and small particles. The problem of trying to catch the diffusion “on the fly” in the flow cell is exacerbated by the long wavelength of the laser source, which leads to long distance scales. The large size of the particles under study, leading to slow diffusion and requisite long acquisition times, also contributes. A detailed study of this problem has not appeared. Quantification of Amino Groups. The quantification of amino groups on the surface of the passivated-functionalized silica particles (P/F particles) was performed using UV−vis spectrometry after reaction with ninhydrin, which is known for yielding a bright purple complex when reacted with primary or secondary amines. Though mostly intended for qualitative analysis,28 ninhydrin can also be used for quantification.43,44 A potential argument against this is that some of the colored product may remain on the surface. Sarin et al. proposed an ion exchange mechanism for the formation of ninhydrin-containing negatively charged colored product.44 Unmodified silica has a large negative zeta potential (∼−50 mV) over a wide range of pH, reflecting a large negative charge due to deprotonated silanol groups. In comparison, the passivated/functionalized particles, with their −NH2, −CH3, and −OH groups may bear a net positive, neutral or negative charge, depending on pH. A faint blue coloration is observed for ninhydrin-treated P/F colloidal silica particles. This suggests the ion exchange mechanism proposed by Sarin et al. operates efficiently to release the colored product to solution, thus validating the use of ninhydrin for the quantification of amino groups. The reaction proceeds in a similar fashion for either primary or secondary amines. An important feature of the ninhydrin reaction is the ability to separate the colored product from the scattering particles, for example by centrifugation or filtration; thus, the blue complex absorption can be measured without interference from scattering. A calibration curve was built using hexylamine as the source of amino groups. It is possible to convert from mmol/L (i.e., mM) to number of amino groups per nm2 on the surface by knowing the total volume of ninhydrin complex solution, total mass and Rh of the P/F particles. Colloidal silica particles of Rh = 103 nm ±2 nm were used (surface area per particle, 4.6 × 106 nm2). From the concentration of the dispersion and the density of colloidal silica (1.96 g/cm3) it is possible to calculate the total surface area of the sample. Appearing in Figure 5 are the results for the surface density (σ, in number of -NH2 groups/nm2) as a function of mol fraction (xAPS) in the APS/ MTMS solution used for surface modification. The straight line is computed from the literature expectation of 4 molecules/nm2 for molecules similar to either APS or MTMS on flat substrates,35,36

Figure 5. Graph of surface density of amino groups vs mole fraction of APS in the solution used for surface modification. The inset is a magnification of the graph at low xAPS.

with the assumption that only a monolayer of functional groups is formed. This assumption is reasonable because only a slight excess of functionalizing mixture was added. The measured values at low mole fraction of APS are in agreement with the expected values for a monolayer of APS and MTMS of about 4 functional groups per nm2. This suggests that APS and MTMS react almost equally toward the colloidal silica surface. The regime of low surface concentration has important spatial implications when initiating and growing rod-like polypeptides such as poly(γ-benzyl-L-glutamate) (PBLG),45 and presumably for other polymer initiations. The measured surface density at low amino group concentrations is approximately equal to the expected values within the experimental error of the measurement, but exceeds expectation at high concentrations. It is likely that monolayers are being formed at low concentrations, while at high concentrations bilayers or multilayers of APS and MTMS are formed,24,25,46,47 resulting in higher-than-expected measured amino group concentrations. This may be because the value of 4 functional groups per nm2 is expected for flat substrates but heavier packing may be possible on spherical surfaces. Additionally, the amino group in APS can, in principle, catalyze the condensation reaction Although the ninhydrin analysis is fairly rapid and seems to work well, according to the initial linearity of Figure 5, an even faster method with the same accuracy was sought. It is known that amino groups are positively charged at low pH. The zeta potential (ζ) of a colloidal particle is related to its surface charge. Therefore, in principle, it is possible to obtain a relationship between zeta potential and surface density of amino groups if all other variables (such as particle size, shape, concentration, ionic strength) are kept constant. A zeta potential measurement is easier and considerably faster (∼1−2 min) than the ninhydrin method (>60 min). The zeta potential of samples was measured at pH = 1 and 1 mM NaNO3 as a function of surface density of amino groups. An empirical relationship was obtained. Figure 6 shows a graph of the zeta potential (ζ) vs surface density of amino groups, σ, defined as the number of −NH2 groups per nm2. A good correlation between the zeta potential and the logarithm of the surface density was observed. The relationship between ζ and σ is described by ζ = 38.7 + 21.6 log10(σ). This result shows that it is possible to measure the surface density of amino groups on amino-functionalized colloidal silica by a simple zeta potential measurement. 5567

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

(2) Bogush, G. H.; Tracy, M. A.; Zukoski, C.F., IV Preparation of monodisperse silica particles: control of size and mass fraction. J. NonCryst. Solids 1988, 104, 95−106. (3) Lindberg, R.; Sundholm, G.; Pettersen, B.; Sjoeblom, J.; Friberg, S. E. Multivariate analysis of the size dependence of monodisperse silica particles prepared according to the sol-gel technique. Colloids Surf., A 1997, 123−124, 549−560. (4) Tan, C. G.; Bowen, B. D.; Epstein, N. Production of monodisperse colloidal silica spheres: effect of temperature. J. Colloid Interface Sci. 1987, 118, 290−293. (5) Bailey, J. K.; Mecartney, M. L. Formation of colloidal silica particles from alkoxides. Colloids Surf. 1992, 63, 151−161. (6) Esquena, J.; Pons, R.; Azemar, N.; Caelles, J.; Solans, C. Preparation of monodisperse silica particles in emulsion media. Colloids Surf., A 1997, 123−124, 575−586. (7) Wang, W.; Gu, B.; Liang, L. Effect of anionic surfactants on synthesis and self-assembly of silica colloidal nanoparticles. J. Colloid Interface Sci. 2007, 313, 169−173. (8) Kim, S. S.; Kim, H. S.; Kim, S. G.; Kim, W. S. Effect of electrolyte additives on sol-precipitated nano silica particles. Ceram. Int. 2004, 30, 171−175. (9) Coenen, S.; De Kruif, C. G. Synthesis and growth of colloidal silica particles. J. Colloid Interface Sci. 1988, 124, 104−110. (10) Brinker, J.; Scherer, G. Sol-gel science: the physics and chemistry of sol-gel processing; Academic Press: Boston, 2004. (11) Green, D. L.; Lin, J. S.; Lam, Y. F.; Hu, M. Z. C.; Schaefer, D. W.; Harris, M. T. Size, volume fraction, and nucleation of Stober silica nanoparticles. J. Colloid Interface Sci. 2003, 266, 346−358. (12) Zhang, J. H.; Zhan, P.; Wang, Z. L.; Zhang, W. Y.; Ming, N. B. Preparation of monodisperse silica particles with controllable size and shape. J. Mater. Res. 2003, 18, 649−653. (13) Csogor; Nacken, M.; Sameti, M.; Lehr, C. M.; Schmidt, H. Modified silica particles for gene delivery. Mater. Sci. Eng., C 2003, C23, 93−97. (14) Kneuer, C.; Sameti, M.; Haltner, E. G.; Schiestel, T.; Schirra, H.; Schmidt, H.; Lehr, C. M. Silica nanoparticles modified with aminosilanes as carriers for plasmid DNA. Int. J. Pharm. 2000, 196, 257−261. (15) Chen, Z.; Hsu, F. C.; Battigelli, D.; Chang, H. C. Capture and release of viruses using amino-functionalized silica particles. Anal. Chim. Acta 2006, 569, 76−82. (16) Fong, B.; Russo, P. S. Organophilic Colloidal Particles with a Synthetic Polypeptide Coating. Langmuir 1999, 15, 4421−4426. (17) Fong, B.; Turksen, S.; Russo, P. S.; Stryjewski, W. Colloidal Crystals of Silica-Homopolypeptide Composite Particles. Langmuir 2004, 20, 266−269. (18) Turksen, S. Synthesis and Characterization of Superparamagnetic Silica-homopolypeptide Composite Particles. Ph.D.Dissertation; Louisiana State University, 2004. (19) Soto-Cantu, E.; Turksen-Selcuk, S.; Qiu, J.; Zhou, Z.; Russo, P. S.; Henk, M. C. Silica-Polypeptide Composite Particles: Controlling Shell Growth. Langmuir 2010, 26, 15604−15613. (20) Heise, A.; Menzel, H.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M. Grafting of Polypeptides on Solid Substrates by Initiation of N-Carboxyanhydride Polymerization by Amino-Terminated Self-Assembled Monolayers. Langmuir 1997, 13, 723−728. (21) Bagwe, R. P.; Hilliard, L. R.; Tan, W. Surface Modification of Silica Nanoparticles to Reduce Aggregation and Nonspecific Binding. Langmuir 2006, 22, 4357−4362. (22) Sugimoto, H.; Daimatsu, K.; Nakanishi, E.; Ogasawara, Y.; Yasumura, T.; Inomata, K. Preparation and properties of poly(methylmethacrylate)-silica hybrid materials incorporating reactive silica nanoparticles. Polymer 2006, 47, 3754−3759. (23) Hozumi, A.; Yokogawa, Y.; Kameyama, T.; Sugimura, H.; Hayashi, K.; Shirayama, H.; Takai, O. Amino-terminated selfassembled monolayer on a SiO2 surface formed by chemical vapor deposition. J. Vac. Sci. Technol., A 2001, 19, 1812−1816.

Figure 6. Graph of zeta potential of amino-funtionalized colloidal silica (Rh = 103 ± 2 nm) vs logarithmic surface density of amino groups (σ, expressed as number of amino groups per square nanometer). Measured in 0.1 M HCl with 1 mM NaNO3 added.

■

CONCLUSION Predilution of reagent grade TEOS without distillation does result in better particle uniformity, as reported by Zhang et al. The improvement, which seemed visible in TEM images, was convincingly revealed by AF4/MALS measurements. A reliable relationship between size of the resulting silica and H2O/TEOS used in a formulation that requires only “as received” reagent grade TEOS was found. Amino content was easily varied by functionalizing the particle surface with a mixture of APS and MTMS, and the surface amino group density was easily determined by ninhydrin test or by zeta potential. The facile variation and characterization of the surface content of amino groups on colloidal silica is expected to add a new level of control to the preparation of certain core−shell hybrid particles. The inability of a simple sphere model to fit the scattering envelopes in the high-angle regime is regarded as an indication that the internal structure of particles can be investigated with improved confidence, now that the uniformity of even reference standard preparations can be improved and quantified by AF4/MALS.

■

ASSOCIATED CONTENT

S Supporting Information *

Includes etching of silica cores for reduced particle size and considerations of core−shell particle form factors for fitting light scattering data. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by NSF-DMR 0606117 and 1005707. The Duke Scientific standard silica particles were a gift from Professor Jayne Garno. The authors thank Ms. Cornelia Rosu for assistance with some of the calculations.

■

REFERENCES

(1) Stö ber, W.; Fink, A.; Bohn, E. Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 1968, 26, 62−69. 5568

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569

Langmuir

Article

(24) Fadeev, A. Y.; McCarthy, T. J. Self-Assembly Is Not the Only Reaction Possible between Alkyltrichlorosilanes and Surfaces: Monomolecular and Oligomeric Covalently Attached Layers of Dichloro- and Trichloroalkylsilanes on Silicon. Langmuir 2000, 16, 7268−7274. (25) McGovern, M. E.; Kallury, K. M. R.; Thompson, M. Role of Solvent on the Silanization of Glass with Octadecyltrichlorosilane. Langmuir 1994, 10, 3607−3614. (26) Wasserman, S. R.; Tao, Y. T.; Whitesides, G. M. Structure and reactivity of alkylsiloxane monolayers formed by reaction of alkyltrichlorosilanes on silicon substrates. Langmuir 1989, 5, 1074− 1087. (27) Vadala, M. L.; Zalich, M. A.; Fulks, D. B.; St.Pierre, T. G.; Dailey, J. P.; Riffle, J. S. Cobalt-silica magnetic nanoparticles with functional surfaces. J. Magn. Magn. Mater. 2005, 293, 162−170. (28) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 1970, 34, 595−598. (29) Felix, A. M.; Jimenez, M. H. Rapid fluorometric detection for completeness in solid phase coupling reactions. Anal. Biochem. 1973, 52, 377−381. (30) Hancock, W. S.; Battersby, J. E. A new micro-test for the detection of incomplete coupling reactions in solid-phase peptide synthesis using 2,4,6-trinitrobenzenesulfonic acid. Anal. Biochem. 1976, 71, 260−264. (31) Schimpf, M.; Caldwell, K.; Giddings, J. C. Field-Flow Fractionation Handbook; Wiley-Interscience: New York, 2000. (32) Roessner, D.; Kulicke, W. M. Online Coupling of Flow Field-Flow Fractionation and Multi-Angle Laser-Light Scattering. J. Chromatogr. A 1994, 687, 249−258. (33) Wyatt, P. J. Light-Scattering and the Absolute Characterization of Macromolecules. Anal. Chim. Acta 1993, 272, 1−40. (34) Berne, B.; Pecora, R. Dynamic Light Scattering; Wiley: New York, 1976. (35) Fadeev, A. Y.; McCarthy, T. J. Trialkylsilane monolayers covalently attached to silicon surfaces: wettability studies indicating that molecular topography contributes to contact angle hysteresis. Langmuir 1999, 15, 3759−3766. (36) Kallury, K. M. R.; Macdonald, P. M.; Thompson, M. Effect of Surface Water and Base Catalysis on the Silanization of Silica by (Aminopropyl)alkoxysilanes Studied by X-ray Photoelectron Spectroscopy and 13C Cross-Polarization/Magic Angle Spinning Nuclear Magnetic Resonance. Langmuir 1994, 10, 492−499. (37) Razink, J. J.; Schlotter, N. E.; Bogush, G.H.; Tracy, M.A.; Zukoski. Correction to "Preparation of monodisperse silica particles: Control of size and mass fraction". J. Non-Cryst. Solids 1988, 104, 95− 106. J. Non-Cryst. Solids 2007, 353, 2932−2933. (38) Wyatt, P. J. Weida, M. J. Method and Apparatus for Determining Absolute Number Densities of Particles in Suspension. U.S. Patent 10/ 640,893(6774994 B1). 8-10-2004. (39) Kratochvil, P. In Light Scattering from Polymer Solutions; Huglin, M. B., Ed.; Academic Press: New York, 1972; pp 333−379, Chapter 7. (40) Bevington, P. R. Data Reduction and Error Analysis for the Physical Sciences; McGraw-Hill: New York, 1969. (41) Note: A model for core−shell structures is available in the Wyatt Astra software, too. (42) Koppel, D. E. Analysis of macromolecular polydispersity in intensity correlation spectroscopy. Method of cumulants. J. Chem. Phys. 1972, 57, 4814−4820. (43) Henry, A. C.; Tutt, T. J.; Galloway, M.; Davidson, Y. Y.; McWhorter, C. S.; Soper, S. A.; McCarley, R. L. Surface Modification of Poly(methyl methacrylate) Used in the Fabrication of Microanalytical Devices. Anal. Chem. 2000, 72, 5331−5337. (44) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 1981, 117, 147−157. (45) Abelow, A. E.; Zharov, I. Poly(L-alanine)-modified nanoporous colloidal films. Soft Matter 2009, 5, 457−462.

(46) Bierbaum, K.; Kinzler, M.; Woell, C.; Grunze, M.; Haehner, G.; Heid, S.; Effenberger, F. A Near Edge X-ray Absorption Fine Structure Spectroscopy and X-ray Photoelectron Spectroscopy Study of the Film Properties of Self-Assembled Monolayers of Organosilanes on Oxidized Si(100). Langmuir 1995, 11, 512−518. (47) Bierbaum, K.; Grunze, M.; Baski, A. A.; Chi, L. F.; Schrepp, W.; Fuchs, H. Growth of Self-Assembled n-Alkyltrichlorosilane Films on Si(100) Investigated by Atomic Force Microscopy. Langmuir 1995, 11, 2143−2150.

5569

dx.doi.org/10.1021/la204981b | Langmuir 2012, 28, 5562−5569