Article pubs.acs.org/ac
Confocal Raman Microscopy Investigation of Molecular Transport into Individual Chromatographic Silica Particles David A. Bryce, Jay P. Kitt, and Joel M. Harris* Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States S Supporting Information *
ABSTRACT: Porous silica is used as a support in a variety of separation processes, including chromatographic separation and solid-phase extraction. The resolution and efficiency of these applications is significantly impacted by the kinetics of partitioning and molecular transport into the interior of the porous particles. Molecular transport in porous silica has been explored previously by measuring chromatographic elution profiles, but such measurements are limited to relatively low retention conditions, where within-particle molecular transport must be inferred from elution profiles of solutes emerging from a packed column. In this work, a measurement of within-particle molecular transport is carried out using confocal Raman microscopy to probe the time-dependent accumulation of pyrene from an aqueous mobile phase into the center of individual C18-chromatographic particles. The measured time constants for pyrene accumulation were much slower than diffusion-limited transport of solute in solution to the particle surface. Furthermore, the accumulation into the center of the particle did not show a time-lag characteristic of slow-transport into the particle interior. The exponential rise of pyrene concentration is, however, consistent with first-order Langmuir adsorption kinetics at low surface coverages. The linear dependence of the time-constant on particle radius indicates an adsorption barrier near the outer boundary of the particle, where the accumulation rate depends on flux across the boundary (proportional to the particle area) to satisfy the within-particle capacity at equilibrium (proportional to the particle volume). The pyrene accumulation kinetics into the porous particle, expressed as a heterogeneous rate constant, were nearly 50-times faster than the pyrene adsorption rate at a planar C18-functionalized silica surface, which demonstrates the impact of multiple surface encounters within the porous structure leading to much greater capture efficiency compared to a planar surface. Monte Carlo simulations of within-particle pyrene diffusion, with the adsorption efficiency estimated from the planar-surface adsorption rate, predict a diffusion-to-capture distance within the porous particle that is within 40% of that observed in the radial dependence of the pyrene within-particle accumulation results.
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surfaces of the porous particles. Therefore, understanding the kinetics of within-particle molecular transport is essential to developing high-efficiency chromatographic and extraction separations. Much of the foundational work to understand molecular transport in stationary phase materials has been carried out as elution measurements in packed columns of silica particles. These experiments have addressed how diffusion in the mobile and stationary phases and resistance to mass transfer between the phases influence peak shapes in chromatographic separations.7−12 Although the impact of these experiments advanced the understanding of transport phenomena in chromatographic separations, investigations carried out in packed beds only allow examination of ensemble behavior, while the nature of transport within individual particles must be inferred. Additionally,
orous silica is a versatile support with applications in a variety of chemical processes. This is due to the structural stability and high surface area of the porous silica structure, as well as the ease with which silica surfaces can be chemically modified using silane chemistry.1 Two common applications of silica supports are as stationary phases in chromatographic separations and in solid-phase extractions. The most commonly employed chromatographic separations are based on reversedphase interactions,2,3 where the surfaces of porous silica particles are functionalized with hydrophobic ligands [typically long-chain hydrocarbons such as octadecylsilane (C18)], allowing retention of nonpolar compounds from polar, aqueous mobile phases. C18-silica can also be used to concentrate nonpolar molecules in solid-phase extractions,4−6 where nonpolar analytes are concentrated from solution into a short extraction column of C18 particles under high-retention (highwater content mobile phase) conditions for subsequent elution and analysis. The efficiency of both chromatographic separations and solid-phase extractions depends on the analyte accessing and partitioning into/onto the stationary phase on the interior © XXXX American Chemical Society
Received: September 5, 2016 Accepted: January 30, 2017 Published: January 30, 2017 A
DOI: 10.1021/acs.analchem.6b03498 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
solute under high retention conditions, typical of those used in solid-phase extractions.
packed bed experiments are slow and accumulation and elution times become prohibitively long when analyte affinity for the stationary phase becomes excessive, as in solid-phase extractions. To develop a better understanding of masstransport phenomena at chromatographic interfaces, fluorescence techniques such as fluorescence correlation spectroscopy (FCS) have been carried out at model (planar) C18-functionalized silica interfaces to measure adsorption and desorption kinetics13 and surface diffusion coefficients.14−16 While these measurements have provided new insight into interfacial dynamics, flat surfaces do not replicate the porous network within chromatographic particles, and they neglect the pore geometry and nanometer-scale wall-to-wall distances that can impact molecular transport phenomena. Thin porous silica sol−gel films have been used to mimic the interior of chromatographic particles in an attempt to address the pore-structure contributions neglected in planar experiments.17−20 However, sol−gel preparation methods for these deposited films generate a pore structure and surface chemistry that differ significantly from that of the xerogels used in reverse-phase chromatographic materials.21,22 Recently, it was shown that fluorescence microscopy can be used to image fluorescent solutes in situ within individual porous silica particles,23,24 eliminating the need for a planar model-system. Fluorescence microscopy within chromatographic particles has been used as a means to investigate the mechanisms and impact of varied separations conditions, which influence chromatographic separations of various analytes and in a variety of stationary phases.25−31 With the development of improved single-molecule fluorescence microscopy capabilities, these techniques are capable of reporting the heterogeneity of molecular transport kinetics within a single chromatographic particle to the limit of single-molecule trajectories.23,24,32,33 These measurements have revealed heterogeneity in micropolarity,24 the impact of defect sites14,34 on intraparticle transport,32 and the influence of the porous surface structure on rates of diffusion within chromatographic particles.33 While fluorescence measurements have provided valuable insight into within-particle transport, they are limited to fluorescent or fluorescently labeled molecules. Furthermore, these experiments to date have not addressed the kinetics of molecular exchange at the particle/mobile-phase boundary. In the present work, confocal Raman microscopy is used to investigate the kinetics of solute accumulation into individual reversed-phase (C18-modified) chromatographic silica particles. Raman spectroscopy has previously been employed on packed-bed samples of reversed-phase silica to characterize the conformational order of the bonded alkyl chains in response to changes in the mobile-phase composition.35−40 Confocal Raman microscopy has been utilized previously to probe the interior of individual chromatographic particles to determine the impact of organic modifiers and surface-wetting hysteresis on the interfacial environment41,42 and to investigate retention mechanisms in ioninteraction chromatography.43 More recently, confocal Raman microscopy was used to probe solid-phase extraction on a femtoliter scale, where a 50000-fold preconcentration of pyrene within individual C18 particles provided 10 nM source-phase detection limits.44 In this work, we extend the use of confocal Raman microscopy into time-resolved experiments to measure kinetics of pyrene accumulation into individual chromatographic particles and thereby investigate the roles of molecular transport and adsorption kinetics on the accumulation of a
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EXPERIMENTAL SECTION Within-Particle Confocal Raman Microscopy. Experiments described here were carried out in spherical, reversedphase chromatographic silica particles obtained from YMC America (YMC ODS-A, Allentown, PA). The particles were monofunctional, C18-derivitized, and end-capped with trimethylchlorosilane. Individual particle radii were measured with bright-field illumination in the microscope and ranged from 1.5 to 10 μm from lots of nominally 1.5-, 2.5-, and 5-μm radii, as specified by the manufacturer. The mean pore diameter (12.3 ± 0.7 nm), specific-surface area (335 m2/g), pore volume (1.01 mL/g), and carbon content (17.3%) were determined by the manufacturer, who also report that the end-capping step contributes ∼0.5% to the carbon content. Correcting the carbon content for the end-capping contribution and dividing the result by the carbon contribution to the molecular weight of the C18 silane and the specific surface area gives a C18 coverage of 2.3 ± 0.1 μmol/m2. The confocal Raman microscope used in this work is previously described;45 details are in the Supporting Information. To collect Raman spectra from within individual particles, the focused laser beam was brought to the coverslip solution interface where visible reflection of the laser spot could be observed. The microscope stage was then translated in the x and y dimensions to center an individual particle above the focused spot. The focused spot was then brought to the center of the particle by translating the objective in the z-dimension (upward) by a distance equal to the particle radius, as measured by the eyepiece camera; this localizes the ∼1 fL (600 nm diameter) confocal probe volume at the center of the particle. Raman spectral analysis was accomplished in Matlab (Mathworks, Natick, MA) using custom scripts (see the Supporting Information). Local baseline corrections were applied to regions of the spectra containing Raman bands of interest. Raman bands used for quantification were fit to Gaussian functions using the highest signal-to-noise spectrum acquired for each kinetic series. These functions were amplitude-scaled to fit all other spectra in the series and integrated to determine peak areas for quantification. A sample spectrum showing the local baseline subtraction and peak-fitting methodology can be found in the Supporting Information. Measuring Pyrene Accumulation into Particles. To measure the time-dependent concentration of pyrene within chromatographic particles, C18-silica particles were suspended in methanol, flowed into the microscopy cell, and allowed to settle on the coverslip surface. Adhesion of particles to the coverslip was achieved by drying the cell at 90 °C for ∼1 h. Adhered particles were rewetted, initially with 100% methanol, and then equilibrated with blank (no analyte) sample solution of 20% methanol in water, where 10% of the water in the aqueous solution was deuterated (D2O) to provide a spectral indicator of solution changeover in the flow channel. Spectra collected from the interior of an individual particle were monitored as blank solution was flowed through the flow channel until D2O and methanol bands were no longer changing, indicating the achievement of equilibrium. At this point, a solution of pyrene in aqueous 20% methanol was flowed into the cell. Spectra were collected at 10 s intervals to monitor pyrene accumulation until the concentration of pyrene in the particle interior reached equilibrium as indicated by constant B
DOI: 10.1021/acs.analchem.6b03498 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
C18 chains as an internal standard, calibrated by standard solution spectra (Supporting Information).44 The time-dependent intraparticle pyrene concentrations thus determined (Figure 2),
pyrene and C18 Raman intensity in successive frames; the timezero point in the accumulation experiment was indicated by the disappearance of the D2O Raman scattering from the solution (see the Supporting Information).
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RESULTS AND DISCUSSION Solute Accumulation into Individual C18-Functionalized Chromatographic Particles. To develop insight into the phenomena that govern solute transport into chromatographic particles under high-retention conditions, time-dependent Raman spectroscopy measurements were carried out to follow the accumulation of pyrene from aqueous mobile-phase solutions into individual, spherical, C18-modified silica particles. To accomplish this, particles adhered to the coverslip inside of a microscope flow cell were equilibrated with a blank solution of aqueous 20% methanol and 10% D2O as a dead-volume marker. The flow was then switched to 2.0 μM pyrene in an equivalent 20% methanol aqueous solution without D2O, and Raman scattering was collected as a function of time from the center of an individual particle using confocal Raman microscopy. Localized excitation and collection of Raman scattering from the particle center was achieved by using a high numerical aperture (NA) objective allowing excitation light to be tightly focused within an individual particle and scattered light to be collected from the focused laser spot with high efficiency. By incorporating a confocal aperture into the collectedlight path, the acquisition was limited to a small confocal probe volume of less than a femtoliter at the center of the particle.44,46,47 Representative spectra of Raman scattering collected as a function of time from the center of an individual (R = 4.0 μm radius particle), as pyrene solution was flowed through the cell, are presented in Figure 1. Pyrene accumulation is observed as
Figure 2. Time-dependent intraparticle pyrene concentration determined from the data in Figure 1 (○), together with least-squares fits to a within-particle diffusion-limited model of eq 2 (red line) and to a partitioning-rate-limited first-order accumulation of eq 3 (blue line).
provide insight into the accumulation process. First, the time to equilibrate the particle can be compared to the rate of diffusional transport of pyrene from the surrounding solution to the particle surface. A free-solution diffusion-limited timeconstant for pyrene accumulation depends on the amount of pyrene that must be delivered to a particle to reach equilibrium and the pyrene concentration in solution that supplies the build-up within the particle. The moles of pyrene within a 4.0-μm radius particle are given by product of the final intraparticle pyrene concentration (∼30 mM) and the particle volume (250 fL) or 7.5 fmol. Dividing this amount of analyte by the solution concentration of pyrene (2.0 μM) gives the volume of source-phase solution (3.8 nL) required to supply the particle with pyrene at equilibrium. This solution volume occupies a hemisphere surrounding the surface-attached particle having a radius, rsoln = 120 μm, which can be used to estimate the characteristic solution-diffusion time for pyrene molecules to reach the particle surface: τ = rsoln2/6Dpyrene = (1.2 × 10−2 cm)2/(6 × 2.4 × 10−6 cm2 s−1) = 10 s. Pyrene accumulates into this particle with a time-constant greater than 200 s, or more than 20-times slower than the solution diffusioncontrolled rate of supplying pyrene to the surface of the C18-modified particle. Thus, diffusion from the source-phase solution to the particle cannot account for the slow accumulation of pyrene in the particle. A possible kinetic barrier to equilibration would be slow diffusion of pyrene from the outer perimeter of the particle into the particle center, where the confocal-volume probes the timedependent pyrene concentration. Slow diffusion into a spherical particle can be modeled by a combination of Fick’s first and second laws, which for a radial geometry is given by
Figure 1. Raman spectra tracking the accumulation of pyrene into the center of a 4.0 μm radius C18 chromatographic particle from a 2 μM solution in 20% methanol over time. Constant scattering intensity from the n-alkyl chains in the silica particle is noted, along with the increasing scattering intensity from pyrene with time.
⎡ ∂ 2C ∂Cr 2 ∂Cr ⎤ ⎥ = D⎢ 2r + r ∂r ⎦ ∂t ⎣ ∂r
an increase in the intensity of ring breathing modes (1241 cm−1 and 1410 cm−1) relative to C18 alkyl chains scattering from CH2-twisting (1300 cm−1) and CH-bending (1410 cm−1) modes, which remain constant throughout the experiment. Quantitative measurement of within-particle pyrene concentrations was achieved by using the CH2-twisting mode from surface-bound
(1)
Note that this model is based on a random walk in a continuous spherical volume, while the actual diffusion of strongly retained molecules occurs on the C18-modified surface of the C
DOI: 10.1021/acs.analchem.6b03498 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
If the rate of adsorption or partitioning is relatively slow then rapid solution-phase diffusion of pyrene in the pores of the particle could outpace capture by the stationary phase (case I), leading to uniform accumulation throughout the particle versus time. A second possibility (case II) is that the partitioning is fast compared to solution-phase diffusion within the pores, so that partitioning occurs at or near the outer particle boundary followed by rapid surface-diffusion10,11,52−54 of the strongly retained solute into the particle interior. Both of these situations lead to first-order accumulation kinetics uniformly throughout the particle; however, their rates would be expected to exhibit a different dependence on particle size. For the first case, capture by the stationary phase would arise from encounters of the solute with the interior surface throughout the porous particle; as the particle size increases, the internal surface area governing the adsorption rate and the stationaryphase capacity would both increase in proportion to the particle volume and the time to achieve equilibrium would be independent of particle size. For the second case, rapid partitioning would occur at or near the outer surface of the particle, and the outer particle area will govern the accumulation rate, which is proportional to the particle radius squared, while the stationary phase capacity is proportional to the particle volume or radius cubed. In this case, the time to reach equilibrium would depend on the ratio of the particle volume (governing the partitioning capacity) to the outer surface area (governing the partition rate); a simple dimensional analysis indicates that the ratio of volume to surface area would vary linearly with particle radius. Solute Accumulation As a Function of Particle Size. To test whether slow partitioning occurs uniformly throughout the particle or at the outer surface, the effect of particle size on time-dependence of pyrene accumulation was measured (see Figure 3). Each of the curves rises exponentially, so that the
porous structure. Recent measurements of single-molecule trajectories within chromatographic silica particles have shown that, on distance scales greater than the resolution limit of the measurement (37 nm), diffusion of strongly retained molecules within the particle follows a continuous random-walk in three dimensions,32 which supports the use of this model. The relevant boundary conditions for solving eq 1 are as follows: the initial concentration inside the particle, Cr
