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Single Molecule Observations of Fatty Acid Adsorption at the Silica/Water Interface: Activation Energy of Attachment† Andrei Honciuc, Alexander L. Howard, and Daniel K. Schwartz* Department of Chemical and Biological Engineering, UniVersity of Colorado, Boulder, Colorado 80309 ReceiVed: June 12, 2008; ReVised Manuscript ReceiVed: July 15, 2008
Total internal reflection fluorescence microscopy was used to obtain real-time images of individual fluorescently labeled hexadecanoic acid molecules as they absorbed at the aqueous-fused silica interface. In contrast with laterally averaged ensemble measurements of surface coverage versus time, single-molecule observations provide direct, model-independent measurements of adsorption kinetics. Additionally, since the observations were made under steady-state conditions at extremely low coverage, effects due to transport kinetics did not influence the results, permitting accurate measurement of the fundamental attachment rate as a function of temperature, that is, the rate at which molecules are transferred to the surface from the subsurface layer. We found that the attachment rate systematically increased with increasing temperature; an Arrhenius analysis gave an activation energy of Ea ) 19 ( 2 kJ/mol. We hypothesize that this energy, which is similar in magnitude to the strength of an OH · · · O hydrogen bond, is associated with the displacement of a water molecule from the silica surface. Introduction The adsorption of amphiphilic molecules at the solid/liquid interface modifies the physical and chemical properties of the solid surface. This has important implications in the environment, where humic and fulvic acids are know to adsorb on mineral surfaces.1,2 It is also technologically important for applications that involve detergency, colloidal stability, corrosion inhibition, etc. The formation of self-assembled monolayers (SAMs)3 represents a special case of this process, where lateral associations between the adsorbed amphiphilic molecules eventually result in a well-organized, stable, and robust surface coating. Several thorough reviews of surfactant adsorption and the structure of adsorbed layers have recently been published.4-8 Both equilibrium thermodynamic measurements (e.g., adsorption isotherms) and kinetic studies of adsorption can provide insight into the mechanisms of the adsorption process.7 In the conventional picture of surfactant adsorption,9-11 the surface layer formation process is divided into a number of conceptual steps: (a) transport (diffusive, convective, or both) from bulk solution to the subsurface layer, (b) attachment from the subsurface layer to the surface, (c) detachment from surface to the subsurface layer, and (d) mobility and (re)organization on the surface. Conceptually, the subsurface is considered to be a physical region within the solution phase in the near-surface region. However, the specific microscopic meaning of the subsurface layer is not well-defined, and there is no good molecular-level picture of how molecules are exchanged between the surface and the subsurface layer. In this paper, we concern ourselves with step b and seek to determine the rate and mechanism by which molecules are attached to the surface from the subsurface. A number of experimental methods can be used to measure macroscopic adsorption kinetics at the solid/solution interface, including solution-depletion studies,12,13 ellipsometry,14-16 opti† Part of the special section “Physical Chemistry of Environmental Interfaces”. * Corresponding author. E-mail:
[email protected].
cal reflectometry,13,17-21 surface plasmon resonance,22-24 and spectroscopic methods.25 In all of these methods, the net amount of surface-bound adsorbate is determined as a function of time starting at some initial time where the surface is considered bare. Thus, the combined effect of steps a-c is measured (and in some cases, step d is also involved). Although kinetic models have been developed to describe this overall process,9-11,26,27 in practice it remains difficult to separate the kinetic parameters associated with individual steps. If the net attachment rate (the attachment rate minus the detachment rate) is fast, the subsurface layer becomes depleted, and models can be used only if the bulk transport is extremely well-characterized. This is particularly difficult in the short-time/low-coverage regime, which is of interest because lateral interactions between adsorbed surfactant can be neglected. Even in the simplified case where the attachment rate is slow enough that bulk transport can be neglected, it is not generally possible to separate the attachment and detachment rates with confidence. Thus one cannot measure rates that are associated with specific molecular-level surface processes, like attachment and detachment, using macroscopic laterally averaging methods. In this paper, we show that total internal reflection fluorescence microscopy (TIRFM) can be used to explicitly count the number of molecules adsorbing with time and thereby directly calculate the rate of attachment from the subsurface layer without the need to compare with any particular model. These measurements are made under steady-state conditions at extremely low surface coverage (10-10-10-9), so bulk transport is not a consideration. In any case, the measured attachment rates are several orders of magnitude smaller than the diffusionlimited flux. These rates increase with temperature, suggesting that attachment is an activated process with an activation energy of ∼19 kJ/mol. Adsorption from solution has not conventionally been considered an activated process; in fact, the results presented here represent the first direct evidence for this concept. However, we argue that this is a sensible hypothesis, since attachment should actually be considered exchange between an
10.1021/jp8051856 CCC: $40.75 2009 American Chemical Society Published on Web 10/09/2008
Fatty Acid Adsorption at Silica/Water Interface
J. Phys. Chem. C, Vol. 113, No. 6, 2009 2079
adsorbate molecule and a surface-bound solvent (water) molecule, which must be displaced in order for adsorption to occur. Experimental Methods Fused silica (FS) wafers were washed with detergent (Micro 90, International Product Corp.), gently wiped with lens paper, and rinsed with 18 MΩ cm-1 water (Millipore Milli-Q UV+). After detergent cleaning, the substrates were immersed in piranha solution for ∼1 h, followed by UV-ozone cleaning for 60 min. Extremely dilute solutions, ∼3 × 10-9 M, of fluorescently labeled palmitic acid (fl-PA) fluorescent probe (BODIPY FL C16, Invitrogen, CA) in water were used in these singlemolecule experiments; this concentration is well below the upper solubility limit reported for palmitic acid (PA) in water, ∼2 × 10-5 mol/L.28 Upon introduction of the solution of interest into the flow cell, the fluorophores were excited with a blue light, 488 nm, from an Ar-ion laser (model 543-A-AO3, Melles-Griot Inc.), and the exposure time was controlled with a Uniblitz shutter (model VMM-D3, Oz Optics Ltd.). A prism-based illumination setup was used to attain the large incidence angle required by the total internal reflection condition. The blue excitation light and the green emission light of the fluorophores entering the objective were separated with a dichroic mirror (cuton wavelength ∼505 nm) and a green filter (band-pass ∼515-555 nm). An electron-multiplied CCD camera (model Cascade-II:512, Photometrics Inc.), cooled at -70 °C, was used as a photon detector. Metamorph 6.3 software (Molecular Imaging, Sunnyvale, CA) was used for the image and movie acquisition, data processing, and shutter control. The images acquired were 512 pixels × 512 pixels, and for a 60× magnification, the corresponding pixel size was 0.28 µm × 0.28 µm. Movies showing single-molecule events were acquired during continuous exposure to excitation light; frames were acquired at 2 s time intervals. The raw TIRFM experimental data consisted of movies in which individual molecules appeared as bright diffraction-limited spots. No fluorescent spots were observed in control experiments with pure water. When fl-PA was added, fluorescent spots appeared due to adsorption. Further details of the TIRF microscope, flowcell, general procedures, and data analysis methods used in the current work were presented previously.29 Results Figure 1 shows a representative time sequence of images illustrating how adsorption data were determined. In the first frame, certain diffraction-limited spots are observed. In contrast with our previous work where hexadecane was used as a solvent,29 we do not observe these spots to move with time within our ability to resolve them. This puts an upper limit on the lateral diffusion coefficient of ∼10-4 µm2/s. In the second frame in Figure 1, obtained 2 s later, some spots remain in their original positions, but one of the original spots (marked by an arrow in the first frame) has disappeared due to desorption or photobleaching. In the third frame, a new spot (marked by a triangle) has appeared due to adsorption. This process continues from frame to frame as a function of elapsed time. In order to determine the raw adsorption rate, we simply count the new spots that appear in each frame. These data are averaged to determine the mean adsorption rate. The spots that disappear are irrelevant to this particular analysis. At this point, we note that while some fluorescent emission from molecules moving in the near-surface region may reach the camera as background
Figure 1. A sequence of movie frames showing single-molecule adsorption events and desorption/photobleaching events as a function of elapsed time. The annotated time is relative to the first frame presented. The triangular markers indicate adsorption events, and the arrows indicate impending desorption.
noise, these molecules in solution are moving far too quickly for us to resolve them under the conditions of these experiments. The cumulative adsorption kinetics data were calculated as the sum of new molecules appearing as a function of time. Data from representative experiments are shown in Figure 2a for measurements made at temperatures ranging from 19 to 45 °C. The increase in slope with increasing temperature is clearly visible. The data are presented in units of molecules per site, where a site is taken as the approximate cross-sectional area of an adsorbed fl-PA molecule, 0.25 nm2. While in principle, one could normalize the adsorption rate by any unit of area, we feel that it is scientifically sensible to normalize the adsorption rate by a physically meaningful surface area rather than by an arbitrary value such as the field-of-view. In this experiment, the only relevant surface area or length scale involves the molecular cross-section. This approach is standard in the theoretical/computational literature, and by presenting data in this way, we hope to facilitate the comparison of data from different laboratories and the comparison of experimental data to theoretical models. Figure 2b shows (on logarithmic axes) the measured data at 30 °C along with the calculated adsorption flux for a hypothetical system where the adsorption is diffusion-limited, using the asymptotic form appropriate at short times, Γ(t) ) 2C0(Dt/π)1/2,11,30 where C0 is the bulk solution concentration of fl-PA and D is the solution-phase diffusion coefficient for fl-PA (on the order of 10-6 cm2/s) calculated using the Stokes-Einstein equation. This figure clearly demonstrates that the observed adsorption is much slower (by about 4 orders of magnitude) than would be expected for diffusionlimited adsorption. Furthermore, the linear dependence of the measured data with time is consistent with an attachmentlimited increase in surface coverage. This suggests that an equilibrium is rapidly reached between bulk solution and the
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Figure 2. (a) Representative cumulative number of molecules adsorbed per site (defined as described in the text) as a function of time for experiments performed at different temperatures as annotated. (b) A log-log plot comparing measured data at 30 °C (symbols) with calculated values associated with diffusion-limited adsorption (line).
lowing experiment to establish that the time constant associated with photobleaching is very long compared with the desorption rate and the integration time for the image acquisition. We measured the apparent mean surface residence time (due to a combination of photobleaching and desorption) as a function of the intensity of the laser excitation. As the intensity was increased by approximately 50%, the apparent surface residence time did not change within experimental uncertainty, suggesting that photobleaching is very slow compared with the image acquisition time (and with desorption). Moreover, it is important to note that even if the photobleaching rate were significant, the calculated activation barrier would not be affected. In such a situation, we would fail to count a certain number of molecules that bleached very quickly; this would result in a reduction of the measured absolute adsorption rate. However, this error due to photobleaching would be a temperature-independent scaling factor that would not affect the activation energy (Arrhenius) calculation. Figure 3. Arrhenius plots for fl-PA on FS. The dashed line is a fit corresponding to an activation energy of 19 ( 2 kJ/mol.
Discussion
subsurface layer and that the adsorption kinetics observed in the experiment are dominated by the rate of attachment from the subsurface layer. As described above, the mean attachment rates were determined by averaging the raw frame-by-frame data. These rates are presented in Figure 3 in the form of an Arrhenius plot, that is, logarithm of the rate is plotted versus the reciprocal of temperature. According to standard transition state theory, the slope associated with these data is equal to -Ea/R, where Ea is the activation energy associated with the adsorption process and R is the gas constant. This analysis gives an activation energy of Ea ) 19 ( 2 kJ/mol. In any type of fluorescence microscopy experiment, photobleaching is an omnipresent issue. In our experiments, it is important to consider the possibility that molecules may bleach after such a short surface residence time that they cannot be distinguished from background fluorescence. It is extremely difficult to quantitatively separate photobleaching from desorption in this system because both are first-order processes and occur simultaneously. However, we have performed the fol-
Over the past decade or so, a number of researchers have measured the adsorption kinetics of surfactants at the solution/ solid interface.12-25 However, as discussed above, at best these macroscopic laterally averaging methods can determine the effective net attachment rate, which is a function of both the attachment rate and the detachment rate. Since these latter two rates have different temperature dependences in general, an Arrhenius analysis of the net attachment rate is not expected to be physically meaningful. Appropriately, most researchers have not reported an activation barrier associated with the effective net attachment rate, since it would not correspond to any particular molecular process or state. The molecule counting approach used here is advantageous because adsorption events are directly observed, and not calculated by fitting kinetic data to a model. Furthermore, one does not measure a net attachment rate-attachment and detachment events are independently observed. In the extremely dilute limit under which these experiments were performed (the surface coverage was ∼10-10), adsorbed fl-PA molecules can be considered noninteracting, and site blockage by other adsorbed fl-PA molecules is negligible. Importantly, we obtained data
Fatty Acid Adsorption at Silica/Water Interface
J. Phys. Chem. C, Vol. 113, No. 6, 2009 2081 at the aqueous-fused silica interface. Under steady-state conditions and at extremely low surface coverage, the rates that were determined provided unambiguous measures of the rate at which surfactant molecule were transferred from the subsurface layer to the surface (i.e., the attachment rate). These rates increased with temperature in a manner consistent with an activation energy of 19 kJ/mol. This suggests that adsorption from solution is an activated process under appropriate conditions. We hypothesize that the activation energy is associated with a transition state involving the displacement of a water molecule that is hydrogen bonded to hydroxyl groups on the silica surface. References and Notes
Figure 4. A cartoon illustrating a hypothetical energy diagram for adsorption of fl-PA at the water/FS interface. Part A represents the solvated fatty acid, and part C represents the state where it is adsorbed on the surface. The activated complex shown as a transition state (part B) has a higher energy due to displacement of the water molecule from a surface hydroxyl.
under steady-state conditions, where total adsorption and desorption rates were equal. Under these conditions, the concentration in the near surface layer is constant (and equal to the bulk concentration) and solution transport becomes irrelevant. Therefore, the measured adsorption rates can confidently be assigned as fundamental values associated with the transfer of fl-PA molecules from the subsurface layer to the surface, that is, the attachment rate. Surfactant adsorption is generally an exothermic process as indicated in Figure 4, so an increase in temperature typically leads to a decrease in the equilibrium coverage of surfactant. Hence, it is not intuitively obvious that adsorption should have a positive activation energy, and one does not often find such speculations in the literature. However, our data clearly suggest the existence of a transition state with an energy 19 kJ/mol higher than the solvated fl-PA. We hypothesize that this transition state involves the displacement of a bound water molecule as illustrated in Figure 4. A series of spectroscopic measurements by Shen, Richmond, and others (reviewed in refs 31 and 32) indicate that water molecules are preferentially oriented at silica and other hydrophilic surfaces. This suggests the existence of specific hydrogen bonding between water and surface hydroxyl groups (protonated or deprotonated depending on pH). Since a water molecule must be displaced to permit the attachment of a fl-PA molecule, the energy associated with an OHsO hydrogen bond (∼21 kJ/mol33-37) is a reasonable upper limit for an activation barrier, consistent with our findings. Future experiments with other solvents and substrates will allow us to test this hypothesis in greater detail. Conclusions Single-molecule TIRFM was used to count fluorescently labeled hexadecanoic (palmitic) acid molecules as they absorbed
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