Anal. Chem. 1994,66, 1708-1712
Lateral Diffusion of an Adsorbate at a Chromatographic &/Water Interface Steven L. Zulli,t John M. Kovaleski,t X. Ron Zhu,* Joel M. Harris,* and Mary J. Wirth'lt Department of Chemistry, University of Delaware, Newark, Delaware 197 16, and Department of Chemistry, University of Utah, Salt Lake City, Utah 84 112
The first measurement of the lateral diffusion coefficient of an adsorbateon a chromatographicsurface is reported. Patternphotobleaching/fluorescence-recoverywas used to measure the lateral diffusion coefficient of acridine orange at the interface of water and a C18 chromatographic surface. Also, the diffusion coefficient of acridine orange in bulk water was measured by the relaxationof a transient holographic grating. The combined results show that the lateral diffusion coefficient of acridine orange at the water/C:lS interface is 35 times smaller than its diffusion coefficient in bulk water: 1.3 (f0.1)X cm2/s for the chromatographicinterface vs 4.2 (f0.5) X 10" cm2/s for bulk water. The slow diffusion at the water/C:l8 surface is interesting in light of previous measurements showing that acridine orange reorients rapidly in the plane of the interface, with an apparent microviscosity comparable to that of bulk water. The rotational and translational diffusion behaviors suggest that the adsorbed acridine orange occupies favored sites at the water/Cls interface which allow it to reorient freely, but these sites exchange slowly due to the slow dynamics of the C18 chains. The study of the dynamics of molecules residing at interfaces is important to the understanding of chromatography, electrochemistry, chemical sensors, and biological membrane processes. Translational diffusion at surfaces and interfaces, where mass transport is restricted to two dimensions, is commonly called lateral diffusion. Fluorescence recovery after photobleaching has been used extensively to characterize the lateral diffusion of fluorescent molecules in biological membrane~l-~ and model membranes."1° Lateral diffusion of molecules on surfaces is a phenomenon important in chromatographic studies because dynamics control separation speed. There have been no previous studies of lateral diffusion at chromatographic interfaces despite the importance of dynamics in chromatography. + University of Delaware. t University of Utah. ( I ) Chahine, J. M.; Cribier, S.;Devaux, P. F. Proc. Narl. Acad. Sci. U.S.A.1993, 90, 447. (2) Bruckert, F.; Chabre, M.; Minh Vuong, T. Eiophys. J . 1992, 63, 616. (3) Boullier, J. A.; Peacock, J. S.;Roess, D. A.; Barisas, B. G. Eiochim. Eiophys. Acta 1992, 1107, 193. (4) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 6739. ( 5 ) Rubenstein, J. L. R.; Smith, B. A.; McConnell, H. M. Proc. Narl. Acad. Sci. U.S.A. 1979, 76, 15. (6)Alecio, M. R.; Golan, D. E.; Veatch, W. R.; Rando, R. R. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 5171. (7) Wright, L. L.; Palmer, A. G.; Thompson, N. L., Eiophys. J . 1988,54, 463. (8) Huang, Z.; Pearce, K. H.; Thompson, N. L. Eiochim. Eiophys. Acra 1992, 1112, 259. (9) Tamada, K.; Kim, S.;Yu, H. Langmuir 1993, 9, 1545. (IO) Smith, B. A.; McConnell, H. M. Proc. Natl. Acad.Sci. U.S.A. 1978,75,2759.
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There are two spectroscopic methods using periodic pattern photobleaching for measuring diffusion. One is the transient holography method,"-l3 where a spatially periodic concentration of molecules is photobleached by an excitation interference pattern; the modulated absorbance and/or refractive index of the sample causes diffraction of a probe beam into a detector. Theefficiency of diffraction decreases with time as the pattern of labeled molecules is washed out by diffusion. The time constant, 7 , for diffusion across a fringe depends on the square of the fringe spacing, d , and inversely on the diffusion coefficient, D.
Since diffraction efficiency depends on the square of the grating amplitude, the time constant for the decay of diffraction is half the time constant for the diffusional decay of the grating amplitude. The other method also involves bleaching with a periodic pattern but uses fluorescence detection of the pattern,I0 which disappears due to translational diffusion. There is a variety of ways of monitoring the disappearance of the pattern fluorometrically, including imaging the probed fluorescence with photographic filmlo or a CCD camera,14 or probing the periodic pattern with a weak beam having the same periodic pattern, while monitoring the total fluorescence.15 In this last case, diffusion of the molecules from the dark fringes to the bright fringes increases the fluorescence signal. Regardless of the method of probing, the recovery of the signal in the bright fringes is an exponential rise with the same timeconstant as that of the transient holography method, given in eq 1. For bulk solution, the transient holographic method is advantageous because it is insensitive to spatial shifts of the photobleached pattern arising from convection. For surfaces, the fluorescence recovery method is advantageous because it provides the high sensitivity of fluorescence spectroscopy, which is the key consideration in surface studies. Convection is not a significant problem at solid surfaces. Acridine orange is a fluorescent molecule that photobleaches irreversibly, eliminating any restriction on the size of the diffusion coefficient that is amenable to experimental measurement. It is well suited as a fluorescent probe for the chromatographic surface because there is a considerable (11) Hervet, H.; Urbach, W.; Rondelez, F. J. Chem. Phys. 1978, 68, 2725. (12) Hervet, H.; Lcger, L.; Rondelez, F. Phys. Reo. 1979, 42, 1681. (13) Zhu, X.R.; McGraw, D. J.; Harris, J. M. Anal. Chem. 1992.64, 710A. (14) Miehlich, R.; Gaub, H. E. Reu. Sei. Insrrum. 1993, 64, 2632. ( 1 5 ) Lani, F.; Ware, B. R. Reu. Sci. Instrum. 1982, 53, 905.
0003-2700/94/0386-1708$04.50/0
0 1994 American Chemical Society
water
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hydrocarbon Figure 1. Structure of acridine orange and its orientation at water/ hydrocarbon interfaces.
body of knowledge about its behavior at this surface. It has been shown that acridine orange is strongly adsorbed to CIS surfaces from aqueous mobile phases, and it is highly oriented at the water/Cls interface,16J7as illustrated in Figure 1. The reorientation about its symmetry axis, termed in-plane reorientation, has a time constant of approximately 500 ps, which is within a factor of 2 of that for acridine orange in bulk water. Its in-plane reorientation is slightly hindered.18 The viscosity of the hydrocarbon phase (CIS, n-hexadecane, decalin, isopropylcyclohexane) has little effect on this reorientation time,lg suggesting that the molecule penetrates little into the hydrocarbon phase. The phase reorientation of acridine orange at the water/ Clg fiterface is indicative of a microviscosity comparable to that of water. Such a fluid environment might lead one to predict its lateral diffusion coefficient at the water/Clg interface to be on the order of its bulk translational diffusion coefficient in water. In this work the lateral diffusion coefficient of acridine orange at the water/Cls interface and its translational diffusion coefficient in bulk water are measured and compared.
EXPER IMENTAL SECTION I. Bulk Diffusion. The diffusion coefficient of acridine orange in bulk water was measured at the University of Utah. The interferometer used for laser-induced diffraction measurements has been discussed elsewhere.20i21For the present experiment, two excitation beams derived from an argon ion laser (Spectra Physics, Model 165) operated at A, = 488 nm were crossed in the sample at an angle, 28, = 4.1 mrad (in air); the spot size (radius) of the excitation beams was 500 Km and the total power in the beams was 300 mW at the sample. The interference pattern produced in the sample had a fringe spacing, d = &/[2 sin(&)] = 59 pm. Probe beam radiation, A, = 488 nm, from a 10-mW air-cooled argon ion laser (Ion Laser Technology) was used to probe diffraction in the sample. The duration of excitation pulses was controlled by an electronic shutter (Uniblitz). The diffracted probe beam was directed into a cooled PMT through a 488-nm bandpass interference filter and pinhole, used to isolate the diffracted (16) Wirth, M. J.; Burbagc, J. D. And. Chem. 1991, 63, 1311. (17) Burbage, J. D.; Wirth, M. J. J . Phys. Chem. 1992, 96, 5943. (18) Piasecki, D. A.; Wirth, M. J. Submitted for publication in Lnngmuir. (19) Wirth, M. J.; Burbage, J. D. J . Phys. Chem. 1992, 96, 9022. (20) McGraw, D. J.; Michaclson, J.; Harris, J. M. J. Chem. Phys. 1987.86, 2536. (21) Zhu, X. R.; Harris, J. M. Chem. Phys. 1991, 157, 409.
plate Figure 2. Optical schematic for lateral diffusion measurement. The lines of the Ronchi ruling were parallel to the ellipsoidal image on the surface. The cell housing was made of Teflon.
signal and reduce scattered light. A second electronic shutter, which was synchronized with the shutter controlling excitation beams, was mounted in front of the PMT entrance to protect the detector during excitation of the sample. The sample was acridineorange (Aldrich) 4.5 X 1@M in water (glassdistilled (Corning) and filtered (Barnstead, Nanopure)). Transient diffraction signals were recorded with a digital oscilloscope (LeCroy) and transferred to a PC for data analysis. The diffraction signals were fit to a single exponential decay model by nonlinear least squares using a Marquardt algorithm** compiled in FORTRAN. 11. Lateral Diffusion. The measurements of lateral diffusion of acridine orange on the cl8 chromatographic surface were made at the University of Delaware. A schematic diagram of the optical arrangement is illustrated in Figure 2. An argon ion laser beam at 488 nm was optically processed to have TEMm mode structure and linear polarization with high extinction. The collimated beam was passed through a Ronchi ruling to generate the desired pattern. Ruling spacings of 50 and 100pm were found to be near the optimum. The beam was spatially filtered to select only the 0 and f l diffraction orders. The resulting sinusoidalgrating was imaged onto the surface through a trapezoidal coupling prism. The optics were designed to provide an image on the surface that was the same as the spacing on the grating, and this was confirmed experimentally. The beam entered the trapezoidal prism at an angle of 72' with respect to the surface normal, which is beyond the critical angle. The evanescent wave was thus used to photobleach and probe. The totally internally reflected beam was directed to a beam stop. The intensity of the beam was switched between bleaching and probing by using a Pockels cell followed by a fixed Glan-laser prism. The fluoresence was collected along the surface normal and passed through a shutter to prevent the bleach pulse from saturating the photomultiplier. A bandpass filter was used in front of the photomultiplier to select the fluorescence emission. Acridine orange does not bleach readily at the water/Clg interface. For the results presented in this work, 5 s was allowed for photobleaching by an unfocused 5 mm diameter beam of 150 mW power. Lower powers and shorter times caused insufficient bleaching, giving noisier data, although the shape of the recovery curves was the same. The opening (22) Press, W. H.; Flannery, B. P.; Tcukolsky, S. A.; Vetterling, W. T. Numerical Recipes; Cambridge: London/New York, 1986.
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of the shutter was synchronized to the electrooptic switching of the beam to less than a 10 mW level for probing. Single photon counting was employed to quantify the fluorescence signal, using Ortec electronics interfaced to an IBM-AT running the ASYST software. Photon counting was performed in 10-ms intervals over the recovery time. The data analysis was performed in ASYST using a Marquardt algorithm. For the surface experiment, it is important to ensure that the chemical systems are very pure. Acridine orange was purified by passage through silica to remove a nonfluorescent impurity. The water was distilled, deionized, and passed through a charcoal filter and, finally, through a Sep-Pak cl8 filter. The temperature of the mobile phase was held at 20 O C using a Neslab temperature bath. The c 1 8 surfaces were prepared as before,16 and this procedure provides surface coverages comparable to chromatographic surfaces, Le., 3 pmol/m2. The surfaces were cleaned with methanol between experiments to prevent possible accumulation of any contaminants. Acridine orange was adsorbed onto the C18 surface from a 6 nM aqueous solution. This has previously been shown to provide an interfacial concentration that is well below the threshold for energy transfer.I6 This low concentration also serves to keep the amount of photolysis product on the surface to very low levels and to minimize the amount of thermal energy deposited during photobleaching. To avoid a possible contribution to fluorescence recovery from readsorption from solution, the 6 nM acridine orange solution was replaced by pure water before the start of each experiment. Due to the very high adsorption coefficient, a negligible amount of desorption occurred during the course of the experiment.
RESULTS AND DISCUSSION I. Diffusion in Bulk Water. Attempts to measure the diffusion coefficient of acridine orange in bulk water using fluorescence recovery after pattern photobleaching were unsuccessful; this was probably due to convection, driven by laser heating of the solution, causing dephasing of the bleached pattern relative to reading-beam pattern. Diffraction from a photobleached grating of acridine orange in water was readily detected, as shown in Figure 3. A 50-ms, 300-mW excitation pulse from a pair of 488-nm excitation beams creates measurable diffraction above the scatter background from the probe beam. A typical bleaching transient shown in Figure 3A is an average of 10 single-shot experiments; the diffraction signal decays, by diffusion of dye molecules in the photobleached pattern, back to the scattered light level within a few hundred milliseconds. Fitting the last 700 ms of the timeresolved signal to a single exponential (Figure 3B), we find a time constant for the decay of t = 210 f 10 ms. The corresponding diffusion coefficient of the dye is D = 4.2 (f0.5) X loa cm2/s, where the error bounds include uncertainty in the fringe spacing. 11. Diffusion at the Water/Cu Interface. The raw data for the fluorescence recovery of acridine orange at the water/ cl8 interface are shown in Figure 4 for grating sizes of 50 and 100 pm. The solid line in each case represents the best fit to eq 1. The diffusion coefficient calculated from the two grating sizes is 1.3 (f0.2) X and 1.3 (f0.4)X lo-’ cm2/s for the 50- and 100-pm gratings, respectively. The error represents 1710
Analytical Chemistry, Vol. 66, No. 10, May 15, 1994
1.27
,
IO
0.87
100
300
500
700
900
Time (ms) Figure 3. Dtffraction from a photobleachedgrating of acridine orange in water. (A) While the detector Is blocked, a 50-ms excltation pulse in an Interferencepattern creates B grating in the sample. Diffraction from this grating decays by diffusion back to the level of background scatter from the probe beam. (B) The decay of dlffracted light Intensity fit to an exponential decay; both signal and best flt exponential are plotted.
the 95% confidence interval, which was determined by replicate measurements. The measurements for the two different grating sizes agree very well with one another, which confirms the absence of effects other than diffusion on the recovery. Pooling the two results gives a diffusion coefficient of 1.3 (*O. 1) X cm2/s. The pattern-photobleaching/fluorescence-recovery measurement thus provides a precise and accurate determination of the lateral diffusion coefficient. These results show that the diffusion coefficient for acridine orange at the water/Cle interface is 35 times smaller than that for acridine orange in the bulk water. Before interpretation, several factors that might make the lateral diffusion coefficient appear to be erroneously small must be considered. The first possibility that would decrease the measured diffusion coefficient of the surface is extreme roughness. Surface roughness would require the adsorbate to travel laterally over greater distances to relax the same photobleached pattern. The surface roughness contribution is readily predictable. For a given roughness feature, the increased distance of travel, E’, and the minimum travel distance, E, are related to one another through the angle, 8, at which the adsorbate is tilted on a roughness feature. For acridine orange, its orientational distribution, P(8), has been determined p r e v i ~ u s l y . ~Averaging ~J~ over the roughness features, the ratio of the two distances is a simple function.
For the c18 surface, E/€’ = 0.97; therefore, the longer distance
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rotational or translational diffusion, the diffusion coefficient is
D = kT/f
.94
(3)
.si
where f is the friction coefficient. The term kT is the contribution from thermal energy, which provides the driving E force for diffusion. The friction coefficient slows the motion B of the molecule. The friction coefficient is proportional to the z viscosity of the environment and the molecular size (radius for translational diffusion and volume for rotational diffusion). The friction coefficient for in-plane rotational diffusion must correspond to that of the lateral diffusion at the interface between two homogeneous media because both are impeded by the same interfacial shear viscosity. In essence, if the molecule rotates in-plane with little friction, then it must .90 translate in-plane with little friction. Both kT andfpoint to a consistency between the in-plane rotational and lateral .82 0 diffusion coefficients. .78 Diffusion will behave according to eq 3 only in the absence of any in-phase potential gradient. Potential gradients are common in out-of-plane rotational diffusion, where the phenomenon of "hindered rotation" has been long known for m e m b t a n e ~ . ~ Acridine ~ ~ * ~ orange has a large potential .62 gradient, which is essentially a potential barrier, for out-ofplane rotation, restricting its orientation to a narrow range of I 3.100 6.bo s.bo 121.0 141.0 1d.0 2d.o 2 d . 0 2 j . o 3 d . o angles.17 Its fast in-plane rotational diffusion is slightly TIME I SECONDS 1 hindered,18 pointing to the presence of a potential barrier in Flgure 4. Fluorescence recovery slgnal for acridine orange at the the plane of the adsorbate, which is encountered by the water/Cle interface: (A) signal using a 50-pm gratlng; (B) signal using a 100-pm grating. molecule as it rotates. In previous work, hindered in-plane reorientation has been associated with surface roughness.19 of travel resulting from interfacial roughness has a negligible The hindered in-plane reorientation on the chromatographic effect upon the diffusion coefficient. surface suggests that surface roughness contributes to the A second possibility might be that lateral diffusion is too slow lateral diffusion. Roughness features would transfer some fast for the data acquisition electronics, and what was observed of thevery high potential for out-of-plane rotation into a higher instead was the recovery of a small subpopulation that was potential in the interfacial plane. A higher potential would strongly adsorbed (at silanols, for example). This possibility exist, for example, if acridine orange diffused to a roughness was addressed early in the experimentation, where 200- and feature that required either that its hydrophobic area be 400-pm gratings were used. N o recovery from faster diffusion exposed to water or its charge be solvated by hydrocarbon. was observed. A third possibility is that the photolysis product The roughness sites would slow the lateral diffusion by virtue from bleaching collects on the surface, impeding the lateral of their higher potentials. transport of acridine orange. In the experiments, typically Since roughness features appear to be responsible for the only 10%of the acridine orange was converted to photoproduct, slow lateral diffusion, this raises the question as to whether as shown by a 10% decrease in the size of the fluorescence the adsorbates overcome the potential barriers, or the barriers signal after recovery. A fourth possibility is that the silica themselves are mobile. The roughness features would migrate plate under study was atypical. This was found not to be the if the underlying chains reconfigured. If this were the case, case; several different silica plates were studied over the course one would observe similar time scales for translational diffusion of the investigation and all gave consistent lateral diffusion of adsorbates at the interface and inside the chains. coefficients. Two indirect measurements of diffusion of sorbates at Clg Now that it has been established that the lateral diffusion silica/solution interfaces have been reported in the literature. coefficient of acridine orange at the water/Clg interface is Bogar et aLZ5and Stahlberg et a1.26observed excimer formation 35X slower than that in bulk water, this result can be rates from pyrene partitioned into C1g ligands on silica in interpreted. It is a rather surprising result in light of the fast contact with solution. Both of these studies reported dynamic in-plane rotational diffusion behavior of acridine ~ r a n g e . ~ ~ J excimer ~ formation, indicating that pyrene diffuses within the Despite the large apparent disagreement between rotational CIS ligands on the surface. Quantitative interpretation of and translational diffusion, both experimental results are these results is difficult, however, since the thickness of the unambiguous, so the interpretation must account for both results. (23) Lipari, G.; Szabo, A. Biophys. J . 1980, 30, 489. (24) Kawato, S.; Kinosita, K. Biophys. J . 1981, 36, 277. To interpret the results requires a careful examination of (25) Bogar, R. G.; Thomas, J. C.; Callis, J. 9 . A n d . Chem. 1984, 56, 1080. the physical processes that underlie diffusion. For either (26) Stahlberg, J.; Almgren, M.; Alsins, Anal. Chem. 1988, 60, 2487. 0
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surface layer is comparable to the size of the pyrene probe. Thus, neither a two-dimensional diffusion model nor a threedimensional model is appropriate for the geometry of the problem. Nevertheless, for the short (2 nm) distances that the probe can diffuse during the 2004s excited state lifetime,27 a three-dimensional diffusion model is a reasonable approximation for a probe such as pyrene that partitions into the c 1 8 p h a ~ e . Using ~ ~ ? a~three-dimensional ~ model for diffusioncontrolled encounters leading to excimer formation, Bogar et al. estimated the diffusion coefficient for pyrene to be D = 2.5 X cm2/s in c18 chains in contact with 75:25 methanol/ water solution.25 Stahlberg et al. measured pyrene excimer formation kinetics in CIS ligands in contact with water containing a small concentration of sodium tetradecyl sulfate surfactant and found a slower excimer formation rate, corresponding to a diffusion coefficient, D = 9 X cm2/ s . * ~ The somewhat smaller value for the latter study is not surprising because surfactants increase the CISdensity30while short-chain alcohols decrease the Cl8 d e n ~ i t y . ~ 'Despite .~~ the 50000-folddifference in distance scales between the pyrene excimer experiment (2 nm) versus the present study of acridine orange lateral diffusion (100 Wm), these results are of comparable magnitude to the pyrene results. The similarity in diffusion coefficients between pyrene, which is inside the (27) Wong, A. L.; Hunnicutt, M. L.; Harris, J. M. J . Phys. Chem. 1991,95,4489. ( 2 8 ) Carr, J. W.; Harris, J. M. Anal. Chem. 1986, 58, 626. (29) Carr, J. W.: Harris, J. M. Anal. Chem. 1987, 59, 2546. (30) Montgomery, M. E.: Wirth, M. J. Anal. Chem. 1992, 64, 1170. (31) Montgomery, M. E.; Wirth, M. J. J . Anal. Chem. 1994, 66, 680. (32) Montgomery, M. E.; Wirth, M. J. Langmuir 1994, 10, 861.
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C18 interphase, and acridine orange suggests that the slow motions of the chain backbones give rise to the slow lateral diffusion out at the interface. This supports the notion that the migration of roughness features controls the lateral diffusion of acridine orange at this interface.
CONCLUSIONS These results demonstrate thevalue in performing a variety of measurements to probe such a complicated and poorly understood phenomenon as chromatographic interaction. Each technique, e.g., measurement of rotational diffusion and measurement of translational diffusion, provides a complementary piece of information. The measurement of lateral diffusion should be added to the set of tools used to probe chromatographic phases because it reveals important dynamic information. It will be interesting to study other chain lengths and to explore CISsurfaces in the presence of other types of mobile phases to study effects of organic modifiers on lateral diffusion. ACKNOWLEDGMENT This work was supported by the Department of Energy under Grant DE-FG02-91ER14187 (to M.J.W.) and by the National Science Foundation under Grant CHE-90103 19 (to J.M.H.). Received for review November 15, 1993. Accepted February 21, 1994. a *Abstract published in Advance ACS Abstracts, April 1,
1994.
