Anal. Chem. 2000, 72, 3725-3730
Lateral Diffusion of 1,1′-Dioctadecyl-3,3,3′3′tetramethylindocarbocyanine Perchlorate at the Interfaces of C18 and Chromatographic Solvents Derrick J. Swinton and Mary J. Wirth*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
The influence of mobile phase on the lateral diffusion of an amphiphilic dye was studied for four chromatographic interfaces using fluorescence correlation spectroscopy. The fluorescent dye was DiI (1,1′-dioctadecyl-3,3,3′3′tetramethylindocarbocyanine perchlorate); the stationary phase was a covalently bonded monolayer of dimethyloctadecylsiloxane on fused silica; and the four mobile phases were acetonitrile, methanol, tetrahydrofuran, and water. Time-resolved measurements at varying focal positions of the microscope confirmed negligible fluorescence from solution. Single-molecule spectroscopy showed that exchange between mobile and immobile adsorbates was negligible. The lateral diffusion of DiI was the fastest for tetrahydrofuran, D ) 5.3 × 10-5 cm2/s, and slowest for water, D ) 2.5 × 10-6 cm2/s. Methanol and acetonitrile gave intermediate rates of diffusion, D ) 3.4 × 10-6 and 4.8 × 10-6, respectively. There was no correlation of the lateral diffusion coefficient with solvent viscosity, a weak correlation with interfacial tension, and a strong correlation with 13C NMR line shapes for the terminal methyl group of the hydrocarbon chain. The increased wetting of the C18 interphase in the order water < methanol < acetonitrile < tetrahydrofuran agreed with data for the miscibility of these solvents with n-hexadecane. It is concluded that the wetting of the hydrocarbon interphase by the mobile phase enhances the lateral diffusion of the amphiphile. Understanding transport of adsorbates at chemical interfaces is critical in many areas of research, including chromatographic retention, electron exchange, biological membranes, DNA hybridization, and recognition at biosurfaces. Transport is important in chromatography because it controls the speed of chemical separations. Lateral diffusion contributes significantly to intraparticle diffusion,1 which is a factor that affects separation efficiency. Lateral diffusion is particularly interesting because it depends on how the adsorbate interacts with the stationary phase, i.e., it provides insight into the fundamental basis of the separation. There is a growing number of physical studies of adsorbates at chromatographic interfaces, particularly for the interface between solvents and the dimethyloctadecylsiloxane (C18) monolayer on silica. The lateral diffusion of pyrene in the C18 interphase (1) Miyabe, K.; Guiochon, G. J. Phys. Chem. B 1999, 103, 11086-11097. 10.1021/ac0000933 CCC: $19.00 Published on Web 07/20/2000
© 2000 American Chemical Society
in contact with water was sensed by excimer formation.2,3 The lateral diffusion coefficient of an amphiphilic fluorophor, acridine orange, at the C18/water interface was shown to be much smaller than its diffusion coefficient in water4,5 and to drop precipitously with hydrocarbon coverage.6 The lateral diffusion coefficient of rubrene, a nonpolar fluorophor, was observed to be 3 orders of magnitude higher (slower) than its value in solution7 and decreased with coverage of the hydrocarbon monolayer, although the decrease was not nearly as steep as that for acridine orange.8 A possible reason is that rubrene is partitioned into the hydrocarbon interphase, and its slow diffusion is enthalpically controlled, rendering the entropic contribution negligible. The lateral diffusion of rubrene at the C18/water interface was found to increase as methanol was added to the water, with a factor of 2 increase at a level of 20% methanol.7 This was attributed to an increase in chain mobility caused by retention of methanol in the hydrocarbon interphase. The study of lateral diffusion at the chromatographic surface and how it is affected by mobile-phase wetting is in its infancy. Chromatographic retention measurements show that mobile phases wet the C18 monolayer in the order water < methanol < acetonitrile < tetrahydrofuran.9,10 Wetting of the hydrocarbon interphase has been probed directly by NMR spectroscopy. Gilpin and Gangoda showed by 13C NMR that the motions of the terminal carbon on the C18 chain are greater for acetonitrile than for methanol,11 consistent with the chromatographic results.9,10 There is mounting evidence that the dry C18 monolayer has liquidlike behavior. Raman spectroscopy of the C18 chains reveals an orderto-disorder transition at 20 °C to give liquidlike disorder at room temperature.12 Zeigler and Maciel showed, from 13C NMR measurements, that the dry monolayer has a broad line shape,13 indicating the motions of the terminal carbon on the C18 chain (2) Bogar, R. G.; Thomas, J. C.; Callis, J. B. Anal. Chem. 1984, 56, 10801084. (3) Stahlberg, J.; Almgren, M.; Alsins, J. Anal. Chem. 1988, 60, 2487-2493. (4) Zulli, S. L.; Kovaleski, J. M.; Zhu, X. R.; Harris, J. M.; Wirth, M. J. Anal. Chem. 1994, 66, 1708-1712. (5) Kovaleski, J. M.; Wirth, M. J. J. Phys. Chem. B 1996, 100, 10304-10309. (6) Kovaleski, J. M.; Wirth, M. J. J. Phys. Chem. B 1997, 101, 5545-5548. (7) Hansen, R. L.; Harris, J. M. Anal. Chem. 1995, 67, 492-498. (8) Hansen, R. L.; Harris, J. M. Anal. Chem. 1996, 68, 2879-2884. (9) Schunk, T. C.; Burke, M. F. J. Chromatogr., A. 1993, 656, 289-316. (10) Yonker, C. R.; Zwier, T. A.; Burke, M. F. J. Chromatogr. 1982, 241, 269280. (11) Gilpin, R. K.; Gangoda, M. E. J. Magn. Reson. 1985, 64, 408-413. (12) Ho, M.; Pemberton, J. E. Anal. Chem. 1998, 70, 4915-4920. (13) Zeigler, R. C.; Maciel, G. E. J. Phys. Chem. 1991, 95, 7345-7353.
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are slow compared with those of the wetted chains probed by Gangoda and Gilpin. There have been no studies of the lateral diffusion of amphiphiles at the interfaces between C18 and any of the organic solvents commonly used in chromatography. Fluorescence measurements have previously been performed for predominantly aqueous mobile phases due to the need for high surface excess. The use of DiI, an amphiphile with long hydrocarbon tails, allows study of an interfacial adsorbate for highly organic mobile phases. The same technological advances in fluorescence spectroscopy that enabled the observation of single molecules14-16 also enable the study of minute concentrations of adsorbates at chemical interfaces by fluorescence correlation spectroscopy, allowing study of the organic mobile phases that are used in chromatography. The research presented here uses fluorescence correlation spectroscopy to characterize the lateral diffusion of an amphiphile, DiI, at the C18/solvent interface, using solvents that solubilize the C18 alkyl chains to various degrees. The solvents include methanol, acetonitrile, and tetrahydrofuran. Confocal microscopy is employed, where the contributions to the signal from DiI in the solvent and from solvent Raman emission are both minimized. EXPERIMENTAL SECTION Chemicals and Materials. The fluorescent dye 1,1′-dioctadecyl-3, 3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Molecular Probes. Methanol (MeOH), tetrahydrofuran (THF), and acetonitrile (ACN) were HPLC grade solvents obtained from Fisher Scientific. n-Hexadecane and trifluoroacetic acid (spectrophotometric-grade) were obtained from Aldrich. Distilled water was purified using a Barnstead E-pure system. The water was additionally treated with ultraviolet radiation and filtered using a 0.2-µm particle filter. The purified water had a conductance of 18 MΩ cm. Fused silica coverslips obtained from ESCO Products Inc. (R425025, 25 × 25 mm) were cleaned in a 50:50 mixture of nitric acid and chemically modified with chlorodimethyloctadecylsilane (United Chemical Technologies) by refluxing in dry toluene, with n-butylamine as a catalyst. The surface coverage achieved in the procedure is typically on the order of 3 µmol/m2. The chemically modified coverslip was mounted in a Teflon flow cell, which was secured to the platform of an inverted optical microscope (Zeiss Axiovert 100). The fluorescent dye, DiI, was adsorbed to the surface by exposing the chemically modified coverslip to a 20 µM solution of DiI in methanol. The coverslip was then rinsed with a solution of 0.1% TFA in methanol and finally rinsed several times with the solvent of interest until the concentration was significantly reduced to allow observation of individual molecules diffusing through the excitation beam. The laboratory temperature was 22 °C. Fluorescence Measurements. The apparatus was similar to the one used previously.17 The 514.5-nm line of a mode-locked argon ion laser was directed into the back port of an inverted microscope (Zeiss Axiovert 100), which had a filter set optimized (14) Soper, S. A.; Shera, E. B.; Martin, J. C.; Jett, J. H.; Hahn, J. H.; Nutter, H. L.; Keller, R. A. Anal. Chem. 1991, 63, 432-437. (15) Nie, S. M.; Chiu, D. T.; Zare, R. N. Science (Washington, D.C.) 1994, 266, 1018-1021 (16) Schmidt, Th.; Schutz, G. J.; Baumgartner, W.; Gruber, H. J.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2926-2929. (17) Wirth, M. J.; Swinton, D. J. Anal. Chem. 1998, 70, 5264-5271.
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Figure 1. Schematic of the optical microscope used to measure the diffusion coefficient of DiI. A pinhole and imaging optics were placed external to the microscope to achieve spatial filtering.
for detection of DiI (Omega Optical, XF32). The laser beam was focused onto the surface using an oil immersion objective having a numerical aperture of 1.40. This allowed illumination and collection of fluorescence originating at the interface. The dimensions of the focused spot were characterized by imaging the fluorescence from a concentrated interface onto a CCD camera (Roper Scientific, TEK512B). The fluorescence image fit very well to a Gaussian with a standard deviation of 2.0 µm. The excitation power, measured at the surface, was reduced to 100 µW using neutral density filters. The focused spot fit very well to a Gaussian with a standard deviation of 2.0 µm. Background fluorescence from immersion oil was reduced by using a band-pass emission filter. Raman emission from solvent was reduced by adding a cutoff filter (610 nm-EFSP) to the filter set. Background signal from immersion oil was further reduced using spatial filtering, as illustrated in Figure 1. The fluorescence was imaged through a 50-µm pinhole and then imaged onto the active area of an avalanche photodiode, which was in the final image plane. Additional reduction in background from Raman emission was accomplished by time filtering using a time-to-amplitude converter (Tennelec 863), where the start pulse was provided by the sync out from the mode locker and the stop pulse from the avalanche photodiode. The output was sent to a multichannel scalar, with the dwell time set to 4 ms, to give an acquisition time of 65.5 s for the 16 384 bins for each scan. 13C NMR Spectroscopy. The chromatographic silica gel, derivatized with C18, (Waters Corporation, Symmetry C18) had a particle size of 5 µm, a median pore diameter of 90 Å, a surface area of 343 m2/g, and a C18 coverage of 3.23 µmol/m2. Using the method of Bliesner and Sentell,18 an HPLC pump (Waters, 590) was employed to equilibrate the mobile phase with the chromatographic silica gel, using a flow rate of 2 mL/min for 30 minutes. The column was then unpacked, and the silica gel was placed into an 8-mm rotor and closed tightly. It was confirmed visually, after the NMR measurement, that the silica gel was still wetted with solvent. (18) Bliesner, D. M.; Sentell, K. B. J. Chromatogr. 1993, 631, 23-35.
Figure 2. Time-resolved emission of DiI for each solvent, with the position of the microscope objective varied to focus alternately between the surface and the solution.
Solid-state NMR spectroscopy was completed using a Chemagnetics-100 spectrometer. Single-pulse excitation was used along with proton decoupling. Single-pulse excitation was shown to be sensitive to molecular motion of the groups on the unbound end of the alkyl chains, and proton decoupling reduced influences associated with heteronuclear dipolar coupling.13 The sample was spun at a rate of 3 kHz at the magic angle to remove linebroadening influences due to chemical-shift anisotropy. Contact Angle and Surface Tension. Dynamic contact angle (DCA) measurements were made using a Wilhelmy-Balance (CAHN Instruments, DCA-322). The surface tensions of the solvents were confirmed using a DuNouy Tensiometer (CSC Scientific). RESULTS AND DISCUSSION The lateral diffusion of DiI was detected using the inverted optical microscope illustrated schematically in Figure 1. The precise positioning of the pinhole, to be confocal with the object plane, was essential for obtaining a sufficient signal-to-noise ratio for these experiments. The focus of the microscope objective was varied between the interface and the solution to establish that there was no significant contribution to the fluorescence from DiI in the solution. For each solvent, the time-resolved emission is shown in Figure 2 for the two focal positions. The signal from the solution approaches the detector response function in each case, indicating that the signal from solution is primarily Raman emission from the solvent. The signal from the surface in each case shows a contribution from the fluorescence decay of DiI. For the signal from the surface, the ratio of fluorescence to Raman decreases in the order H2O > ACN > MeOH > THF, which is primarily a consequence of the strength of the Raman signal for a given solvent. In each case, the ratio of fluorescence to Raman emission significantly exceeds unity at a time of 0.75 ns after the
peak of the Raman emission; therefore, time-filtering was used to reject Raman signal by adjusting the time-to-amplitude converter to reject counts falling before this time. Examples of the time-filtered burst data are shown in Figure 3 for each solvent, with the focus at the surface vs in the solution. For every solvent, the number and intensity of bursts are both significantly higher when the objective is focused at the surface than when it is focused in solution, as expected. The offset in the signal (counts per 4 ms) is due to Raman emission from the solvent, and this offset is shown to be worse for solution than for surface, as expected. The burst data establish that the signals being acquired are for DiI at the surface, not in solution. A possible complication in the interpretation of lateral diffusion is reversible strong adsorption of DiI to silanols, as revealed by single-molecule spectroscopy.17 If the adsorbate stops frequently at strong adsorption sites in its passage through the laser beam, the lateral diffusion coefficient would be underestimated. Strong adsorption is identified by the fluorescence remaining constant due to the molecule remaining at a fixed position in the Gaussian beam profile. Strong adsorption was previously shown to be rare for DiI at the water/C18 interface, with 99% of the molecules not being adsorbed at any time in their passage through the beam.17 Figure 3 shows comparable behavior, where strong adsorption is observed only occasionally, and an arrow denotes a strong adsorption event in each panel of the figure. As examples, a strong adsorbate is seen for water in Figure 3a at 55 s, lasting 600 ms; one for methanol is observed at 54 s, lasting 100 ms; and one for acetonitrile occurs at 61 s, lasting for 100 ms. For tetrahydrofuran, the signal-to-noise ratio is inadequate for observing strong adsorption by single-molecule spectroscopy, but it would be surprising if this solvent behaved very differently. The conclusion is that most single-molecule events are described by Brownian motion rather than strong adsorption. Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
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Figure 4. Plot of the autocorrelation of the fluorescence counts, again alternating the focus between surface and solution, for DiI at the solvent/C18 interface.
Figure 3. Plot of counts per 4-ms bin as a function of time, showing bursts due to single molecules of DiI diffusing into the beam. The focus of the microscope objective was again alternated between surface and solution.
Figure 5. Autocorrelation of DiI for each solvent with the focus of the microscope at the surface.
A and B are constants, and σ2 is the beam variance in the object plane. Autocorrelations were performed for data sets of 655 s, 10 times greater (longer) than the data sets shown in Figure 3, to obtain a high signal-to-noise ratio. Figure 4 shows the autocorrelations of the burst data for all four interfaces, including data for both the focus at the surface and the focus into the solution. In each case, the corresponding autocorrelations are consistently much noisier and decay faster for focusing into solution compared with those for focusing at the surfaces. For shot noise from Raman emission, the autocorrelations for the solutions would decay after the data point at time ) zero; however, these autocorrelations have a noninstantaneous decay, possibly from 1/f noise of the laser. The solid line in each plot of Figure 4 is the best fit of eq
1 to the autocorrelation of the data corresponding to focus at the surface. The fit is excellent in every case, consistent with the conclusion that the motions of DiI molecules are due to simple diffusion. The autocorrelation decays for each solvent, obtained by focusing at the surface, are normalized and compared on the same scale in Figure 5. Talley and Dunn have shown, from singlemolecule measurements of DiI in membranes, that its fluorescence emission fluctuates, apparently due to twisting motions within the chromophore.20 These fluctuations are expected to affect our diffusion measurements negligibly because the time scale for these fluctuations is more than an order of magnitude more (slower) than the autocorrelation decays in our work. The comparison shows that solvent has a substantial effect on lateral diffusion of DiI. The lateral diffusion coefficients, recovered from the nonlinear regression of the autocorrelations with eq 1, are listed in Table 1. The solvents cause the diffusion coefficient of DiI to change by over a factor of 20. Diffusion is slowest for water, fastest for tetrahydrofuran, and intermediate for methanol and acetonitrile, with the latter giving faster diffusion. Lateral diffusion increases in the order H2O < MeOH < ACN < THF. The simplest models for diffusion relate the diffusion coefficient to the viscosity of the medium. Table 1 lists the viscosities of the
(19) Elison, E. L.; Magde, D. Biopolymers 1974, 13, 1-17.
(20) Talley, C. E.; Dunn, R. C. J. Phys. Chem. 1999, 103, 10214-10220.
The lateral diffusion coefficient of DiI at each interface was determined by fluorescence correlation spectroscopy.19 The autocorrelation function, G, of burst data is related in a straightforward fashion to the diffusion coefficient, D, for two-dimensional diffusion.
G(τ) ) A/(1 + Dτ/σ2) + B
3728 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
(1)
Table 1.
mobile phase water methanol acetonitrile tetrahydrofuran
diffusion coeff. solubility in interfacial × 10-6 viscosity line width hexadecane tension 2 (cm /s) (mPa s) (ppm) (g/mL) (dyn/cm) 2.5 ( 0.1 3.4 ( 0.2 4.8 ( 0.1 5.3 ( 0.4
1.0 0.54 0.43 0.49
No data 2.5 1.1 0.9
immiscible 0.0376 0.0473 miscible
26.76 16.37 16.72 12.84
solvents at room temperature, showing that these change little compared with the changes in the diffusion coefficients with solvent. There is also no correlation between solvent viscosity and lateral diffusion coefficient of DiI. The solvents wet the stationary phase by different amounts, and since DiI is an amphiphile, its diffusion coefficient could be sensitive to wetting of the solvent/C18 interface. Interfacial tension is the free energy required to increase the area of the interface. A lower interfacial tension would lower the barrier for creating an adjacent open space in which the adsorbate could diffuse. The surface and interfacial tensions are listed in Table 1. The experimental values obtained for the surface tensions of the organic solvents are in close agreement with values obtained by Carr and Cheong.21 The interfacial tensions are also listed in Table 1, calculated from the dynamic contact angle22 and using 26 dynes/ cm as the surface tension of the C18/air interface.23 The results show that there is some correlation between lower interfacial tension and faster lateral diffusion; however, methanol/C18 and acetonitrile/C18 have nearly identical interfacial tensions, yet the lateral diffusion coefficients of DiI are distinctly different for the two cases. Wetting of the interface does not appear to be the factor that controls the lateral diffusion of DiI. Since the alkyl tails of DiI might penetrate into the C18 layer, it is possible that the wetting of the C18 interphase reduces friction with the hydrocarbon medium to allow faster lateral diffusion of DiI. The 13C NMR spectra of the stationary phase for all three organic solvents are shown in Figure 6 for the Waters SymmetryC18 silica gel. This material is similar in hydrocarbon coverage to the fused silica sample for which the lateral diffusion measurements of DiI were made. NMR measurements were not performed for the case of water as solvent because it was expected that the pores would not be wetted, on the basis of the work of Rutan.24 The height of each spectrum was normalized to the height of the resonance near 33 ppm, which corresponds to the chain backbone. For the chromatographic silica gel studied here, the band for the terminal methyl group of the C18 chain for each solvent is denoted. In each case, the band is much narrower than that for the C18 monolayer in contact with air.13 A narrower line indicates faster motion; therefore, motion increases in the order MeOH < ACN < THF. The resonance for the terminal methyl group of tetrahydrofuran is almost as narrow as the resonance for liquid tetrahydrofuran, denoted by an asterisk. The resonance for the chain backbone is beginning to be resolved into two different resonances (21) Cheong, W. J.; Carr, P. W. J. Liq. Chromatogr. 1987, 10, 561-587. (22) Rame, E. J. Colloid Interface. Sci. 1997, 185, 245-251. (23) Jasper, J. J.; Kring, E. V. J. Phys. Chem. 1955, 59, 1019. (24) Li, Z.; Dong, S.; Rutan, S. Anal. Chem. 1996, 68, 124-129.
Figure 6. Solid-state 13C NMR spectra for a silica gel, to which C18 is covalently bonded, wetted with the respective organic solvent.
for tetrahydrofuran; there is a shoulder for acetonitrile and no resolution for methanol. This reinforces the conclusion that motion increases in the order MeOH < ACN < THF. This order is in agreement with chromatographic data on chain wetting.9,10 This order also agrees with the miscibilities of these solvents in n-hexadecane, which we measured and listed in Table 1. The NMR data thus reveal that wetting of the hydrocarbon interphase correlates with the diffusion coefficient of the amphiphile. The simplest interpretation is that the lateral diffusion of DiI is controlled by friction between its alkyl tails and the C18 interphase. Since the lateral diffusion coefficient of DiI is 20× slower for contact with water than with tetrahydrofuran, this might suggest that the viscosity of the stationary phase decreases by a factor of 20 when wetted by a miscible solvent. The alkyl tails are not necessarily a reliable viscometer because one does not know how far they penetrate into the monolayer nor whether they penetrate the same amount for all solvents. With the lateral diffusion coefficients for rubrene, pyrene, and DiI spanning three orders of magnitude, it may be hard to imagine that they are probing the same interphase. For rubrene, it was suggested that the slow diffusion could be due to the nonplanarity of rubrene.7 It is also possible that the size of rubrene is comparable to the size of the interphase, which guarantees that there is significant interaction with the stationary ends of the chains. Pyrene and DiI can avoid the stationary ends of the chains, and DiI could interact mainly with the free ends. The three compounds, therefore, may be reporting viscosities in three different regions of the interphase. It is thus possible that the alkyl tails of DiI penetrate into the C18 interphase, yet allow fast diffusion. The results reveal the trend that a wetted interphase enables faster lateral diffusion, but a microviscosity of the interphase cannot be reported. The lateral diffusion behavior of DiI is very different from that of another amphiphile studied earlier, acridine orange. Despite being a smaller molecule, acridine orange has a lateral diffusion coefficient that is 20× slower than that for DiI for the same interface and the same temperature.4 The difference might be explained by the rigidity of acridine orange vs the flexibility of DiI. The lateral diffusion of acridine orange was found to have a large entropic barrier, suggested to arise from molecular-scale roughness of the interface,5 where the highly oriented acridine orange would have to tilt unfavorably to cross these rough regions, exposing its hydrophobic side to water.5 Acridine orange laterally Analytical Chemistry, Vol. 72, No. 16, August 15, 2000
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diffuses an order of magnitude faster at the interface of water and liquid hydrocarbon,6 with a diffusion coefficient comparable to that of DiI at the water/C18 interface. This behavior indicates that DiI does not encounter the entropic barriers experienced by acridine orange. The flexibility of DiI may allow multiple ways of passing over sites that are inaccessible to acridine orange at the water/ C18 interface. The large lateral diffusion coefficient of DiI at the tetrahydrofuran/C18 interface is approximately what would be expected for DiI in solution. This raises the question of whether DiI is adsorbed in the electrical double layer rather than straddling the interfacial boundary between tetrahydrofuran and the hydrocarbon. These two circumstances can be distinguished by calculation. The surface excess due to the double layer could be calculated from the surface potential, if it were known. If the surface potential were assumed, as an extreme case, to be as large as that for bare silica in contact with water, which is 0.14 V,25,26 the calculated ratio of surface concentration to bulk concentration is only 200. The double layer would have to be many micrometers thick to attribute the confocally resolved signal to adsorption into the double layer. For tetrahydrofuran, which has a dielectric constant of only 7.6, it is likely that there are fewer surface charges. The presence of the C18 monolayer would reduce the number of ionizable groups (25) Kohr, J.; Engelhardt, H. J. Chromatogr. 1993, 652, 309-316. (26) Huang, X.; Kovaleski, J. M.; Wirth, M. J. Anal. Chem. 1996, 68, 41194123.
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further, so the surface excess would be even lower. It is reasonable, therefore, to conclude that DiI is adsorbed at the interfacial boundary rather than into the electrical double layer. CONCLUSIONS The lateral diffusion of an amphiphile, DiI, at a chromatographic interface for a varying mobile phase occurs through Brownian motion. For the water/C18 interface, the lateral diffusion of DiI is much faster than that of a previously studied amphiphile, acridine orange, and it is also much faster than that of previously studied hydrophobic probes, pyrene and rubrene. For organic mobile phases, the lateral diffusion of DiI increases with wetting of the C18 monolayer. ACKNOWLEDGMENT This work was supported by the National Science Foundation under Grant CHE- 9610446. Derrick J. Swinton is grateful to the University of Delaware for support by a Presidential Fellowship. Dr. Shi Bai is credited for the NMR data. We are grateful to Ms. Melody D. Ludes for obtaining additional information and optimizing the instrument used in the diffusion experiment as well as for informative discussions. Received for review January 31, 2000. Accepted April 20, 2000. AC0000933
