And. C h m . 1992, 64, 1170-1175
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Orientational Dynamics of a Hydrophobic Guest in a Chromatographic Stationary Phase: Effect of Wetting by Alcohol Malcolm E.Montgomery, Jr., M. Anthony Green,and Mary J. Wirth* Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware 19716
The orlentatlonal distribution of the alkyl chalns of a C1, chromatographlc surface was sensed Indirectly from tho orhtatlonal dktrlbutlon of a long hydrophobic probe, 1,4bk(omethyMyryl)benzene. Frequency-domah fluorescence anlootropy meaeurements were used to dotermhe the orlent a t h and roorhtatkn of the fluorescent probe. When pure water Is In contact with the C1, surface, the orientational dktrfbuth of the probe is centered clo8e to the plane of the surface. The effects of two dttferent wetting rdvents were expkred: 20% methanol and 5% lgropanol. When the C18 aurfaco kin contact wlth etther dthme,there Is Mtb change in the orlentatlonal distribution of the probe.
INTRODUCTION Covalently bonded alkyl monolayers are under active investigation in the areas of advanced materiala, sensors, and chromatography. Characterization of the detailed structures and dynamics of these monolayers has been the goal of a diverse group of scientists. A question of frequent interest is the configuration of the alkyl chains. Very dense monolayers of alkyl chains have been well characterized for LangmuirBlodgett films,'alkanethiols on gold,24 and dense, horizontally polymerized alkylsilanes on For each of these densely packed systems, where packing is on the order of 22 A2/chain (7.6 pmol/m2),the chains tilt approximately 30' with respect to the surface normal, achieving a density approaching that of polyethylene. Lower density monolayers of alkylsilanes bonded to silica,e.g., 60 A2/chain (2.8 pmol/m2),'8 are widely used in chromatography because of their ability to solubilize analytes. These systems are not as well understood as the dense monolayers, but one would expect a greater degree of disorder for low-density monolayers. So far, there have been no direct measurements of chain orientation for chromatographic phases. The configuration of the alkyl chains in chromatography has been discussed in the context of wetting and maas transfer between mobile and stationary phases. For example, the slow equilibration of monomeric C18 stationary phases upon a sudden change of mobile phase composition from water to methanol has been explained as owing to a change in conformation of the chains?JO The chains are depicted as lying flat on the surface for an aqueous mobile phase but are extended toward the surface normal in the presence of a wetting mobile phase. By suddenly changing the mobile phase from methanol to water, the idea is that the collapsed chains trap methanol, which is observed to escape very slowly. This picture is widely employed in informal discussions about wetting. This idea of collapsed vs extended chain configurations has not yet been documented by direct experimental measurement.
* Corresponding author. 0003-2700/92/0364-1170$03.00/0
Wetting is important to the dynamics of chromatography. For example, Cole and Dorsey showed that the addition of only a few percent of 1-propanol to an aqueous mobile phase increases chromatographic efficiency almost 2-fold.ll The improved maea transfer was interpreted as owing to increased wetting of the chromatographic surface by the mobile phase due to significant adsorption of 1-propanol onto the hydrophobic surface. As supporting evidence cited, Scott and Simpson had measured the adsorption isotherms for a variety of alcohols in water with a polymeric C18surface, and their results showed that short-chain alcohols achieve saturation coverage on the chromatographicsurface even at concentration levels of only 5% of 1-propanol in water.12 Higher concentrations were shown to be required for methanol and ethanol. Results obtained by McCormick and Karger are in general agreement.13 The faster dynamics are thus associated with surface adsorption of the alcohol. The term wetting is quite familiar from a chemical viewpoint, but it is instructive to point out that wetting has a strict observational definition based on the spreading of a drop of liquid on a ~urface.'~J~ If a drop spreads to such an extent that ita contact angle becomes virtually zero, the liquid is said to wet the surface completely. If a drop interacts so little with the surface that its contact angle exceeds No, the liquid is said not to wet the surface. Adsorbed alcohol presumably has its -OH groups directed toward the aqueous solution, thus fostering a hydrophilic surface that can be wetted, at least partially, by water. This is quite reasonable because, even in the absence of a hydrophobic surface, alcohol collects in ex= at a water/air ~urface.'~J'The observational definition of wetting thus provides insight into the way alcohol covers the surface. This discussion of the role of alcohol in wetting suggests the possibility that alcohol could coat the surface with a layer of -OH bonds to allow wetting without significantly affecting the dynamica of the alkyl chains. N M R spedrometry has been used to addreas the effect of solvent on the dynamica of carbon atoms in the alkyl chains. Bayer et al.'* measured spin-lattice relaxation times, Tl, for the intermediate carbons of a polymeric C18surface as a function of percentage of acetonitrile in water. Tl decreased by about 20% as the fraction of acetonitrile in water increases from 10% to 100%. By contrast, Gilpin and G a n g ~ d a 'used ~ deuterium labeling of the second carbon from the terminus and found that Tl increased 20% and 80% for 60:40 methanol and 100% methanol, respectively, compared to the dry surface. To our knowledge, no NMR studies of wetting by small amounts of alcohols have been reported. Wetting of a hydrophobic bonded phase, (3-pyrenyldecy1)silane on silica, was synthesized and studied by Lochmuller and Hunnicutt.20 The results showed that solvent affects the proximity of adjacent pyrenyl groups: the pyrenylalkylsilane groups are collapsed together when water is the solvent but are farther apart when methanol, hexane, or tetrahydrofuran is the solvent. Stahlberg and Almgren2I 0 1992 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
1171
a Flgure 1. Structure of bis-MSB.
showed that the polarity of CI8,as probed by pyrene, decreased with increasing methanol fraction over the range 0-30%. Cam and Harrisz2observed the opposite behavior over a range of methanol fractions from 50% to 80%: the apparent polarity steadily increased with methanol fraction. Wong, Hunnicutt, and Harrisz3showed that pyrene covalently bonded to the surface sensea no change in polarity as the amount of methanol is changed from 75 % to 100% . These pyrene studies combine to show that methanol might play a different role at low and high concentrationsand that methanol may not be uniformly distributed with respect to distance from the silica substrate. The question of how short-chain alcohols affect the orientations of surface-bound alkyl chains remains open. The purpose of this work is to use spectroscopy to probe the orientation and reorientation of a hydrophobic solute residing in the chromatographic stationary phase. Fluorescence anisotropy is very sensitive to molecular orientation. The probe solute chosen for this work, 1,4-bis(o-methylstyryl)benzene, is referred to as bis-MSB, and ita structure is illustrated in Figure 1. This solute was chosen because its geometry favors preferential alignment with the octadecylsilane chains. A premise behind this work is that any changes in the orientational distribution of the chains will cause related changes in the orientational distribution of the probe solute. To study wetting, three different mobilephase compositions are used: pure water, 20% methanol, and 5% 1-propanol The latter two compositions are expected to wet the surface significantly without affecting the optical properties of the solution. A flat silica substrate is used to allow contact angle measurements and spectroscopic studies with polarized light. THEORY A. Contact Angle. A drop of mobile phase on a flat substrate, which is chemically identical to the stationary phase, will spread until it reaches an equilibrium contact angle, 8. Once the surface, the drop, and the vapor of the mobile phase equilibrate, Young's equation" applies, provided there are no unusual complications such as autophobicity.26 (1) Ysv - Ysl = Ylv COB e This relation represents the balance of forces controlling the drop geometry. The y terms represent the interfacial forces of adheaion between surface and vapor (sv),surface and liquid (sl), and liquid and vapor (lv). The values of ylv are tabulated,%and the value of ysvcan be estimated from that of the alkane liquid in air. The contact angle thus allows calculation of ysl, which would be expected to decrease with increasing coverage of alcohol on the hydrophobic surface. B. Fluorescence Depolarization at Interfaces. The theory needed for designing and interpreting fluorescence depolarization experiments at interfaces has been developed previou~ly.~~ Figure 2 shows the coordinate system used to describe the reorientation of the adsorbate with respect to the surface normal. A trapezoidal coupling prism is used to bring the incident light to the surface. The anisotropy decay for rotation with respect to the surface normal, re@),is defined as27
I, - Iy r&) = I, + 21, where the subscripts indicate that the polarization of the
\ Figure 2. Coordinate systems and optical schematic diagram. The laser beam is coupled into the substrate through a trapezoidal prlsm. The beam totally Internally reflects and exits through the opposite prlsm face. The point at which the beam totally Internally reflects is on the derivatized surface, which is In contact with the mobile phase. Light polarized in the plane of incidence provides raxispdarlzed excitation; light polarized perpendicular to the plane of IncMence provldes yaxis-polarized excitation.
evanescent wave is alternated between the z and y axes. The fluorescence is observed along the z axis without ry polarization discrimination. The z axis is the surface normal. Precautions are needed when evanescent wave excitation is used. First, the evanescent wave penetrates a distance of about the wavelength of light. This is counterbalanced by using a fluorescent probe that is many orders of magnitude more concentrated at the surface compared to the solution. Second, the electric field of the evanescent wave depends on polarization and the refractive indices of the media. Harricka described the electric field, E,at the surface for each polarization vedor x , y, and z, as a function of the angle of incidence and the relative refractive indices of the substrate and solution. It can be shown from Harrick's equations that the intensity is weakly dependent upon polarization: Ey2f E,2 = 0.9 for the refractive indices of 1.46 for silica and 1.33 for water, for an angle of incidence of 72'. For input light polarized along the surface normal, there is also a nonzero E, component of 5%. Since the refractive indices of long-chain alkanes are similar to that of silica,there is some question as to whether bis-MSB should be treated as though it were in the substrate or in the evanescent field. It is assumed that bis-MSB resides in a medium of refractive index of 1.46,rather than in the evanescent field. Although the corrections would be rather small anyway, this assumption will be considered again in the interpretation of the data. C. Spectroscopic Polarization Measurements. Chrcmatographic surfaces are inherently dynamic; this is essential for achieving high column efficiency. Time-resolved polarization studies are therefore necessary for characterizing the orientational distribution. The method of frequency-domain fluorescence anisotropy has been applied to the study of reorientation of solutes on surfaces?' Spherical polar coordinates are used here, where 0 represents the azimuthal angle. For probing reorientation through angles 8, the time dependence of the anisotropy would therefore reveal the extent to which the solute reorients. The limited range of angles through which the solute reorients reveals the orientational distribution. The geometry of hindered rotation for a simple case of wobbling-in-a-coneis illustrated in Figure 3. The reorientation of the ensemble of solutes would be restricted in this case to a limited range of angles, 8, < P(0) < e,. For a system undergoing hindered rotation, the fluorescence anisotropy, re@),does not reach
r&) = [r&O)- r&m)l W
+ rg(m)
(3)
F ( t ) is some monotonically decaying function that is usually an exponential decay. The subscript B is omitted hereafter.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
a
have been described p r e v i o ~ s l y . ~ ~ ~ ~ ~
x+
Flguro 3. Qeometry of hlndered rotation. For illustration, a probe whose o r l e n t a t h are restricted between angles 8, and 8, would be observed to have a fluorescence anisotropy that does not decay to zero.
For any arbitrary functionality of P($,the experimentally determined values of r(0) and r(-) can be related to P(8,) and P(O,), where the subscripts 0 and represent the respective times. QJ
1.5SP(B0)cos2 8, sin2 8, sin Bo deo r(0) =
SP(8,) sin2 8, sin 8, deo
- 0.5 (4)
r ( - ) = l.SSP(8,) cos2 8, sin 8, de, - 0.5
(5)
If the chemical system is homogeneous, meaning that all probe molecules are statistically equivalent, and if excited states behave chemically the same as ground states, then the equilibrium distribution of excited states would be the same as the equilibrium distribution of ground states, i.e., P(8,) = P(8,), provided that the fluorescence lifetime is longer than the reorientation time. To characterize the functionality of the distribution would require many parameters: to use only two parameters, it is assumed that P(8)is Gaussian with mean Or and standard deviation 8,.
There are physical constraints on the possible values of r(0) and r ( m ) because they both have the same equilibrium distributions of probes. For example, r(0) cannot be more negative than -0.5, which corresponds to all molecules lying flat in the plane of the surface. If r(0) = -0.5 then one would expect. r(-) = -0.5 because the probe molecules cannot reorient with respect to 8 if the probes are, by definition, all oriented in the plane of the surface. Similarly, r(0) cannot be more positive than 1,which corresponds to all molecules oriented along the surface normal, and this must be associated with a value of r ( m ) = r(0) again. Further, if the distribution is isotropic,then r(0) = -0.2 and r(-) = 0. Finally, for hindered rotation, as illustrated in Figure 3, the values of r(0) and r( -) are controlled by P(8,) and P(8,), respectively. Inconsistencies in the values of r(0) compared to r ( - ) would mean that P(0,) # P(B,), which would be evidence that the probes were not statistically equivalent on the time scale of the fluorescence experiment. In this work the anisotropy decays are determined using frequency-domain s p e ~ t r o s c o p y In . ~ t~hi~s ~ ~~ technique, the excitation source is modulated, and the phase shift between the fluorescence excited along the surface normal vs in the surface plane is measured for each modulation frequency. Also, the ratio of the amplitudes of the fluorescence signals for the two polarizations is measured at each frequency. The phase shift is termed At$ and the amplitude ratio is termed M,. The Fourier transform relations between these parameters, At$ and M,, and the time-domain parameters of eq 3
EXPERIMENTAL SECTION Methanol and 1-propanolwere obtained from Fisher Scientific, and bis-MSB was obtained from Aldrich. The alcohols were distilled and further purified by passage through a t-C18Sepak column. The water was distilled, deionized, and further purified by passage through a bC18Sepak column. The bis-MSB spectrum showed no obvious impurities and was used without further purification Polished silica plates, cleaned with boiling concentrated nitric acid, followed by ultrapure water, were derivatized with chlorodimethyloctadecylsilaneusing a hexadecane reflux for 3 h. The plates were end-capped with trimethylchlorosilane. nButylamine was used as the catalyst for both derivatizations. Both silanizing agents were purchased from Aldrich. The coverage of the plates was determined by FTIR spectrometry to be 60 f 10 A2/CI8chain (2.8 0.5 pmol/m2). The amount of end-capping was not quantitated. Contact angle measurements were performed using an enclosed housing at room temperature. The volume of the housing was approximately 1L, the droplet volume was 20 pL, and a time of 15 minwas allowed for the plate, droplet, and vapor to equilibrate. The contact angle of the mobile phase on the chromatographic surface was measured by imaging the drop, with high magnification, onto a pair of cross hairs. The angle of the cross hairs was rotated with respect to the surface plane to measure the contact angle to within &lo. A schematic of the optical experiment for studies of bis-MSB in the C18 film is explained with reference to Figure 2. For fluorescence excitation, the laser was frequency-doubled to 330 nm and the incident power was approximately 50 pW. The angle of incidence with respect to the surface normal of the silica plate was set to 7 2 O by retroreflection of the incident radiation from the surface of the 7 2 O trapezoidal coupling prism. The polarization of the excitation beam was controlled by a Pockels cell, and it was confiimed that the polarization extinction ratio was always at least 101. For fluorescence emission, the detection angle was verified to be along the surface normal by observing that scatter disappears for z-axispolarization. The emission was intentionally collected without ry polarization discriminaton. The fluoreecence was filtered using a combination of glass, Corning BG-3 and 400-600 band-pass fiters. The fluorescence was imaged onto the photocathode of a Hamamatau 1635 photomultiplier tube. For steady-state polarization measurements, the photomultiplier output was directed to photon-counting electronics. For timeresolved measurements, the photomultiplier output was directed to frequency-domain electronics, which have been described previously.34 Introduction of the solute bis-MSB into the stationary phase is complicated by the very low solubility of bis-MSB in water. The equilibrationtime of bis-MSB solutions of nanomolar concentration levels was found to be unduly long. Instead, a method for rapidly introducing the solute was devised. The stationary phase was equilibrated with a flowing lo-' M solution of bis-MSB in methanol. The methanol solution was then pumped out by air. The remaining droplets of methanol solution were then rinsed away with pure methanol to avoid colloidal dispersion of bis-MSB in water. The surface was then allowed to equilibrate with the mobile phase of interest. The mobile phase was passed through a guard column to avoid contamination of the surface during the experiment. A period of 30 min was allowed for equilibration with the mobile phase in each cme. It was found that the surface concentration of bis-MSB was not measurably diminished over the time duration of data acquisition,which was typically 1 h. The very high partition coefficient (-105) favors most of the bis-MSB to remain in the stationary phase. The sample housing was constructed such that all parta that come into contact with the solution are Teflon. The bis-MSB adsorption was found to be reversible: the solute was rapidly and quantitatively removed from the surface by using pure 1-propanol as the mobile phase. No evidence of energy transfer or concentration dependence of the fluoreacence was observed. Quantitative desorption of bis-MSB, followed by fluorometric analysis,revealed that the surface coverage of bis-MSB never exceeded 1 molecule/1300 A2. The critical energy-transfer radius of bis-MSB was calculated from ita absorption and emission spectra%and found
*
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Table I. Anisotropy Decay Parameters and Gaussian Orientational Distribution Parameters for bis-MSB as a Function of Mobile-Phase Composition ~(0)
water
I 6.00
I
I iB.0
I
I 30.0
I
I 42.0
I
I 54.0
Alcohol (XI Fbure 4. Dependence of the Interfacial tenslon on the moblie-phase composition. The Interfacial tension, yd,between the C,, surfaceand the moblle phase was determlned by contact angle measurements.
to be 16 A, which is consistent with the observation of negligible energy transfer. The mode-beats of a synchronously-pumped,frequency-doubled dye laser are used as the modulation frequencies. The base frequency is 82 MHz, and the f i t , second, and fourth harmonica are used in these experiments. This frequency range is applicable to subnanosecond to nanosecond anisotropy decays. For each experiment, the fluorescence anisotropy data acquisition was repeated six times. Analpis of the data is accomplished by a grid search to recover the time-domain parameters of eq 3. The fluorescence of the blank surface (no bis-MSB) was 6% for the measurements reported. The blank was further characterized and found to affect the anisotropy data by 1% .
RESULTS AND DISCUSSION A. Contact Angle Information. The contact angle of pure water on the C18surface was measured to be 93O, which indicates that water does not wet the surface. For 20% methanol in water and 5% 1-propanol,the contact angles were measured to be 65 and 69O, respectively, which indicates that the surface is partially wetted by these mobile phases. Contact angles were measured for a range of methanol and 1-propanol compositions and were used to calculate the solid-liquid interfacial tension, ysl, as described by eq 1. A plot of ysl vs percent alcohol in water is given in Figure 4. For the case of 1-propanol, the behavior of yslis consistent with the conclusions of Scott and Simpson, whose chromatographic retention studies showed that 1-propanol achieves saturated coverage on the C18surface above 5% l-propanol.12 For the case of methanol, the behavior of ysIis also consistent with that reported by Scott and Simpson: their chromatographic studies showed that methanol achieves much lower than saturation coverage for concentrations up to 10% methanol. These contact angle studies confirm that the surface is partially wetted by the alcohol solutions and the behavior is generally the same as that observed in high-pressure chromatographic separations on porous silica. For both methanol and 1-propanol, the presence of alcohol significantly affects the interfacial tension. B. Characterization of bis-MSB. To utilize bis-MSB as a probe of the orientational distribution dynamics of the chains, its spectroscopy must first be characterized. It was observed that bis-MSB aligned strongly in a stretched polyethylene film, using the method described by Michl et al.36 Fluorescence was excited 10 times more strongly for the polarization aligned along the film axis. The fluorescence
20% methanol 5% 1-propanol
-0.30 -0.22 -0.18
water 20% methanol 5 % 1-propanol
-0.34 -0.25 -0.20
~ ( m )
x2
e,, deg
e,. deg
Day 1 -0.24 -0.15 -0.10
1.2 1.6 2.4
73 63 60
20 16 18
1.1 1.6 2.3
85 68 61
25 20 18
Day 2 -0.28 -0.18 -0.12
emission polarized along the film axis was 10 times stronger than that of the perpendicular polarization. The extinction ratio of the polarization optics was approximately 10. These results show that the transition moment of bis-MSB is along the long axis of the molecule and that the transition moment for the emission is parallel to that of the excitation. Finally, the fact that bis-MSB aligns strongly in a polyethylene film, which has the same alkyl functionality as the C18 chains of the chromatographic surface, supports the idea that bis-MSB will have preferential alignment with the C18 chains. C. Orientational Behavior of bis-MSB in the C18 Layer. The orientational behavior of bis-MSB was probed using frequency-domain fluorescence anisotropy measurements. A difficulty in carrying out these experiments is that the stationary phase slowly hydrolyzes, which is a well-known problem with monomeric C18 surface^.^' The results detailed here are from a set of experiments designed to ensure that hydrolysis does not affect the interpretation: (1)measurements were made within a single day, where the mobile-phase composition was changed from water to 20% methanol to 5% 1-propanol, and (2) the cycle of measurements was repeated the following day for the same surface. To ensure that the surface was representative, surfaces prepared independently were also studied, and it was found that the trends for the surface described in detail here are representative. Figure 5 summarizes the raw frequency-domain anisotropy data obtained for the fist day the surface was studied. The data show that the behavior of bis-MSB is qualitativelysimilar for the three different mobilephase compositions. The phase shifts are very similar, while the amplitude ratios are distinguishable. Further interpretation requires analysis of the frequency-domain data to recover the timedomain anisotropy parameters. Analysis of the raw frequency-domain data through the Fourier transform relations yields the results summarized in Table I. The data fit well to the hindered rotor model of eq 3; therefore, reorientation is strongly hindered on the time scale of about 3 fluorescence lifetimes, or about 7 ns. The regression was found to be more sensitive to r(0)and r(=) and rather insensitive to the orientational decay constant. The estimated error in each value reported in Table I is expressed by the number of significant figures, with the error in the last significant digit. Just as the raw data show small but measurable differences with differing mobile-phase composition, so do the analyzed data show qualitative similarity but quantitatively resolvable differences with mobile-phase composition. In the regressions, F ( t ) fit to a single-exponential decay; the orientational decay constants were found to be subnanosecond but were neither quantitatively characterized nor needed for the investigation. The Gaussian orientational distribution parameters, 8, and 8, are also shown in Table I. These parameters are calculated from r(0) and r ( m ) through eqs 4-6. The results reveal that there is some day-to-day change in the anisotropy parameters and, consequently, the recovered orientational distribution.
1174
- .BOO
-
-
p-2.40
0
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
tt
t
Y
i
-t
r(
6-4.00 El
m
n z
-5.60
t
,300
f
I
I
-7.20
t: I
I 40.0
I
I 120.
I
I
I
I
1
280.
200.
1
I 360.
Angle ( 0 ) Flgura 6. Calculated orlentatitmi distrlbutkns of bls4SB as a functkn of mobile-phasecomposition. The means and standard deviations of the truncated Gausslans are llsted In Table I.
1 I5% PrOH
O r(
c,
Ratio
.640
I
-
0.61
LT
m
0.53
0
2 .480 r(
a
-
4 E
.320
0.36
-
.160
I
I
I
I
I
I
c
I
Frequency (MHz)
.BOO
.500
n
I
I
1
1
However, this possible origin of the change in amplitude ratio is rejected because the changes in the amplitude ratios measured experimentally are 3 times greater than the change in intensity ratio would be if the environment of bis-MSB changed completely from packed n-alkane to water. The changes in the amplitude ratios must be due to adsorbate orientational changes rather than refractive index changes. Second, it is possible that the distributions are isotropic but decay on a much longer time scale than the fluorescence lifetime. For isotropic distributions, the anisotropy eventually decays to zero and the initial anisotropy is greater than or equal to -0.2. Since any fluorescence experiment has a limited time scale, it is better to make an evaluation from the initial anisotropies. Because r(0) < -0.2 for water as the solvent, one can conclude that the equilibrium orientational distribution is not isotropic in this case. It therefore must be true that its rotation is hindered. The results for either 20% methanol or 5% 1-propanol could be attributed individually to an isotropic equilibrium distribution; however, they cannot both be isotropic. The r(0)value was consistently observed to be less negative for 5% 1-propanolthan for 20% methanol over the course of this investigation. If one of these were isotropic, its anisotropy would have to decay to zero through some timedependent function, F’(t),having a fractionalcomponent, f , instead of decaying to r ( m ) .
r ( t ) = (r(0)- f ) F(t) + f F’(t) (7) It would be hard to support an argument for the applicability of eq 7 for the case of one alcohol, while eq 3 was used to describe the case of water and the other alcohol. One would have to explain why the mechanism of rotational diffusion changed from single exponential to double exponential and then explain why the experimental data so closely resembled one another while the physical interpretation is so different. The argument for hindered rotation for all three mobile-phase compositions is compelling. Third, it is possible that the orientational distributions are inhomogeneous. Time-resolved fluorescence anisotropy measurements are unusual in their ability to sense orientational distributions of surface-bound species. To sense even more detail, such as the homogeneity of the distribution, one could employ two-photon-excited fluorescence anisotropy to create a COS4 0 distribution of excited states.% However, in the case at hand, it is not the details of the orientational distribution that are relevant; it is the fact that the transition
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
moments are clustered near the plane of the surface and are affected little by wetting. Inhomogeneity would not alter the interpretation. The fluorophor bis-MSB was chosen for these experiments because its geometry favors it to align significantly with the chains, and spectroscopic polarization measurements are extremely sensitive to alignment. To the extent that bis-MSB reflects the orientational distributions of the alkyl chains, it can be concluded that the orientations of the chains lie strongly in the plane of the surface when water is present. Also, there is no evidence that they change their orientations significantly when small amounts of alcohol are added to water. The data reject the possibility that the chains are extended toward the surface normal in the presence of small amounts of alcohol: for 8, to be 45O or smaller with a eo of 16O, r(0) would be positive. The negative signs of the anisotropies can only be explained by substantial in-plane projections of the transition moments of the bis-MSB molecules in the ensemble. The addition of small amounts of alcohol has a much more obvious effect upon the interfacial tension, as measured by contact angle, than it has on the orientation of bis-MSB. The effect of wetting on the reorientation of surfaebound acridine orange has been studied.39." Acridine orange resides at both the water/ C18and the waterln-hexadecane interfaces, and interfacial wetting was found to increase its orientational distribution for both interfaces.
CONCLUSIONS The results of this work support the widely held notion that the cl8 chains lie flat on the surface when water is the mobile phase, although the term flat now has an experimental estimate of e,, = 75O. The results do not support the idea that a small amount of alcohol causes the c18 chains to become extended toward the surface normal. The small effect of wetting might at first appear to contradict the observation that methanol is trapped when the mobile phase is abruptly switched from methanol to watersgHowever, the key concept is to make a distinction between wetting the interface and solvating the cl8 chains. At low concentrations, alcohol primarily wets the interface, while at high concentrations of methanol, and presumably 1-propanol,there is evidence that the chains are solvated.'@ The distinction between solvation and interfacial wetting should be used instead of the often used phraseology "alcohol wets the chains". If the interaction of alcohols With is not significantly affected by pressure or pore size, as the agreement between Figure 4 and ref 12 suggests, then these conclusions apply to operating chromatographic systems. ACKNOWLEDGMENT This work was supported by the National Science Foundation (Grant CHE-8814602) and the Department of Energy
(Grant DF-FG02-91ER14187). Registry No. Bis-MSB, 13280-61-0; methanol, 67-56-1; 1propanol, 71-23-8.
REFERENCES (1) Lengmuk-8Mgett Fllms; Roberts, G., Ed.; Plenum Press: New York. 1990. (2) Nuuo. R. G.; DuBols. L. H.; Allara, D. L. J. Am. Chem. Soc. 1000, 112, 558.
(3) Chidsey, C. E. D.; Lolacono, D. N. Lengmuk 1000, 6,682. (4) Wldrlg, C. A.; Alves, C. A.; Porter, M. D. J. Am. Chem. Soc. 1001, 173, 2805. (5) Moez. R.; Saglv, J. J. W . Interface Scl. 1084, 100, 485. (6) Wasserman. S. R.; Whltesldes, 0. M.; Tldswell, I.M.; Ocko, B. M.; Pershan, P. S.; Axe, J. D. J . Am. Chem. Soc. 1080, 7 7 7 , 5852. (7) Unger. K. K.; Becker, N.; Roumeliotls, P. J . Chromatcgr.1078, 125, 115. (8) Roumellotls, P.; Unger, K. K. J . Chromatop. 1078, 749, 211. (9) Scott, R. P. W.; Slmpson, C. F. J . Chrome-. 1080, 197, 11. (10) Glbln. R. K.: Ganaoda, M. E.: Krlshen. A. E. J. c)wwnetoa.Scl. . 10'82. 20, 345. (11) Cole. L. A.; Dorsey. J. G. Anal. chem.1000, 62, 16. (12) Scott, R. P. W.; Slmpson, C. F. Fafaday Svmp. Chem. Soc. 1080,
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RECEIVED for review November 18,1991. Accepted February 21, 1992.
