Anal. Chem. 1882, 64, 2566-2569
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Effect of Sodium Dodecyl Sulfate on the Orientational Behavior of a Hydrophobic Probe in a CI8 Monolayer Bonded to Silica Malcolm E. Montgomery, Jr., and Mary J. Wirth' Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
The orlentatlonal dlstrlbutlon of a hydrophoblc probe, 1,4blr[~ethylrtyryl]benzene,In a monolayer of covalently bonded dlmethyloctadecylslloxane chains has been studled at varlous concentratlons of sodium dodecyl sulfate (SDS) below and above the critlcal micelle concentratlon (cmc). Fluorescence anisotropy measurements were made uslng frequency-domalnspectroscopyfor a macroclcopkaliyoriented sllka plate. For any amount of added SDS, probe reorlentatlon was found to become less hlndered than that measured for a pure water moblle phase. The effect of SDS Is therefore to make the hydrophoblc envlronment on the surface more fluid. Near the cmc, unusual behavlor was observed: the probe reorlentatlon was even less hlndered than it was either above or below the cmc.
INTRODUCTION Retention and selectivity of a chromatographic system can be manipulated by altering the mobile-phase composition. The addition of sodium dodecyl sulfate (SDS) as a mobilephase modifier for reverse-phase HPLC has been utilized to obtain unique solute selectivity. In ion-pair chromatography, the SDS concentration is kept below its critical micelle concentration (cmc) and the SDS molecules act as hydrophobic counterions, forming complexes with charged solutes. The retention behavior of the complex is different from that of the solute alone, thus achieving enhanced selectivity of the solute.' It is known that there is adsorption of the SDS molecules onto the stationary phase below the cmc; however, the role that the stationary phase plays in the retention mechanism has not been elucidated in detail. Micellar liquid chromatography (MLC),which utilizes SDS above the cmc, was first described by Armstrong2 and has been the subject of active research. Micellar liquid chromatography has the advantages of providing unique selectivity, lower cost of operation, and reduced toxicity when compared to organic solvents used for mobile-phase modification.3 The role of the stationary phase in micellar liquid chromatography has been examined by several methods,4v5 and it is generally thought that the amount of SDS adsorbed onto a C18 surface increases until the cmc is reached, after which the stationary phase concentration becomes constant. Above the cmc, therefore, the stationary-phase concentration of SDS would be independent of the bulk SDS concentration. Since there is no further stationary-phase modification, Dorsey et aL4proposed MLC as a good method for performing gradient elutions. The use of micellar gradients would eliminate the need for column reequilibration time, which is
* Corresponding author.
(1)Ion-Pair Chromatography; Hearn, M. T. W., Ed.; Marcel Dekker Inc.: New York, 1985. (2) Armstrong, D.W.; Faruk, N. Anal. Chem. 1981, 53, 1662. (3) Berthod, A,; Girad, I.; Gonnet, C. Anal. chem. 1986,58, 1359. (4) Dorsey, J.G.;Khaledi,M. G.;Landy, J.S.;Lin,J.-L.J. Chromatogr. 1984,316, 183. ( 5 ) Berthod, A,; Girad, I.; Gonnet, C. Anal. Chem. 1986, 58, 1356.
needed when gradient elutions are performed with organic modifiers. It was observed that the efficiency was reduced compared to organic modifiers, but the addition of 3% 1-propanolwith the temperature elevated to 40 OC gave similar separation efficiency.6 Different approaches have been taken to determine the adsorption isotherms and behavior of SDS on various stationary phases, including CIS. Using frontal chromatography, Dorsey and Landy7 have determined the adsorption isotherm and reported a coverage of 1.8 pmol/m2 in 3% 1-propanol. Berthod et al.5 obtained similar isotherms by desorbing with methanol and subsequently titrating for the SDS concentration, although the maximal coverage was found to be 4.5 pmoVm2. Borgerding et al.8determined the isotherm by measuring the increase in carbon content of the packing material and reported a maximal coverage of 1 pmol/m2. T o further elucidate the role of the stationary phase in separations employingSDS, more information is needed, such as how the orientational distribution and the dynamics of a retained solute are affected by the presence of SDS. Also, a spectroscopic study of the dependence of the solute behavior on SDS concentration could independently confirm the isotherm shape in situ, Le., under equilibrium conditions. In this work, the orientation and reorientation of a hydrophobic probe molecule are examined in the presence of SDS at various concentrations below and above the cmc. The fluorescent probe, 1,4-bis[o-methylstyryllbenzene (bis-MSB),is depicted in Figure 1. Its orientation and dynamics are measured by frequency-domain fluorescence anisotropy measurements.
THEORY The theory for performing anisotropy measurements at surfaces has been previously d e r i ~ e d .The ~ anisotropy decay for rotation of the probe from the surface normal, r&), is defined as
where z and y denote the polarizations of the excitation, with the z-axis being the surface normal. The fluorescence is collected along the z-axis without xy polarization discrimination. Spherical polar coordinates are used to describe the reorientation of the probe, where 0 is the polar angle. The time dependence of the anisotropy exhibits the degree to which the probe reorients through the angles of 0. The limited range of angles through which the solute reorients reveals the orientational distribution. Adsorbed speciestypically behave as hindered rotors, because the chemical potential gradient (6) Dorsey, J. G.; DeEchefaray, M. T.; Landy, J. S. Anal. Chem. 1983, 55, 924.
(7) Dorsey, J. G.; Landy, J. S. Anal. Chim. Acta 1985, 178, 179. (8)Borgerding, M. F.; Hinze, W. L.; Stafford, L. D.; Fulp, G. W., Jr.; Hamlin, W. C. Anal. Chem. 1989, 61, 1353. (9) Wirth, M. J.; Burbage, J. D. Anal. Chem. 1991, 63, 1311.
0003-2700/92/0364-2566$03.00/0 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
A
Flguro 1. Structure of bis-MSB.
limits rotation to a restricted range of angles.gJ0 For a system behaving as a hindered rotor, the fluorescence anisotropy, r&), can be described by the following equation.
r&t) = [r&O)- re(-)] exp(-t/.r,) + re(-)
(2) For any arbitrary functionality of the orientational distribution of transition dipoles, P(@,the experimentally determined values of r(0) and r ( - ) can be related by
r(0) =
1.5sP(8)cos' 8 sin' 8 sin 8 d8
-0.5
(3)
s P ( 8 )sin' 8 sin 8 dB r(=) = l.BsP(8)cos' 8 sin 8 d8 - 0.5
(4)
where it is assumed that the ground-state equilibrium distribution is the same as the excited-state equilibrium distribution. If P(0)is assumed to be a Gaussian distribution, it is characterized by two parameters: a mean of 0, and a standard deviation of e,,
(5)
EXPERIMENTAL SECTION Sodium dodecyl sulfate and 1,4-bis[o-methylstyryllbenzene (bis-MSB) were obtained from Aldrich. The bis-MSB was determined spectroscopically to be of sufficient purity and was used without further purification. The SDS was extracted with diethyl ether to remove fluorescent impurities. Someof the SDS was further purified by passage through Sep-pak C18 columns several times until it no longer exhibited an anomalous surface tension near the cmc.ll Surface tension measurements at the liquid/air interface were performed with a CSC-DuNouy tensiometer. The water was distilled, deionized,and passed through a t-Cls Seppakcartridge. The substratesused in the experiments were optically flat silica plates. The plates were cleaned with boiling nitric acid overnight, rinsed with water, and derivatized by chlorodimethyloctadecylsilane in a hexadecane reflux. The plates were then endcapped with trimethylchlorosilane in a hexadecane reflux. Both derivatization steps were catalyzed by n-b~tylamine.~JO The silanes were purchase from Aldrich. The coverage of C18 chains was estimated to be 60 f 10 A2/C18(2.8 f 0.5 rmol/m2) by FTIR spectroscopy. The optical arrangement has been described previously.1° The derivatized surface was placed in a Teflon flow cell where 330nm light from a frequency-doubled synchronously-pumped dye laser was focused onto the surface. The polarization of the exciting evanescent wave was controlled with a Pockels cell. The evanescent wave intensity and the negligible contribution from the bulk solution have already been addressed.lOJ2 The fluorescence from the bis-MSBwas collectedalongthe surface normal, after which it was passed through filters and detected by a photomultiplier. The anode signal was quantitated by photon counting. Aqueous solutions of 0, 2, 6, 8.4, 12, and 24 mM SDS were prepared. Each of these solutions was saturated with bis-MSB to ensure that none of the probe would be leachedfrom the surface chains during the experiments. The solutions were prepared by (10) Montgomery, M. E.,Jr.; Green, M. A.; Wirth, M. J. Anal. Chem. 1992,64, 1170.
(11) Rosen, M. J. J. Colloid Interface Sci. 1981, 79, 587. (12) Internal Reflection Spectroscopy; Harrick, N. J., Ed.; Marcel Dekker Inc.: New York, 1970.
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adding small amounts of bis-MSB in methanol to a flask and evaporating the methanol with a flow of nitrogen. The appropriate SDS solution was added to the flask, and the solutions were stirred for up to 3 days to ensure saturation. The solutions were then filtered to remove any colloidal suspensions of bisMSB and equilibrated to 26 "C in a water bath before being introduced into the flow cell. The solutions were flowed past the surface for the duration of the experiment, which was normally about 1 h. In addition, the mobile phases were analyzed for bis-MSB content by fluorometry and found to correspond to concentrations that provide negligible background contribution. Due to its extremely high hydrophobicity, bis-MSB was introduced into the flow cell in a lo-' M methanol solution where it was allowed to equilibrate with the surface for about 4 min. The methanolic bis-MSB solution was then removed by pressurized air, and the surface was rinsed with a small quantity of pure methanol to remove any droplets of the bis-MSB/methanol solution. Also, it has been previously determined that energy transfer was not occurring on the surface.9 The surface was then equilibrated with the saturated solution of bis-MSB in the SDS micellar solution. The bis-MSB was washed off the surface and reapplied prior to each experiment. Anisotropy measurements were made after a 20-min equilibration period, which was more than enough time for the fluorescenceintensity to reach a stable value. For each solution,the entire experiment,including removal and reapplication of bis-MSB, was repeated six times, and the results of the six experiments were averaged together. The experimentswere repeated in random order of SDS concentration. Time-resolved polarization measurements are required for determining the probe behavior within the CIS chains, since chromatographic surfaces are dynamic. Frequency-domain anisotropy measurements were used to determine the orientational and reorientational behavior of the bis-MSBwithin the CISchains. The instrumentation for acquiring the anisotropy decay data has been previously described.13 For frequency-domain fluorescence spectroscopy, the mode beats of a frequency-doubled synchronously-pumped dye laser were used for modulation of the excitation intensity. The phase shift, A$, of the emission upon excitation with z vs y polarization was measured at each modulation frequency. Also, the ratio, M,,of the emission intensities from z vs y excitation was measured at each frequency. A grid search was used to recover the time-domain parameters.
RESULTS AND DISCUSSION The raw data from the frequency-domain fluorescence anisotropy measurements are shown in Figure 2. The phase shifts for the different SDS concentrations are similar, but large differences can be seen among the amplitude ratios. The lowest amplitude ratios are observed for the pure-water mobile phase, but with the addition of any amount of SDS, the amplitude ratios increase. Near the cmc, unexpected behavior is observed; there is a maximum in the amplitude ratio just below the cmc (8.3mM SDS).Above the cmc, the 12 and 24 mM SDS solutions have similar amplitude ratios. A comparison of the amplitude ratios with an adsorption isotherm is made in Figure 3,which illustrates the constant behavior above the cmc and the anomalous behavior near the cmc. The amplitude ratios have been artificially scaled for this illustration. I t was further determined that the excited-state lifetime for bis-MSB on the surface in the presence of either water or the SDSsolutions was the same as that in a hexane solution, which is 1.24 138.14 Similar data were obtained upon very careful purification of SDSby the Cls Sep-pak columns. Therefore,the phenomenon cannot reasonably be attributed to a surface-active impurity in SDS. To obtain physical insight into how the probe behavior changes with SDSconcentration,the time-domain parameters can be determined by their Fourier transform relations to (13) Wirth, M. J.; Chou, S.-H. J.Phys. Chem. 1991, 95, 1786. (14) Berlman, I. B. Handbook of Fluorescence Spectra; Academic Press: New York, 1971.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
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-2.90 -4.70
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.750
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Flgure 3. Plot of adsorption isotherm obtained from ref 5 and amplitude ratios. The amount of SDS adsorbed onto the stationary phase, as measured by the chromatographic method, Is the solid line, and its scale is shown on the left y-axis. The circles are the frequencydomain amplitude ratios (factored to the same scale) measured at 82 MHr, and their scale is shown on the right y-axis. Both are plotted vs the concentration of SDS in the mobile phase. Table I. Anisotropy Decay Parameters and Guassian Orientational Distribution Parameters for Bis-MSB as a Function of Mobile-Phase Composition
4
a m
n
5
.550
4 4
P
0 2 6 8.4 12 24
4
,450
-0.30 -0.21 -0.16 -0.16 -0.18 -0.18
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0.2 0.8 1.1 1.0 1.5 1.1
1.2 13.2 6.1 8.9 3.5 2.3
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Flgure 2. Raw frequencydomainfluorescence anisotropy data. The differential phase shifts for z-axis vs y-axis excited fluorescence and the amplitude ratios for z-axis to paxis excited fluorescenceare plotted vs the mode beat frequency. The data points are connected by lines so it will be easier to distingulsh between the different solutions. the frequency-domain data, as derived pre~iously.'~J6The recovered time-domain parameters are given in Table I. The values of x2 are high for some of the SDS concentrations; therefore, these time-domain parameters lend insight rather than quantitative interpretation. The same trends are observed as in the raw data. The time-domain parameters r(0) and r ( - ) were fit to the orientational distribution, P(8), through eqs 3-5. The resulting orientational distribution parameters, 8, and 8,, are listed in Table I and plotted in Figure 4. These results show that water causes the probe distribution to lie near the plane of the surface, which is in agreement with previous work.1° The addition of 2 mM SDS widens the distribution, tilting the average away from the surface plane. Well above the cmc, at 24 mM SDS,the distribution is similar to, albeit wider than, that for 2 mM SDS. The time-domain parameters reveal that the hydrophobic environment of bis-MSB at the Cl8 surface becomes more fluid when SDS is adsorbed. This supports a picture where the SDS is adsorbed as a bilayer (15) Klein, U. K. A.; Harr, H.-P. Chem. Phys. Lett. 1978,58, 531. (16) Lakowicz, J. R.; Cherek, H.; Maliwal, B. P.; Gratton, E. Biochemistry 1985, 24, 376.
-m
a
9.00
27.0
45.0
Angle
Flguro 4. Calculated orientationaldistributionsof bis-MSB as a function of mobile-phase composition. The means and standard deviations of the truncated Gaussians are listed in Table I. rather than significantly interpenetrating among the CIS chains. Significant interpenetration would make the environment of bis-MSB more dense, which would impede, rather than enhance, the rotational diffusion. Bilayer adsorption would provide a fluid hydrophobic region accesible to the surface-bound bis-MSB, thus enhancing the rotational diffusion.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
Near the cmc, at 6 and 8.4 mM SDS, the distributions are measurably more directed toward the surface normal (where e,, = 0). At these intermediate SDS concentrations, r ( a ) = 0; therefore, it is mathematically possible that the reorientation is not hindered a t all. Analysis of the time-domain parameters to obtain P(6)thus confirmsthat higher amplitude ratios shown in the raw data indicate that the reorientation of the probe is less hindered near the cmc when compared to the behavioral above and below the cmc. This observation of anomalouslyless hindered rotation near the cmc is reproducible: six independent experiments showed the same behavior, without exception. The anomaly near the cmc is real: no readily apparent artifact would be responsible. The most obvious potential problem would be a contribution from the bulk solution. The absence of bulk contribution was carefully confirmed by determining the signal level from the 10-7 M solution of bis-MSB in methanol upon evanescent wave excitation, and comparing the steadystate fluorescence intensities of the saturated bis-MSB/ micellar solutionswith that of the bis-MSB/methanol solution. Further, a bulk contribution would not account for the raw data for the 12 and 24 mM solutions being virtually the same. For the fluorescence anisotropy of bis-MSB to undergo a hiatus near the cmc means that the Cle surface must be undergoing some type of change. Specifically,the amount of
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adsorbed surfactant must be changing. Despite ita being sensitive to the amount of SDS adsorbed, reorientation behavior does not allow a calculation of the amount of SDS absorbed. There are no direct means of measuring selectively the amount of SDS adsorbed on the C u surface while it is in equilibrium with the SDS solution. For example, infrared or NMR detection of adsorbed SDS would be badly hampered by the bulk contribution. One cannot remove the SDS solution without disrupting the equilibrium, which is what happens in the chromatographic measurement. Surface second harmonic generation avoids the bulk contribution but is indirect in that it is sensitive to orientation rather than just to the amount absorbed. Other experimental approaches are being considered to study this phenomenon further.
ACKNOWLEDGMENT This work was supported by the Department of Energy under Grant DF-FG02-91ER14187.
RECEIVEDfor review March 31, 1992. Accepted July 24, 1992. Registry No. SDS, 151-21-3;vitreous silica, 60676-86-0;1,4bis[o-methylstyryllbenzene, 13280-61-0.
