Anal. Chem. 1991, 63,1311-1317
should provide a means of studying time fluctuations in materi& that span a time window of 108-107 orders of magnitude. By determining the fast time fluctuations via infrared VRCFcurveB,it be possible to better intermediate motional models that lead to NMR relaxation for chemically anchored groups. LITERATURE CITED Gengoda, M. E.; Gllpln, R. K. J . Mgn. R e m . 1983,53, 140. GIlph~,R, K.; Qangoda, M. E. J . ctwometog*. &I. 1983,21, 352. Gwpln, R. K.: G e e , M. E. AMI. Chem. 1984,56, 1470. GHph, R. K.: Geengode, M. E. J . Mgn. Rem. 1985,64. 408. Gengoda, -. M. E.; Gllpln, R. K.; Fung, 8. M. J . Mp.Reson. 1987,74, 134.
Albert, K.; Evers, B.; Beyer. E. J . Magn. R e m . 1985,82, 428.
S M ,D. W.: Maclel. G. E. J . phys. Chem. 1982,86, 5208. Slndorf. D. W.: Macbl, G. E. J . Am. Chem. Soc. 1983, 105, 1848. Slndorf, D. W.; Macbl, G. E. J . Am. Chem. Soc. 1983, 105, 3787. clmmbl, G. S.; Leyden, D. E.; Quintlng, G. R.; Maclel. 0. E. Anal.
m.1988,80, 1776. Jk", K. J . -tog*. Scl. 1989,2 7 , 729. PfbldWer, B. A.; A W , K.; Lork, K. D.; Unger, K. K.; Bruckner, H.; Beycrr, E. Angew. Chem., Int. Ed. Engl. 1989,28, 327. BOddenbw, B.: Grosw, R.; Breunlnger, U. Surf. Scl. 1986, 173, L655. K e b W , E. C.: Fyfe, C. A. J . Am. Chem. Soc. 1988, 108, 1746. Gengoda, M. E.: CUlpln. R. K.; Flguelrlnhas, J. J . phys. Chem. 1989. 93.4815. angoda, M. E.; Gllpln, R. K. Lengmuk 1990,6,941
1311
(28)
Gordon. R. G. J . Chem. phys. 1984,40, 1973. WdOn, R. 0. J . Chetn. phys. 19M,42, 3858. Gordon, R. 0. J . Chem. phys. 190,44, 1830. Gordon. R. 0. A&. M g . Res. 1968,3, 1. Ewing, 0. E. J . Chem. phys. 1982. 37, 2250. van Woerkom, P. c.; de s k i m , J.; de Zwart, M.; Bwgers, p. M. J.; Leyte, J. c. aer. Besunges. phys. chem. 1974,78, 1303. Lynden-Bell, R. M. Md. phys. 1977,33,907. Evans, M. E.; Evans, G. J.; Coffey, W. T.; Grigolinl, P. Mkc&r Dynamlcp and Thsory of Bmed Band Spectroscopy; John Wlley 8 Sons: New York, 1982; Chapter 6. Rothschlld, W. 0. J . Chem. phys. 1972,57. 991. Rothschlld, W. 0. @"ks ofkWi?c&r L@u&fs; John Wlley 8 Sons: New York, 1984. Rothschild, W. G. Vlbratbnel Specira and Mohular Stnrcfwe; Dudg, J. R.. Ed.: Elsevier: Amsterdam, 1988; Vol. 15. D 57. Gangoda, 19, 283. M. E.; Gllpln. R. K. J . LebeM Compd: Rad-rm. 1982,
(29) (30) (31) (32) (33) (34)
Suffolk, B. R.; Gilpln. R. K. Anal. Chem. 1985,57, 596. Orbva, N. D. Opt. Spectrosc. 1983, 15, 112. Bulenln. M. 0.; Suffolk, B. R.; Gilpln, R. K. Anal. Chem. Acfa 1986, 181, 259. Rothschlld, W. 0. J . Chem. phys. 1978,65. 455. Morel, D.; Secplnet, J. J . Chrometugr. 1980,200, 95. Hann, C. J.; Gllpln, R. K. Anal. Chem. 1989,61, 1534.
(17) (18) (19) (20) (21) (22) (23) (24) (25) (28) (27)
RECEIVED for review September 28,1990. Revised manuscript received February 28,1991. Accepted March 7,1991. This work was supported by US. Army Research Office Grant DAAL03-90-G-0061.
Adsorbate Reorientation at a Water/(OctadecylsilyI)silica Interface Mary J. Wirth* and John D. Burbage Department of Chemistry & Biochemistry, University of Delaware, Newark, Delaware 19716
Mathematkai reiatlons are derived to allow experimental study of adsorbate reorientation at a MfaceAquM Intetrface. It k shown from these reiatlons that experiments can be devbed to probe separately the rotations In the plane of the wrlaco and the rotations away from the plane of the surface. Thb new technique k applkd to the study of acrkline orange IntracMng with an (octadecyMyi)dllca surface in equllklun with an aqueous soiutlon. Frequencydomain spectroscopy k used to characterize the fluorescence anisotropy decays of acrldlne orange. The experlments reveal that acridine h ~ orangeka ~ h l n c k r d r o t o r w l t tooutofplsne rotational motions but is able to rotate slowly in the surface plane. The interpretation is that acridine orange resides at the interface of water and octadecyisliane.
INTRODUCTION An understanding of adsorbate interactions with molecular
films is valuable for the development of sensors, opto-electronic devices, and molecular-scale architecture and for describing the molecular basis of chromatographic selectivity. Fluorescence spectroscopy has the high sensitivity required to probe dilute submonolayer adsorbates. Applications of fluorescence spectroscopy to the study of probes interacting with dimethyloctadecylsilyl(ODS) groups covalently bonded to silica have revealed heterogeneity of solvation energies (1-3), ODS polarity as probed by pyrene symmetry perturbations
* Author to whom correspondence should be addressed. 0003-2700/91/0363-1311$02.50/0
( 4 , 5 ) and the solvatochromic effect (61, and average viscosity as probed by pyrene excimer formation (7,8). A variety of other techniques has been used to study ODS and similar alkylsilyl surfaces. Contact angle measurements assess coverage of alkylsilanization on flat surfaces (91, and ellipsometry sensea film thickness (10). NMR details the correlation times of the motions of constituent atoms of alkylsilyl groups, which are related to viscosity changes and phase transitions (11). Thermodynamic measurements have been applied to alkylsilane-bonded phases to document temperatures and enthalpies of phase transitions (12). Reorientation measurements offer new information. First, the geometry of the adsorbate/surface interaction can be explored. The orientations of the adsorbate molecules and the rotation rates about the adsorbate axes comprise detailed information about the adsorbate/film interaction. Second, the adsorbate dynamics are sensitive to both the friction and the short-range structure of the microscopic environment. Adsorbate dynamics are important to the speed of sensors and molecular communication channels and the efficiency of liquid chromatography. This is the first detailed study of the reorientation of an adsorbate on a surface. The angular directions of the adsorbate rotations and the time scales of these motions are heretofore unknown. In this work, the (dimethyloctadecylsily1)silica surface is chosen because it is widely used and studied. Acridine orange is selected as the adsorbate because it has a high affinity for the chemically modified surface and its Cz,, symmetry simplifies the interpretation of the reorientation behavior. The structure of acridine orange, its van der Waal's dimensions, and the coordinate system used to refer @ I991 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991
?+ =
decay of the fluorescence intensity is designated as F(t). The intensities I j k rely upon the directions of the absorption and emission transition dipole moments, p: and p,, respectively, where p, is time-dependent. By using the spherical polar coordinate system of Figure 2 and representing the angular averages as brackets, the functions I j k are straightforwardly expressed. For simplicity, it is approximated that p: = p:.
H
f Y:20.3 f X: 4.0 f Flguro 1. Structure of acridine orange at neutral pH. Z:9.4
Zyy = F ( t )(cos2 4: cos2 4,) (sin2 e: sin2 e,)
(1)
Iyx= F(t)(cos2$2 sin2 #e)(sin2 e: sin2 e,)
(2)
Izy = F ( t )( (cos2 4:
In-plane rotations
Out-of-plane rotations
Figure 2. Surface Coordinate system. The surface normal is along the z axis, and the surface plane is the xy plane. In-plane rotations occur through angles 4 . Out-of-plane rotations occur through angles 8, where rotation toward the y axis is depicted as an example.
to its axes are illustrated in Figure 1. The direction of the transition dipole moment of acridine orange has been shown by stretched-film work to lie primarily along the y axis (13). The (octadecylsily1)silica surface is in equilibrium with an aqueous solution of acridine orange at neutral pH. Both the ground state and excited state of acridine orange are protonated at neutral pH in water. No alcohol modifiers are used in this work.
THEORY The commonly used relations for fluorescence anisotropy experiments were derived for macroscopically isotropic systems. These are not applicable to the surface experiment because the surface-bound solutes may have an orientation with respect to the laboratory coordinates. The anisotropy relations applicable to the surface experiment are derived here. These relations differ from the isotropic case, necessitating revised equations used for analysis of anisotropy decays in frequency-domain spectroscopy, which are also provided in this section. The laboratory coordinate system chosen is illustrated in Figure 2, which shows that the surface normal is the azimuth in spherical polar coordinates. Rotations through angles 4 are referred to as in-plane rotations and those through angles 8 as out-of-plane rotations. The choice of coordinate system makes use of the compelling assumption that the orientations of solutes are isotropic in the plane of the surface: there is no chemical potential gradient in the plane of the surface to destroy the planar symmetry. A Cartesian coordinate system is also provided to define polarization vectors. The basis of fluorescence anisotropy measurements is that, upon excitation of the sample with polarized light, the polarization of the emission intensity is related to molecular orientation. The surface sample can be excited with light polarized either in-plane (along the y axis) or out-of-plane (along the z axis) by using evanescent waves. The detector is positioned along the surface normal and can detect emission polarization along either the y axis or the x axis. It is thus possible to perform three independent types of polarization experiments, where the intensity of the fluorescence Ijk. results from j-axis excitation polarization and k-axis emission polarization. These three experiments sense I,, Iyx,and I*,,.The
+ sin2 $:)cos2
4e)(cos2 e: sin2 e,) (3)
In these equations, 4O and eoare the angles of both the excitation and emission dipoles at time zero and 4 and 8 are the angles of the emission dipoles at an arbitrary time. A. Isotropic Systems. The relations of eqs 1-3 are general: they apply to both isotropic and oriented systems. Application to the isotropic system is not new but is presented here for completeness. For an isotropic system, the anisotropy is represented as rh(t), which can be obtained from any Ijj and I j k , such as Iyyand Iyx. riso(t) = (Zyy - Iyx)/Uyy + 2Zyx) (4) Upon integration over 4 from 0 to 2 r and integration over 8 from 0 to r/2, it is readily shown that ~ ~ (=00.4) and rlo(m) = 0, as expected. Izy can be substituted for Iyx to obtain the same numerical results. This illustrates the general applicability of eqs 1-3. It also illustrates that it is artificial to associate particular angular motions, 4 and 8, with particular intensity components in isotropic systems: the same results are obtained regardless of which parallel and perpendicular components are used. Instead, one averages over the laboratory coordinates to obtain the familiar expression ris0(t)= (2/5) (1.5 COS' y t - 0.5) (5) where y t is the angle between the excitation and emission polarization vectors as a function of time. Equation 4 is implemented in frequency-domain fluorescence anisotropy by using the relations for the intensities. I,,= F ( t ) [ l+ 2r&)] (6)
m)[l
I, = - riE,(t)l (7) Using the mathematical Fourier transforms of these intensities gives the differential phase shift, A4, and the amplitude ratio, M,,for the parallel and perpendicular intensities as a function of modulation frequency. This application of Fourier transform relations for exponential functions has been outlined in detail previously (14, 15) and has been reviewed (16). Application of eqs 1-3 to macroscopically oriented systems is the novel aspect of this work. The anisotropy relations for oriented systems are derived in the remaining parts of this section. B. Anisotropy Decay for Motions about 4. In the case of solutes interacting with surfaces, it becomes possible to associate particular intensity components with angles 4 and 8 because the surface coordinates are defined with respect to laboratory coordinates. One can use eqs 1-3 to design an experiment that isolates motions about angles 4: alternate detection of Iyyand Iyx.The anisotropy decay is designated as r+(t).
Substitution of eqs 1 and 2 into eq 8 yields a correlation function containing only angles 4. r+(t) = 4(coS2 $2 cos2 4) - 1 (9)
ANALYTICAL CHEMISTRY, VOL. 63,NO. 13, JULY 1, 1991
The physical picture associated with eqs 8 and 9 is that motions about angles 8 do not contribute to the decay because ZW and Zyx sense outsf-plane motions equally. Experimentally, fixing the excitation polarization in the y direction and alternating the emission polarization between y and x isolate the anisotropy decay for angular motions in the plane of the surface. Two limits for the values of r&) are of interest. First, the value of r4(0),i.e., the initial value, is 0.5 in the limit of parallel excitation and emission dipoles. Second, the value at time infinity, rt(=), is zero if rotation is allowed over all angles 4. C. Anisotropy Decay for Motions about 8. To study angular motions in 8,Zzy can be detected alternately with (I,,, + Zxy)/2,with the anisotropy defined as r&).
Substitution of eqs 1-3 into eq 10 gives a correlation function containing only angles 8.
r&) = 1.5(C0S2 0O sin2 8)/(sin2 0)- 0.5
(11)
The physical picture associated with eqs 10 and 11 is that motions about 4 are not sensed because,first, excitation along z populates all angles 4 equally and, second, detection of both y and x components senses all angles 4 equally. Experimentally, Zzy is detected by exciting with the polarization vector along the surface normal and the emission vector along the y axis. The quantity (Z,, + Zyx)/2is detected by exciting with the polarization vector along the y axis and the emission vector at 45O from the y axis, in the plane of the surface. An equivalent measurement of re( t ) is obtained by alternating between (Zzy + Zaz)/2and (I,,,+ Zp)/2, which requires varying only the excitation polarization. The limits for the value of re(0) and re(-) are -0.2 and 0, respectively, for rotation over all angles 8. To interpret frequency-domaindata, the functionalities of the decays for r&) and r,(t) must be known. In isotropic systems, the anisotropy decay is exponential because the mathematical solution to the diffusion equation is exponential (17, 18). It has been shown for hindered rotation that the anisotropy decay is also exponential (19). It is assumed here that the functionality of the anisotropy decay in oriented media is also exponential. The anisotropy decay functions for the oriented system, eqs 9 and 11,are different from that for the isotropic system, eq 5. This changes the equations for which the Fourier transforms are applied in frequency-domain spectroscopy. In the case of r4, the appropriate relations are
IYy = F ( t )( sin2 eosin2 e)[ 1 + r,(t)]
(12)
Iyx = F(t)(sin2eosin2 e ) [ i - r&)]
(13)
and The appearance of the term (sin2eosin28)in these relations necessitates that the study of re(t)be completed before analyzing the behavior of r&). The physical picture for the appearance of this term is that both Zyy and Zyz decrease as the electronically excited adsorbates tilt away from the surface plane. For re, the relations are I,, = F(t)[l + 2r&)l (14) and Uyy
+ ZYX)/2= F(t)[l - r o w 1
(15)
Fortunately, there is no dependence on 4 in these relations. The functional form of re(t ) is straightforwardlyrelated to (sin2 eosin2e),allowing r,(t) to be studied from the results of re@).
1913
The modifications to existing software for data analysis with frequency-domain spectroscopy are thus required only for the case of r+(t)and are very minor. D. Hindered Rotation. In the interpretation of the measured anisotropy decays for species interacting with surfaces, the possibility of hindered rotation must be anticipated. Hindered rotation occurs when there is a specific directional interaction between the solute and the medium, such as a membrane or a micelle, that restricts the range of angles through which the solute can rotate. Hindered rotation thus prevents the anisotropy from decaying to zero; therefore, the anisotropy decay has the form
r ( t ) = r(0) exp(-t/.r)
+ r(m)
(16)
The value of r(-) contains information about the range of angles through which the solute can rotate. The particular form of r(-) for hindered rotation in isotropic media has been derived by several groups (1S21). These relations do not apply to the problem of a solute reorienting on a surface. In this case, hindered rotation can potentially occur for motions associated with either 4 or 8. Here, the forms of the anisotropy decays for hindered rotation about 4 and 8 for the surface-bound solute are derived. For motions about 4, hindered rotation would occur if there were a restricted range of angles in the surface plane through which the solute could rotate before encountering a barrier. For example, covalently bound octadecylsilyl groups are stationary at the point of attachment to silica, and a solute encountering this region could experience a high barrier to rotation. The anisotropy decay function would be described by r?(t), where the superscript h denotes hindered rotation. For rotation through a range of angles h$with , respect to a given angle 4, at infinite time the correlation function is a convolution of the initially excited distribution with the final angular distribution. r+h(m)
=
4 / ( 2 7 r 4 , ) ~ 2 r ~ + r c obo s 2cos2 (4O
+ 4) d d ddo - 1 (17)
Upon integration, this relation simplifies to the product of the initial anisotropy and a sinc function in 4., ~ + ~ ( m ) / r + (=o sin ) (24,)/(24,)
(18)
From the anisotropy remaining at time infinity, the range of angles in the surface plane through which the solute rotates can thus be calculated from the experimental data by using eq 18. For hindered rotation through angles 8, the problem is different because the ground-state equilibrium distribution is not necessarily isotropic with respect to 8. In this model, the solute is allowed to tilt only through a range of angles of ~ / to2 7r/2 - 8, with respect to the surface normal. Physically, this would correspond to a directionally favored interaction between the solute and the surface. The correlation functions are normalized by p-' = Jsin 8 de.
reh(t) = 1.5
~ ; ~ ~ Opo ol=/r /P2 sin2 8 sin 8 de sin eodeo 2-8,
*/2
l/2-8sin2eosin
- 0.5
eodeo
(19) The initial anisotropy depends on e,, and the anisotropy at infinite time is a simple function of 8,. rgh(-)
= -0.5 cos2
e,
(20)
Equation 20 can thus be used for calculating from the ex-
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 13, JULY 1, 1991
Aqueous solution
I Figure 3. Optical arrangement for evanescent wave excitation of
fluorescence.
perimental data the degree to which the solute can tilt away from the plane of the surface. It is noted that the range 7r/2 - 8, to r / 2 is chosen, rather than the range 0 to 8, for the particular case of acridine orange. The opposite circumstance, where the transition moment is preferentially directed toward the surface normal, is described simply by changing the limits of integration to 0 to 8,. EXPERIMENTAL SECTION Acridine orange, chlorodimethyloctadecylsilane, and chlorotrimethylsilanewere obtained from Aldrich. Acridine orange was further purified by column chromatography using silica and methanol as the stationary and mobile phasea. Fused silica plates, obtained from Quartz Products, were used as substrates. These plates originate from a silica ingot that is cut into plates, and each plate is polished to achieve optical flatness. The surfaces of the plates were derivatized by procedures adapted from the literature (22-24). The plates were refluxed in boiling concentrated nitric acid for 24 h, rinsed in distilled, deionized water, and dried at 200 OC for 24 h. The plates were transferred in a glovebag into 500 mL of anhydrous toluene solution containing 5 mL of chlorodimethyloctadecylsilaneand 1mL of pyridine, which catalyzes silanization (25). The mixture was refluxed for 24 h. The ODS-derivatized plates were rinsed with toluene, hexane, and then methanol and were warmed to evaporate the solvents. The derivatized plates were stored under nitrogen until use. For end capping, the ODs-derivatized plates were dried over dessicant for 24 h, transferred under glovebag to 500 mL of anhydrous toluene containing 5 mL of chlorotrimethylsilane and 1 mL of pyridine, and refluxed 24 h. The plate WBB preas-fit into a Teflon housing. Thermodynamic equilibrium between the surface-bound acridine orange and the aqueous acridine orange solution was maintained by continuously flowing the solution through the housing. The solution was deoxygenated and a positive presaure of nitrogen was maintained in the flow system. The optical arrangement for the experiment is illustrated in Figure 3. The 476-nm line of a mode-locked argon ion laser was passed through a band-pass filter to eliminate superradiance at other laser lines and was focused, using cylindrical optics, to a 1 0 ” by 1-mm line onto the inner surface of the plate. A fused silica,trapezoidal coupling prim, obtained from Precision Optical, and silica index matching fluid from Cargille Laboratories were used. The laser power was limited to 1 mW to minimize photolysis. The excitation polarization was generated by using a Glan-Thompson prism and was controlled by a Pockels cell. The emission was collected along the surface normal by using cylindrical optics. The polarization extinction, which was measured by using scatter, was in excess of 100. The emission polarization was selected by a high extinction Polaroid. The intensity of the evanescent wave at the surface was calculated from the relations of Harrick (26)for each polarization and was used to normalize the intensity measurements. The rotation angle of the Polaroid was controlled by a stepping motor. The fluorescence was isolated by using a band-pass filter. A Hamamatsu 1635 photomultiplier was used. For steady-state measurements, the photomultiplier output was directed to a picoammeter. For reorientation studies, the output was directed to the frequency-domain electronics, which are described in detail
in previous work (27). The basis of these electronics is a highfrequency lock-in amplifier to sense the phase and amplitude of the modulated emission signal. The linearity of the phases and amplitudea of the Photomultiplier current were carefully calibrated with reapect to signal intensity. The experimental conditions were found to lie within the linear regions of the photomultiplier response. Background subtraction was accomplished by mechanical chopping of the laser beam, whereupon the in-phaseand quadrature outputs of the high-frequency lock-in amplifier were directed to low-frequencylock-in amplifiers tuned to the chopping frequency. The data acquisition and polarization control are accomplished by using a Keithley 500 Seriea system, and IBM-AT, and ASYST software. The data analysis is the same as described previously (27). RESULTS AND DISCUSSION I. Adsorption Isotherm of Acridine Orange in (Octadecylsilyl)silica/ Water. Acridine orange has a high affinity for the ODS surface when water is the mobile phase and is very quickly removed from the surface by using a pure methanol mobile phase. The adsorption of acridine orange from aqueous solution is quantitatively reversible. Indistinguishable behavior is observed from different plates derivatized in different batches and for different positions on the same plate. A readily detectable fluorescence signal is observed for acridine orange on the ODS surface by using the optical scheme illustrated in Figure 3. The evanescent wave penetrates into the solution by a distance on the order of the wavelength of light. The signal from acridine orange in the bulk solution was found to be negligible by using an underivatized silica plate, and no signal above the blank is expected for the submicromolar concentrations used. For all surface studies, a blank signal larger than the dark current was observed. Its origin was not investigated. The nonzero blank limits the lowest concentration studied in this work to 4 nM, for which the blank is 6% of the signal size. Below this concentration,the blank contribution significantly af€ecta the interpretation of the data. The adsorption isotherm of acridine orange for the (octadecylsilyl)silica/water system was measured by quantitative desorption of acridine orange from the surface for a series of solution concentrations. The fluorescence intensity for each concentration, divided by that for maximum coverage, gives the fractional surface coverage, 8. The data are fit to a Langmuir isotherm, 0=
KCu/ c v 1 + KCu/C,
where C , and C, are the concentrations of solute and solvent for the aqueous solution and K is the partition coefficient. Both K and the maximum coverage were adjustable parameters. The form of eq 21 allows a unitless expression of K, which is the ratio of mole fraction of acridine orange in solution to mole fraction of acridine orange on the surface. The best fit, illustrated in Figure 4, corresponds to a partition coefficient of 7 X 108. The possibility that the data could be described better by a Frumkin isotherm (28)was explored, but no improvement was observed. The chief source of error in the isotherm was in reproducibly preparing the very low concentration aqueous solutions. The glassware was preequilibrated for each solution to minimize this error. The Langmuir isotherm in Figure 4 is linear from 0 to approximately 20 nM in solution concentration. The large value of K is consistent with the conclusion that the fluorescence is attributable only to surface-bound acridine orange, with negligible contribution from the solution. At the high end of the scale of surface coverages in Figure 4,the intermolecular distances become very short. For example, a 100 nM solution concentration provides a surface
ANALYTICAL CHEMISTRY, VOL. 63,NO. 13, JULY 1, 1991 1311 F
,900
I
0
-
200 nu
>
IU
.700 I-
-
E
100
Y '
30.0
I
1
90.0
I
I
150.
I
I
210.
I
I
270.
1
1 2.00
I
I
SOLUTIDN CONCENTRATION InUI
Flgwe 4. Adsorptkn isotherm for acridine orange on ODs In contact wlth water. The circles represent data obtained by quantitative desorption and subsequent analysis. The solid line Illustrates the best fit
to a Langmulr isotherm.
coverage of 9 X 10-l2mol/cm*, assuming a flat surface. This corresponds to a 43-Aaverage separation between adsorbate molecules. For such short intermolecular distances, energy transfer can occur. In an experiment where the fluorescence is measured directly from the surface-bound species, energy transfer would cause the intensity to be nonlinear in concentration. Energy transfer would also distort the fluorescence anisotropy decay; therefore, thispoesibility is explored further. The Forster theory of energy transfer (29) shows that the probability of energy transfer per unit time, km, is related to the sixth power of the intermolecular distance, R. q is the fluorescence lifetime, and Ro is the distance between the excited- and ground-state fluorophorea at which the energy transfer and fluorescence rates are equal. Ro can be calculated from the overlap integrals of the excitation and emission spectra and the refractive index of the medium (29). For acridine orange, Ro is calculated to be 30 1A, for a refractive index range from octadecane to water and an isotropic solution. In the limit of parallel alignment of the adsorbed acridine orange molecules, Ro approaches 41 A. Energy-transfer theory thus predicts that nonlinearities in fluorescence as a function of surface concentration could be significant a t surface coverages of 100 nM and above. Experimentally, to investigate energy transfer on the surface, aqueous solutions of varying acridine orange concentrations were flowed into the surface housing and the fluorescence signal from the surface was measured as a function of time. These measurements track the accumulation of acridine orange on the surface as the equilibration is approached. A t equilibrium, the fluorescence is proportional to surface concentration established by the isotherm, if no energy transfer occurs. A fluorescence intensity lower than this proportionality indicatea energy trader. The laser power was monitored and used to correct the measured fluorescence intensities, to minimize error due to drift in laser power. The resulting accumulation profiles are plotted in Figure 5. The data show that there are two types of behavior. At concentrations below 100 nM, the final fluorescence intensity increases with solution concentration. Also, equilibrium is achieved faster with increasing solution concentration. This
*
I
1
6.00
I
10.0
1 14.0
I
1
I
10.0
TIME lmin.)
Flgure 5. Accumulation cuwes for acridine orange on (octadecyisilyl)slka as a function of concentratkn. The fluorescence emlssbn from surface-bound acridine orange Is plotted as a function of time after the introduction of the acridine orange sokrtkn Into contact with the surface.
Table I. Fluorescence Lifetime of Surface-Bound Acridine Orange as a Function of Solution Concentration concn, nM it. na
100 1.1
50 1.2
10 1.7
6 1.9
4 1.9
is the simple behavior one would expect. At concentrations of 100 nM and above, the fluorescence intensity reaches a maximum and then decreases with time, and the final level shows a decrease with increasing solution concentration. The maximum is the same for each solution. This complicated behavior is the expected artifact at high concentrations due to nonradiative energy transfer. The results show that energy transfer on the surface is considerable for solution concentrations above 100 nM. Energy transfer affects the fluorescence lifetime, which is a parameter that needs to be known for analysis of the anisotropy decays. The fluorescence lifetimes were measured by using frequency-domainspectroscopy using 82- and 184MHz modulation frequencies. This technique has very high precision, which makes it valuable for discerning small changes in lifetime. A complication to lifetime measurements is that the magic angle of 54.7O applies only to isotropic systems. The magic angle can be as small as 45O for a system of planar symmetry when emission is collected along the normal to the plane. In this work, it was assumed that the lifetime is single exponential, and the angle of the emission polarizer was varied until the lifetimes determined from both modulation frequencies agreed. This angle was found to be 49O. The experimentally determined lifetimes are summarized in Table I. These show a significant dependence upon concentration down to a 6 nM solution concentration. The results are reported to 0.1 ns to represent the estimated accuracy of these measurements; the precision corresponds to 0.01 ns. At the concentrations of 6 and 4 nM, the lifetimes were found to be within 0.01 ns of one another: thus, it is concluded that the lifetime is approaching a constant value at the lowest surface coverages for these solutions. Energy transfer must be avoided in studies of molecular reorientation. Energy transfer distorts anisotropy decays because excited solutes can transfer energy to solutes of differing orientations, thereby causing the anisotropy to decay
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ANALYTICAL CHEMISTRY, VOL.
63,NO. 13, JULY 1, 1991
Table 11. Raw Frequency-Domain Data frequency 82 M H z
164 M H z
A P Mlb
-1.63 f 0.08 0.3491 f 0.0007
-2.1 f 0.1 0.347 f 0.002
AY
4.72 f 0.04 1.341 f 0.003
8.2 f 0.2 1.406 f 0.006
rAt)
328 M H z
574 M H z
838 M H z
-2.1 f 0.3 0.342 f 0.002
-4.6 f 1.5 0.33 f 0.01
-3.5 f 2.3 0.33 f 0.01
8.6 f 0.5
8.1 f 1.5
13.1 f 1.9
r,W
M: 1.580 f 0.009 1.579 f 0.05 1.64 f 0.06 ODenotes phase difference of signal for 11 and 1 polarization. bDenotes demodulation ratio of signal for 11 and I polarizations. Table 111. Anisotropy Decay Parameters for Out-of-Plane, r , ( t ) ,and In-Plane, r,(t),Rotations Calculated from Data of Table I by Nonlinear Regression rl,PS
r&) r,(t)
130 740
72,
ns
m
m
f
r(O)
x2
0.09 0.75
-0.3 0.3
2.2 3.9
independent of whether the molecules are adually reorienting. Solution concentrations no higher than 6 nM are therefore required to interpret fluorescence anisotropy decays of surface-bound acridine orange. A solution concentration of 6 nM, which corresponds to approximately 7% of saturated coverage, was used to maximize signal and minimize energy transfer. 11. Fluorescence Anisotropy Decays. The anisotropy decay of surface-bound acridine orange via rotations in the molecular plane, r&), and rotations away from the surface plane, r&), was measured as prescribed by eqs 8 and 10, reapectively. For these studiea, the phase shifts and amplitude ratios for the two polarizations were measured for five modulation frequencies: 82, 164,328,574, and 738 MHz. Aging effecta were obeerved: the frequency-domain data for a given plate change noticeably over a period of about 1week. The origin and detailed behavior of this phenomenon was not investigated. Inatead, a newly synthesized plate, cleaned with methanol, was studied. The raw data and 95% confidence limits are summarized in Table II. These data were analyzed for double-exponential behavior, r(t) = r(O)V exp(-t/.rJ + (1- f ) e x p ( - t / ~ ) I (23) and the best fit parameters for both r&) and re(t)are summarized in Table 111. The results show that the behavior is quite different for the two angular motions. This indicates that the surface is sufficientlyflat to separate the two motions. If the surface were so rough that the system was nearly isotropic, then both experiments would constitute measurements of I,,- I, and the results would be identical. A. Rotation Normal to the Surface Plane. For re@),the regression provides a best fit to a double-exponential anisotropy decay dominated by a component having an arbitrarily large value of TO*. While no experiment can truly characterize an infinitely long decay, 70, is interpreted as infinite because any value greater than 60 ns has a negligible effect on x2. The physical interpretation of an infinitely long anisotropy decay component is hindered rotation. The hindered rotation with respect to 8 can be examined quantitatively, with some approximations. If it is assumed that there is a uniform potential well, then the range of angles through which the solute is able to tilt can be calculated by using eq 20 and the value off. It has been shown that the shape of the potential well in hindered rotation has little effect on the calculated range (30). The maximum range of angles through which the adsorbate can tilt is calculated to be 1 8 O . It can be concluded that the rotation of acridine orange adsorbed to ODS is strongly hindered with respect to rotation about
e.
water
\fi
Figwo 6. Interpreted geometry of adsorption of acrldine orange on the (octadecylsllyl)sillca surface. The ODs monolayer Is Illustrated schematically and is intended not to Imply any conformational state.
The symmetry axls of the adsorbate is restricted from rotam through angles 8 beyond 18' from the svface normal. The a-te rotates
about Its symmetry axls through angles 4 .
The chemical interpretation of the hindered rotation about 8 is that the charged group on acridine orange favors the
aqueous environment. This directs the transition moment, as depicted in Figure 6, along the surface plane. The potential barrier for changing the solvation environment of the charged part of acridine orange from water to octadecane would correspond approximately to the free-energy change upon extraction of acridine orange from water to an n-alkane. The free energy of extraction is in excess of 7kT, based upon the extraction ratio in excess of 1OOO: no detectable acridine orange extracted into an n-hexane solution. A value on the order of 7kT is a very high barrier. The conclusion that acridine orange resides at the interface with ita charge directed toward the aqueous solution is consistent with all of the experimental data. First, the reorientation experiment shows that the transition moment is restricted to lying within 1 8 O of the plane of the surface, which would position the charged group normal to the surface. Second, residence at the interface explains why acridine orange has an extremely high partition coefficient for ODs, as shown earlier, but is insoluble in hexane, a solvent that would appear otherwise to have the same functionality as ODs. Third, methanol removes acridine orange rapidly and quantitatively from the ODS surface, consistent with the idea that the water/ODS interface is essential for acridine orange adsorption. B. Rotation in the Surface Plane. For r&), the decay fits to a double exponential,although the high value of x2 indicates that the data are not described quantitatively by this functionality. A 700-pscomponent comprises 75% of the decay and an infinite component describes the remaining 25% of the decay. Two possible interpretationsof this twecomponent behavior are offered. The first is that there is hindered rotation through a range of angles dr, as described by eq 18. From the value off, which is 0.75, the corresponding value
ANALYTICAL CHEMISTRY, VOL.
of #t would be 80’. While this would seem surprisingly large, it is cautioned that little is known about the surface structure of dimethyloctadecylsilane on silica and the adsorbate environment to discard this interpretation. The chains are covalently bonded, rather than liquid, and hindered rotation is not understood in such an environment. A second possible interpretation is that there are two distinct types of solvation sites: 25% of the sites hold acridine orange almost stationary in both 0 and 4, and the other 75% allow acridine orange to rotate completely about 4 but little about 8. The notion that there may be two different sites is developed further. The possibility that some of the acridine orange is adsorbed to residual silanols was investigated by end capping the plates. It was found that both the isotherm and the frequency-domain data were unchanged. The behavior of r&) cannot be attributed to adsorption by residual silanols. Another way of invoking different adsorption sites is to consider the surface to be heterogeneous. There are two possible origins of heterogeneity. First, the silanizing reagent is only 95% pure, presumably causing defects. Second, domain boundaries may also be sites for adsorption. To elaborate, the chains of alkyl monolayers tilt to achieve dense packing, as is known from studies of Langmuir-Blodgett films (311, alkylthiols on gold (32,331,and dense, polymeric alkylsilanes on silica (34,361.The domain boundaries can be pictured as the parting of chains tilted in different directions. It is known that the dynamics of the chains themselves depend upon chain density (36)and distance from the surface (37‘). Heterogeneity of the adsorption site with respect to either density or depth, due to the presence of defects, could be responsible for the two different decay constants of r&). In a previous study, acridine orange attached to sodium dodecyl sulfate (SDS)micelles was found to exhibit a double-exponentiel anieotropy decay due to hindered rotation (38). For both the SDS and ODS studies, the data are consistent with acridine orange residing at the aqueous/hydrophobic interface. For the SDS micelle, a decay constant associated with rotation of the fluorophore’s symmetry axis was measured to be %oopsand was hinderedto atilt angle of 46O. This decay is analogous to r&) for the ODS surface, for which the reorientation is much more strongly hindered and decays on the same time scale. The interfaces are very different for these two systems. More investigations are required to explain quantitatively the difference in hindered rotor behavior.
CONCLUSIONS A new measurement was designed to probe the reorientation of an adsorbate at an interface. It was shown that adsorbate rotations for in-plane and out-of-plane angles can be examined separately. While the out-of-planerotations of acridine orange
83,NO. 13, JULY 1, 1991
1317
at the ODS/water interface are strongly hindered, the in-plane rotations give rise to a double-exponential anisotropy decay of unknown physical origin. This unresolved question underscores the fact that the basic principles of loosely packed, covalent monolayers and their adsorptive properties have not yet been established. Basic research on more structurally controlled surfaces is needed to build an understanding that can be applied to chromatographically important surfaces. LITERATURE CITED Lochmuller, C. H.; Marshall, D. B.; Wilder, D. R. Anal. Chkn. Acta 1981, 730. 31. Lochmuller, C. H.; Marshall. D. B.; Harris, J. M. Anal. CMm. Acta 1981, 731, 263. Lochmuller. C. H.; colbvn,A. S.; Hunnkutt, M. L.; Harris, J. M. J . Am. 0. Soc. 1984, 706, 4077. Stahlberg, J.; Almgren, M. Anal. Chem. 1985, 57. 817. Can, J. W.; Harris, J. M. Anal. Chem. 1988, 58, 626. 1989, 67, 590. Shaksher. 2. M.; Seltz, W. R. Anal. 0 . Bogar, R. G.; l”s. J. C.; Callis, J. B. Anal. 0 . 1984, 58, 1060. 1988, 60, 2487. Stahlberg. J.; Almgren, M.; Alsins, J. Anal. 0 . Maoz, R.; Sagiv, J. L a m 1987, 3, 1034. Wasserman, S. R.; Tao, Y.-T., Whitesldes, Q. W. L a w 1989, 5 , 1074. GUpln. R. K. Anal. Chem. 1985, 57, 1485A. van Miitenburg, J. C.; Hammers, W. E. J . Ctmnatug. 1983, 268, 147. Matswka, Y.; Yamoka, K. Bull. Chem. Soc.Jpn. 1979, 52, 3163. Klein, U. K. A.; Hear, H.P. Chem. phys. Lett. 1978, 58, 531. Lakowkz, J. R.: Cherek, H.; Mallwal, B. P.; Oratton, E. Bkdwmdsby 1965, 24, 376. Wrth, M. J. Prog. Anal. Spectrosc. 1988, 1 7 , 381. Favro, L. D. phys. Rev. 1960, 119, 53. Chuang, T. J.; Elsenthal, K. B. J . Chem. phys. 1972, 57, 5094. Llparl, 0.: Szabo, A. 6bphys. J . 1980, 30, 489. Klnosita, K.; Sugwu, K.; Ikegaml, A. B(0phYs. J . 1977, 20, 289. Szabo, A. J . Chem. h y s . 1985, 87, 150. Klnkel, J. N.; Unger, K. K. J . chsometogr. 1984, 316, 193. Sander, L. C.; Wlse, S. A. Anal. 0 . 1984, 56, 504. Danlekm, N. D.; Kirkland, J. J. Anal. Chem. 1987, 59, 2501. Fehrer, F. J.; Newman, D. A. J . Am. Chem. Soc. 1990, 172, 1931. Internal RerIscbbn Specircwcopy; Hanlck, N. J., Ed.; Haarrlck ScbnwC Corporatiom New York, 1979. Wirth, M. J.; Chou, S.H. J. phys. Chem. 1991, 95, 1786. MoMkrer, D. M. In EleCtr0enel)rtrcelChedHry; Bard, A. J., Ed.; Marcel Dekker: New York, 1966; Vol. 1. MtcyJtWcs of Aroma& - A s ; Blrks, J. B., Ed.; Wlley-Intersd en-: New York. 1970. Klnosb, K.; Ikegaml, A.; Kawato, S. Blophys. J . 1982, 37, 461. h n g m u k - m t t FwmS; Roberts, G., Ed.; Plenum Press: 1990. N w o , R. 0.; Dubols, L. H.; Allara, D. L. J . Am. Chem. Soc. 1990, 772, 558. ChMsey, C. E. D.: Ldecono, D. N. Langmuk 1990. 6, 682. Moaz, R.; Saw,J. J . CcWd Int. Scl. 1984, 700, 485. Wasserman, S. R.; Whitesides, G. M.; Tldswell, I. M.; Ocko, 8. M.: Pershan. P. S.: Axe. J. D. J . Am, Chem. Soc. 1989. 1 1 1 , 5852. (36) Bayer, E.; Paul&, A:; Peters, 8.; Laupp, 0.; Relners, J.: Albert, K. J . -tug. 1986, 364. 25. (37) Gangode, M. E.; GUpln, R. K. J . M g n . Reson. 1983, 53. 140. (38) Wlrth. M. J.; Chou, S.H. J . phys. Chem. 1989. 93, 7694.
RECEIVED for review October 9,1990. Accepted March 17, 1991. This work was supported by the National Science Foundation under Grant CHE-8814602.
