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Anal. Chem. 1989, 61, 2394-2398
facotr (a)for caffeine and theophylline increases with the application of a negative potential. The mechanism of electrochemically controlled HPLC is currently under investigation using these new stationary phases. It is envisaged that various modes of interaction including ion exchange, exclusion, and hydrophobic interactions can be controlled electrochemically.
LITERATURE CITED
I
.
-2.0
-1.0
0.0
+1.0
+2.0
EN)
Flgure 7 . Capacity factor vs potential between packing and auxiliary electrode: (1) caffeine, (2) theophylline, (3) benzoic acid, (4) m-toluic acid; eluent, 50150 CH,CN/O.2 M NaOAc buffer, pH = 4.5; flow rate, 1 mllmin.
returned to the original value when the negative potential was removed. Application of a more positive potential (up to +LOO V) decreased the capacity factor for the acidic compounds while increasing capacity for the basic compounds. Beyond +LOO V the capacity factor for all compounds decreased markedly. It is well-known that polypyrrole is irreversibly oxidized beyond these potentials (11). Note that the selectivity
(1) Ge,Hailin; Wallace, G. G. Anal. Chem. 1989, 61, 198. (2) Handbook of Conducting Polyms; Skotheim, T. A,, Ed.; Marcel Dekker: New York, 1986 Voi. 1 and 2. (3) Yuping, L.; Wallace, G. G. J . Electraanal. Chem. 1988. 247, 145-156. (4) Imisides, M. D.; Wallace, G. G. J . Ebctroanal. Chem. 1988, 246, 181-191. ( 5 ) Qiin, R.; Oiu, J. P e m . J . 1987, 19, 157-172. (6) Buckley, L. J.; Roylance, D. K.; Wnek, 0. E. J . Polym. Sci., P e m . Fhys.€d. 1987, 25, 2179-2188. (7) Salmon, M.; Dlaz, A. F.; Lcgan. A. J.; Krounbl. M.; Bargon, J. Mol. Cryst. Liq. Cryst. 1982, 83. 265-276. (8) Umaiia, M.; Waller, J. Anal. Chem. 1988, 58, 2979-2983. (9) Zinger, 6.; Miller, L. L. J . Am. Chem. Soc. 1984, 706, 6861-6863. (10) Miller, L. L.; Zinger, 6.; Zhou, 0.-X. J . Am. Chem. Soc. 1987, 709, 2287-2272. (11) Yuping. L.; Wallace, G. G. Anal. Lett., in press. (12) Fujinaga, T.; Kihara, S. CRC Cln. Rev. Anal. Chem. 1977, 6. 223-253. (13) Antrim, R. F.; Scherrer, R. A.; Yacynch, A. M. Anal. Chkn. Acta 1984, 764, 283-286. (14) Hern, J. L.; Strohl, J. H. Anal. Chem. 1978. 50, 1954-1959. (15) Goldberg, A. P. Anal. Chem. 1982, 5 4 , 342-345.
RECEIVED for review April 7,1989. Accepted August 4,1989. A patent is pending on this work.
Detection of a Transient Solvent-Solute Complex Using Time-Resolved Pump-Probe Spectroscopy G . J. Blanchard Bell Communications Research, Inc., 331 Newman Springs Road, Red Bank, New Jersey 07701
The rotational dffluslon properties of the probe molecule oxazine 4 have been studied In the methand-acetonitrile M a r y solvent system. For [MeOH] 2 7.35 X M, exclted oxat h e 4 reorlents more slowly than ground-state oxarlne 4. State-dependent reorlentation Is not observed below thls methanol concentration. These data measure a translent solvent-exclted solute complex. The formation constant for the methanol-exclted oxazine 4 complex Is determined to be 35 M-l < K, < 393 M-' for [MeOH] = 7.35 X lo4 M and 27 M-' < K , < 311 M-' for [MeOH] = 2.45 X lo-' M. The formatlon of this solvent-exclted solute complex dominates the exclted solute dynamlcal behavlor, yet lt Is mantfested as only a small change in the measured reorientatlon time. Careful consideration of such translent phenomena is rqdred H reorlentatlon measurements are to be used for quantitative analytical applications.
INTRODUCTION Analytical optical spectroscopies have traditionally employed frequency domain resolution to achieve selectivity. While many techniques, such as atomic absorption/emission, UV-visible absorption, and Raman scattering, have proven to be sensitive and selective, there are certain applications for
which spectral overlap interference renders frequency domain selectivity inadequate. Recently, time- and phase-resolved techniques have been used to advantage in obtaining an extra dimension of spectroscopic selectivity (1-4). Resolution of spectroscopic signals based on their temporal characteristics has found application in separating different processes as well as in distinguishing between different origins of the same process. Direct time-domain (5) and phase-resolved (6) discrimination have been used to reduced fluorescence background interference to spontaneous Raman scattering. Emission spectral overlap interference between fluorescein and billirubin has also been overcome by taking advantage of the different fluorescence lifetimes of the analyte and interferent (3). These examples demonstrate the utility and versatility of time-resolved techniques in achieving spectroscopic selectivity. In addition to achieving selectivity based on different spectral lifetimes, time-resolved techniques also offer selectivity in the liquid phase because of their ability to sense the dynamical properties of an absorptive moiety. Rotational diffusion measurements, for instance, offer selectivity based on lifetime as well as on the unique microscopic environment surrounding the analyte. Reorientation measurements sense the complex interactions between the solute and its environment, which depend critically on the specific chemical identities of the analyte and the matrix. This sensitivity has
0003-2700/89/0361-2394$01.50/00 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989
been employed in the selective detection of two (tagged) macromolecules of different molecular weight (7,8) as well as in the understanding of the different environmentsoccupied by acridine orange in micellar and submicellar systems (9). The measurement of analyte molecular motion offers greater potential selectivity than lifetime measurement alone. Because of the detailed information contained in dynamical data, however, making quantitative measurements and predictions requires a correspondingly detailed knowledge of the many subtle chemical effects that can contribute to the observed signals (10). The rotational diffusion characteristics of polar solventsolute systems have been examined extensively, especially since the development of the mode-locked laser (10-14). The primary focus of most of these works has been on understanding liquids from a molecular, rather than a continuum, perspective. Current models of rotational diffusion usually relate the microscopic solute behavior to some macroscopic solvent property. The modified Debye-Stokes-Einstein (DSE) model (15-17) T,,
= qVF/kTS
(1)
relates the solute orientational relaxation time, T , ~ , to its volume, V , shape, S, the solvent bulk viscosity, q, and a solvent-solute "friction" term, F. While not quantitative, it is useful for making relatiue comparison between similar systems (14,18). An addition to this model, the inclusion of dielectric friction 7,,
= ( q V / k T ) + 7a
(2)
where Tdf
+
= p2(t - l ) ~ ~ / k T a ~ ( 2 t
(3)
relates "friction" between the solvent and solute to the solvent dielectric constant, c, the solvent Debye dielectric relaxation time, TD, and the solute permanent dipole moment, p. While this model has been quite successful for some systems (19, ZO),it does not work well in all cases, and more importantly, it fails to address site-specific molecular-levelprocesses that determine the solute reorientation characteristics. The recent observation of state-dependent, site-specific solvent-solute interactions has served to reaffirm the importance of molecular processes in determining reorientation dynamics (10,14,18). On excitation, oxazine dye molecules exhibit a characteristic change in valence electron density at their heterocyclic nitrogen site (21). The state-dependent change in electron density causes the Lewis base character of this specific site to change, and the solute interaction with the surrounding solvent is altered accordingly. The effect on the solvent cage is determined primarily by the excitationdependent change at this site rather than by an accommodation on the part of the solvent (14). If the oxazine state dependence is to be used as a sensitive probe of its local environment, it will first be necessary to quantify the (state dependent) change in the nature and degree of the solventsolute interaction. It is the purpose of this paper to determine more quantitatively the characteristics of this state-dependent solventsolute interaction and to highlight the necessity of understanding how subtle chemical changes can affect measured rotational diffusion times. Oxazine 4 was used as the probe molecule because its state-dependent properties have been well characterized (21)and its absorption and emission spectra lie in a spectral region convenient for access by our spectrometer (see Figure 1). While oxazines contain several heteroatom sites where polar interactions with the solvent occur, the experiments reported here have been designed such that the solute heterocyclic nitrogen is the only site that contributes to the state-dependent dynamics. Acetonitrile,
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3 0
v
c .-
1.00
c ffl
._ 0.75 c
._ ffl .E
s
0.50
2
:.e 0.25 ffl
P n nn 0 V.YY
450
550
500
600 650 700 wavelength (nm)
750
800
Flgure 1. Absorption and emission spectra of oxazine 4 in acetonitrile. The emission profile has been normalized to match the absorptkn. The structure of oxazine 4 is shown at top.
a weak Lewis base, was chosen as the solvent, and methanol, a weak Lewis acid, was added in varying amounts. The magnitude of the oxazine 4 rotational diffusion state dependence was measured as a function of added methanol to determine the extent of methanol-excited oxazine 4 association. From these data bounds can be established on the value of the formation constant, Kf, of the methanol-excited oxazine 4 complex. The determination of Kf is limited by uncertainty in the measurement of the ground- and excited-state reorientation times of oxazine 4.
THEORY
-
The association reaction of interest here is Ox4* MeOH {Ox4*-MeOH]
+
(9
where Ox4* represents the excited probe molecule and (Ox4*-MeOH) is the solvent-excited solute complex. The equilibrium between the free Ox4* and the complexed species is related to the MeOH concentration by
Kf = [(Ox4*-MeOH)]/ [Ox4*][MeOH]
(4)
assuming that the (Ox4*-MeOHJ complex has 1:l stoichiometry. Owing to the form of eq 4, it is possible to express both [Ox4*] and [(Ox4*-MeOH)] in units other than molarity. Because it is the ratio of the complexed Ox4* to free Ox4* that is important, eq 4 may be expressed equivalentlyin terms of the fraction of Ox4* that is complexed and free. Kf[MeOH] = N c / N f
(5)
where N , is the fraction of Ox4* that is complexed and N f is the fraction that is free. Since all of the molecules excited are necessarily either free or complexed, N , + N f = 1. The solvent attachment approximation of the DSE model will be used to relate the observed state-dependent reorientation times to the free and complexed species in solution (18). We measure experimentally a ground-state-recovery reorientation time, T ~and , a stimulated emission reorientation time, T , ~ . Within the DSE approximation 7gsr =
qV/kT
V = Vox4
(6)
T~ measures the reorientation of ground-state (uncomplexed) oxazine 4. The stimulated emission measurement, however, detects the reorientation of both free and complexed excited oxazine 4. ~ , b = qVT/kT VT = NfV NcV* (7) V* = Vox4 + V M ~ O H
+
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989
Table I. Experimental Ground-State Recovery and Stimulated Emission Reorientation Times for Oxazine 4 in Acetonitrile-Methanola XM~H 0
0
1.38 X lo4 4.14 x 10-4 1.38 x 10-3 1.36 X 0.315 0.580 0.805
1.0
[MeOH],M 2.45 x 10-3 7.35 x 10-3 2.45 X 0.245 6.12 12.2 18.4 24.5
7, cp
Tgarr
0.440 f 0.002 O.43gb 0.43gb 0.43gb 0.438 f 0.003 0.438 f 0.003 0.454 f 0.002 0.493 f 0.002 0.572 f 0.004
PS
Tnter
57 f 2 49 f 1 48 f 1 48 f 1 51 f 1 55 f 2 61 f 1 69 f 1 88 f 2
PS
6T/T
57 f 3 49 f 1 51 f 1 52 f 1 56 f 1 63 f 1 67 f 2 74 f 2 97 f 2
0.0 f 0.091 0.0 f 0.042
0.063 f 0.043 0.083 f 0.042 0.098 f 0.040 0.145 f 0.057 0.098 f 0.050 0.072 f 0.058 0.102 f 0.046
"For each solution, R,,(O) = R,,(O) within the experimentaluncertainty. *Viscosityvalues were inferred from experimentaldata for neat acetonitrile and solutions with XMsOH = 0.0136 and 0.315. 0 25
The reorientation times are determined experimentally from the function R ( t ) = [I,,(t)- ZL(t)]/[Ill(t)+ 2I,(t)] and are combined to give the quantity 6 i / r - T ~ J / T ~ which J , can be related to the fraction of complexed excited solute through eq 6 and 7: 6 7 / ~ = N,(VM~OH/VOXJ (8) An important feature of eq 8 is that 6 7 / T is not dependent on any model-specific boundary conditions. Thus, by measuring 6 ~ / and 7 using a self-consistent means of approximating the solvent and solute hydrodynamic volumes (22),one can determine the fraction of excited complexed oxazine 4 in solution and, hence, Kf.It should be noted that eq 8 is valid only when the lifetime of the (Ox4*-MeOH) complex is longer than is%.
EXPERIMENTAL SECTION a. Laser. The picosecond laser spectrometer used here has been described previously (23). Briefly, a mode-locked argon ion laser (Spectra-Physics Model 171-09)pumps synchronouslytwo dye lasers (Coherent Model 701-3). Radio frequency triple modulation, shot-noise-limiteddetection is used (24-261, and the time delay between the pump and probe pulse trains is controlled mechanically. For these experiments the pump dye laser was operated at 615 nm (120-mW average power, 5-ps fwhm pulses, Rh610 dye). The probe dye laser was set to 615 nm (100-mW average, 5-ps pulses, RhGlO/DCM mixed dye) for measurement of rprand to 650 nm (65-mW average, 5-ps pulses, Rh61O/DCM mixed dye) for measurement of 7,. Stimulated emission was from the first excited singlet state of oxazine 4. The average pump power at the sample was typically 20 mW, and probe power was maintained at 1150 pW. For all measurements the sample was flowed and its temperature controlled at 27.0 f 0.1 "C. b. Chemicals and Sample Preparation. Oxazine 4 (LD 690) perchlorate was purchased from Exciton Chemical Co. and used without further purification. Anhydrous, gold label grade acetonitrile and methanol were obtained from Aldrich Chemical Co., and both were used as received. All samples were made immediately prior to measurement and were changed daily. Solutions were prepared by using grade A volumetric glassware and, where necessary, with calibrated Eppendorf pipets. c. Steady-State Spectroscopy and Viscosity Measurements. Absorption measurements were made with 51-nm resolution using a Varian Model 634 scanning UV-visible spectrometer. Emission profdes were obtained with -1-nm resolution by using a Princeton Applied Research Model 1453 optical multichannel analyzer (615-nm excitation). Viscosity measurements were made with a falling ball type viscometer in a temperature-controlled water bath.
RESULTS AND DISCUSSION The value of eq 8 lies in its simplicity. When no complexation occurs, no state dependence is predicted. When complete complexation is achieved, the state dependence is expected to reach a limiting value equal to the so1vent:solute volume ratio. Both of these limiting cases are observed experimentally. The measured rgsr,ish, and ~ T / Tdata are
I
7
0.20
-0 Oo0 05 10
4
Il_i 100E-3
100E-4
0 01 log
3 10
' 00
x MeOH
Figure 2. Experimental 67/7 vs X,. The abscissa is logarithmic for clarity of presentation. Not shown is the data for [MeOH] = 0. For neat acetonitrile, ~ T / T= 0 f 0.091.
presented in Table I. Quantitation of the complexation reaction (i) is possible with the experimental data reported here and an estimate of the solvent and solute volumes. Because eq 8 uses the volume ratio rather than the absolute volumes, any uncertainty in such an estimate will be cancelled if the solvent and the solute volumes have been calculated by using the same formalism. Edwards has published a systemic method for the determination of molecular hydrodynamic volumes (22). With this algorithm, Vb4 = 350 A3 and V M ~ , = 36 A3. Thus (VM&H/V&4) = 0.103, in excellent agreement with the experimental 6 7 / 7 value of 0.102 f 0.046 in neat MeOH. Kf can be determined with maximum accuracy when N , 0.5. As N , approaches unity, 6 7 / 7 attains its limiting value and Kf is underestimated. Similarly, as N , 0, Kf 0. In these experiments, two solutions are observed to have a value of 6 7 / 7 intermediate between 0 and its limiting value, indicating N , 0.5. These are [MeOH] = 7.35 X M and 2.45 X M (see Table I). By use of the experimental 6 T / T values and (vM&),/ Vox4) = 0.103, N , can be obtained from eq 8. Substituting N , and [MeOH] into eq 5 gives Kf = 214 f 179 M-' for [MeOH] = 7.35 X M and Kf = 169 f 142 M. M-' for [MeOH] = 2.45 X The uncertainty in these Kf values may seem rather large. This is due solely to the fact that ~ T / is T a normalized difference between two similar values. The small uncertainty in both rw and 7- contributes equally to produce the reported uncertainty in ~ T / T . the rgsrand values are themselves the averages of at least six individual determinations. Despite the uncertainty in Kf,the reported values demonstrate clearly that association between the excited solute and methanol is strongly favored. M for all of The concentration of oxazine 4 was -2 X the reorientation experiments. The excited oxazine 4 concentration, relevant to the complexation studied here, is, however, significantly less. From the parameters listed in the Experimental Section and an absorptivity of ceI5 = 1.09 X lo5
-
- -
-
2397
ANALYTICAL CHEMISTRY, VOL. 61,NO. 21, NOVEMBER 1, 1989 12
I
o’60
5
0.55 . h
??OS0 .
0
20
40
60
100
80
0.401 0.0
.o
’
’
’
0.5
0.6
0.7
’
’
0.8 0.9
1 1.0
The line is a guide
to the eye.
h
v c
-C
”
0.3 0.4
Flgure 4. Solution viscosity as a function of X,.
-1.5
[L
I
0.2
’MeOH
scan time (ps)
-1
‘
0.1
-2.0
-2.5
0
20
40
80
60
delay time (ps)
Flgue 3. (a) Experimental parallel and perpendicular signals for X, = 4.14 X (b) Experimental anisotropy decay presented with the
lo4.
best fit to a single exponential decay. Inset: residuals of the fit.
L/(mol cm) (27),the total concentration of excited oxazine 4 (complexed plus free) is estimated to be -3.5 X lo4 M. Because of the complexity of the solvent-solute interactions that give rise to the observed data, it is appropriate to evaluate three of the approximations employed in the data analysis and interpretation. First is the approximation that T~~ = T , ~ for free oxazine 4. This assumption is consistent with earlier work (10, 14,18) where state-dependent reorientation was shown to arise from excitation-dependent changes in the solvent-solute interaction rather than a change in the solute shape or volume. In addition, T~~~ = T~~ in neat acetonitrile (Table I). Thus, this first approximation is valid. A second assumption, implicit in eq 7, is that the reorientation time of the excited complex will not be very different than that of the free molecule. An alternative way to express this is V0.4 >> VMe0H. For concentrations of MeOH where N, N,, two exponential decays are expected to contribute significantly to the stimulated emission anisotropy decay. If the time constants of these decays are similar, it may not be possible to resolve them experimentally. N, Nf near [MeOH] = 7.35 x M. The experimental data for this MeOH concentration are presented in Figure 3a, and the anisotropy decay and the residuals of its fit to a single exponential decay are presented in Figure 3b. Within the experimental uncertainty only one decay can be resolved. For cases where N, is significantly different than Nf, ratewill be dominated by only one exponential decay. The experimental observation that T~~ = 97 f 2 ps and T~~~ = 88 f 2 ps in MeOH demonstrates the validity of this second assumption. The third assumption is that the stoichiometry of the {Ox4*-MeOH) complex is 1:l. If the stoichiometryis n:l, then ~ T / T= N,(nVMaH/ Vh4). The value of n can be bounded by using the calculated value of ( v ~ e o Hv/0.4) and the experimental value of ~ T / Tin neat MeOH. When ( V M ~ H / V ~ ~ ) and ~ T / T= 0.102 f 0.046 = 0.103 are used, 0.54 In I1.44. The “solvent attachment” model used here is based on the modified DSE model. Because this model does not address the molecular-level aspects of solvation, it is perhaps fair to
-
-
question its utility in this application. Within the framework of the modified DSE model, differences between experiment and calculation can be viewed as reflecting a lack of knowledge regarding the solvent-solute boundary conditions. Because these boundary conditions are very similar for the groundand excited-state probe molecule (10,14, 18), an absolute difference between the experiment and calculation will be factored out in the term ~ T / T .The relative predictions made by this model are instead limited by our ability to estimate the so1ute:solvent volume ratio correctly. If the solvent and solute volumes are approximated by a self-consistent method (22))this model can provide useful predictions. The solvent attachment model has been applied successfully before to state-dependent changes in dipolar solvent-solute interactions between nitriles, alcohols, and methylene blue (a thiazine) (18). A testament to its validity here is the agreement between data and model for high MeOH concentrations, where ~ T / Thas reached its limiting value (see Figure 2). The comparisons between experiment and theory have been made in such a way that relative differences are considered rather than absolute quantities. This has been done because there remains considerable uncertainty in the determination of absolute quantities such as solvent-solute “friction”, solute shape, and solvent and solute hydrodynamic volumes. Despite these limitations, however, it is instructive to examine the experimental T~~~ and T~~ data directly. The bulk and microscopic properties of mixed solvent systems have been examined in detail before (28-31). Polar mixed solvent system viscosities typically show significant deviation from Raoult’s law. This deviation is seen for the methanol-acetonitrile system as well. The viscosity of this system is presented as a function of XMeOH in Figure 4. The important feature to note in these data is that the solution viscosity remains constant within experimental uncertainty for 0 < XMaH I0.315. For X M ~ O>H0.315, the viscosity-to-XMeoH relationship is curvilinear. Based on these viscosity data, the DSE model indicates a constant T~~ for XMeOH 5 0.315. Experimentally, both T~~ and ratedo not remain constant over this range (see Table I). Perhaps most striking is the difference in T~~ for [MeOH] = 0 and [MeOH] = 2.45 x lo9 M. No state dependence is observed at either of these methanol concentrations, but T~~~and T~~ both differ by -8 ps, well outside the experimental uncertainty. These data demonstrate the important state-independent role that methanol also plays in the solvation of oxazine 4. Another important consideration is the time scale of the complexation process. In order for the complexation to be observed via state-dependent reorientation, it must occur on a time scale shorter than the reorientation time. This places an upper bound on the “complexation time” of -50 ps, and this complexation time could be considerably shorter. Recent measurements of the time-delayed fluorescence Stokes shift of polar solutes in polar solvents have shown that the solvation
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 21, NOVEMBER 1, 1989
time of an excited solute occurs on the order of the solvent longitudinal relaxation time, TL (32,33). For acetonitrile, TL = 0.2 ps (34). Thus, a fast complexation time is not necessarily surprising. A relatively rapid complexation might also be expected on the basis of the data presented in Table I. State-independent effects, as discussed above, suggest the presence of methanol in the immediate environment of the solute. The rapid complexation seen here may be reflective of a rearrangement of the solvent cage subsequent to solute excitation. The fact that oxazine 4 displays a methanol concentration dependence in its state-dependent reorientation behavior gives an indication of the relative Lewis base character of its heterocyclic nitrogen site. In the ground state, the oxazine 4 heterocyclic nitrogen is a weaker Lewis base than acetonitrile. On excitation, this moiety becomes a stronger Lewis base than acetonitrile, in agreement with modified neglect of diatomic overlap (MNDO) calculations of oxazine 4 (21) and acetonitrile. In the ground state, the oxazine 4 heterocyclic nitrogen is essentially neutral; when it is excited, its valence electron density increases by -0.25 e. The acetonitrile nitrogen is calculated to have an excess valence electron density of -0.08 e. The value of Kf will depend on the Lewis base character of the (aprotic) solvent used. The choice of a sufficiently strong Lewis base solvent should preclude the occurrence of a solvent-excited solute complex.
CONCLUSION In conclusion, the application of picosecond resolved pump-probe spectroscopy to the determination of the associative properties of transient species has been presented. For chemical systems where excitation-induced changes in sitespecific valence electron density occur, subtle changes in solvent-solute interactions can contribute significantly to the measured orientational relaxation time. The association between solvents and excited solutes can be quite strong, as reported here. Rotational diffusion thus shows promise as a highly selective system property in analytical applications of time-resolved spectroscopy. This work serves, however, to underscore the importance of understanding molecular-level processes before quantitative analytical applications can be developed.
ACKNOWLEDGMENT The author is deeply indebted to Dr. C. J. Weschler for several stimulating discussions and a valuable critical reading of the manuscript. LITERATURE CITED Cline Love, L. J.; Upton, L. M. Anal. Chem. 1980, 52, 496. Bright, F. V.; McGown, L. B. Anal. Chem. 1985, 57, 2877. Bright, F. V.; Vickers, G. H.; Hleftje, G. M. Anal. Chem. 1986, 58, 1225. Russo, R. E.; Hieftje, G. M. Anal. Chim. Acta 1982, 134, 13. Harris, J. M.; Chrisman, R. W.; Lytle, F. E.; Tobias, R. S. Anal. Chem. 1978, 4 8 , 1937. Wirth, M. J.; Chou, S.-H. Anal. Chem. 1888, 60, 1882. Knutson, J. R.; Davenport, L.; Brand, L. Bhxhem/stry 1988. 2 5 , 1805. Davenport, L.; Knutson, J. R.; Brand, L. Biochemistry 1986, 2 5 , 1811. Chou, S.-H.; Wirth, M. J. submitted for publication in J . Phys. Chem. Blanchard, G. J.; Cihal, C. A. J. Phys. Chem. 1988, 92, 5950. Fleming, G. R.; Morris, J. M.; Robinson, G. W. Chem. Phys. 1978, 77, 91. Eisenthai, K. B. Acc. Chem. Res. 1975, 8 , 118. Millar, D. P.; Shah, R.; Zewall, A. H. Chem. Phys. Lett. 1979, 66, 435. Blanchard, G. J. J. Phys. Chem. 1988, 92, 6303. Debye, P. Polar Molecules; Chemical Catalog Company: New York, 1929; p 84. Perrin, F. J. Phys. Radium 1934, 5 , 497. Hu, C.-M.; Zwanzig, R. J. J. Chem. Phys. 1974, 60, 4354. Blanchard, G. J. J. Phys. Chem. 1989, 93, 4315. Kivelson, D.; Spears, K. G. J. phvs. Chem. 1985, 8 9 , 1999. Philips, L. A.; Webb, S. P.; Clark, J. H. J. Chem. Phys. 1985, 83, 5810. Blanchard, G. J. Chem. Phys., in press. Edward, J. T. J. Chem. Educ. 1970, 47, 261. Blanchard, G. J. J. Chem. Phys. 1987, 8 7 , 6802. Bado, P.; Wilson, S. B.; Wilson, K. R. Rev. Sci. Instrum. 1882, 5 3 , 706. Andor, L.; Lorincz, A.; Siemlon, J.; Smith, D. D.; Rice, S. A. Rev. Sci. Instrum. 1984, 5 5 , 64. Blanchard, G. J.; Wlrth, M. J. Anal. Chem. 1988, 58, 532. Kodak Laser products Catalog, Publication JJ-169; The Eastman Kodak Company: 1982; p 16. Beddard, G. S.; Doust, T.; Hudalas, J. Natwe 1881, 294, 145. Gudgin Templeton, E. F.; Qultevis, E. L.; Kenney-Wallace, G. A. J. Phys. Chem. 1985, 8 9 , 3238. Gudein TemDleton. E. F.; Kennev-Wallace. G. A. J. Phvs. Chem. 1886, 90, 2896. Gudgin Templeton, E. F.; Kenney-Wallace. G. A. J. Phys. Chem. 1988. .- - -, 9 - -0 ,. 5441 - . . .. Castner, E. W., Jr.; Maroncelli, M.; Fleming. G. R. J. Chem. Phys. 1987. 86. 1090. Maroncell/, M.; Fleming, G. R. J . Chem. Phys. 1987, 86, 6221. Simon, J. D. Acc. Chem. Res. 1988, 21, 128.
RECEIVED for review June 15,1989. Accepted August 7,1989.
