Anal. Chem. 1997, 69, 2136-2142
Spectroscopic Determination of Pressure-Induced Shifts in Inclusion Complexation Equilibria Shirley M. Hoenigman and Christine E. Evans*
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055
The effect of modest hydrostatic pressure (99%) served as the host molecule in all cases, and solutions were freshly prepared in ultrapure deionized water (Millipore; >18 MΩ). All materials were used without further purification. The fluorophore concentration was maintained constant at 1.00 × 10-5 M, while the βCD concentration range was chosen in accordance with BenesiHildebrand constraints (vide infra). Both the solute and host concentrations were systematically controlled to ensure only 1:1 (14) Ringo, M. C.; Evans, C. E. Anal. Chem. 1997, 69, 643-649. (15) Ruiz Silva, B. E.; Burtnick, L. D.; Turro, N. J. Biochem. Int. 1991, 23, 905913. (16) Mock, D.; Langford, G.; Dubois, D.; Criscimagna, N.; Horowitz, P. Anal. Biochem. 1985, 151, 178-181.
Figure 2. Schematic of high-pressure fluorescence spectroscopy apparatus.
host-guest complexation and to eliminate any self-association. After thorough mixing, the probe-cyclodextrin solutions were allowed to equilibrate for 60-90 min prior to fluorescence measurements. High-pressure fluorescence measurements were conducted using the apparatus depicted in Figure 2. Sample pressurization was accomplished in a 110-cm length of fused-silica capillary (Polymicro Technologies; 320 µm i.d. × 440 µm o.d.) connected between a pressurization valve (High Pressure Equipment; Model 11-11AF1) and an injection valve (Valco; Model C6WY). This configuration creates a system that can be operated in both flowing and static modes. The sample was introduced into the capillary using a syringe pump (Applied Biosystems; Model 140A) together with a 1-mL injection loop. After the capillary was completely filled with sample, the pressurization valve was closed, and the system slowly attained the desired pressure. Using ultrapure water as the pressure transmitting fluid, pressures up to 350 bar were readily achieved. Measurements at multiple pressures for a single injection of equilibrated sample solution were identical to those performed using an individual injection at each pressure. As expected, the sequence of pressure values during each experiment had no affect on the resultant measurement. In this configuration, the determination of steady-state fluorescence in the high-pressure regime was facilitated by the optical transparency of the fused-silica capillary. A window was made in the capillary 60 cm from the injection valve by removing the polyimide coating. This window region was then mounted in a light-tight enclosure and fitted with Teflon sleeves to ensure capillary stability during pressurization. Fluorescence excitation was accomplished using a mercury/xenon arc lamp (Xenon Corp.) and a 10-nm band-pass filter centered at 313 nm. Excitation radiation was transmitted to the capillary window using a 500-µm optical fiber. Emission radiation was collected in a 90° coplanar geometry with another 500-µm optical fiber, filtered, and focused onto a PMT (Hamamatsu; Model R1463). For 2,6-ANS measurements, a 10-nm band-pass filter centered at 450 nm was used, while a 10-nm band-pass filter at 470 nm was used for 2,6-MANS measurements. The resulting photocurrent was then digitized (National Instruments; Model AT-MIO-16-L-9), and the signal was Analytical Chemistry, Vol. 69, No. 11, June 1, 1997
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averaged over 50 s (rate ) 20 Hz) using the LabView software program (National Instruments; Version 3.1.1). Exposure times of up to 10 min showed no measurable photodegradation. Data Analysis. Complex formation constants were determined using the method of Benesi-Hildebrand, the derivation of which has been described in detail elsewhere.17,18 In brief, this method uses simple mass balance arguments to determine the molar complexation constant (Kc) based on signal measurements under systematically varying cyclodextrin concentration conditions. For conditions where the analytical concentration of cyclodextrin (CβCD) is very much greater than the equilibrium concentration of the complex ([probe‚βCD]), the Benesi-Hildebrand relationship is given as
Cprobe ) (KckiQcomplexCβCD)-1 + (kiQcomplex)-1 Fcomplex
(1)
When the fluorescence intensity of the complex (Fcomplex) is measured as a function of the analytical cyclodextrin concentration, the complexation constant is determined from the interceptslope ratio of a double-reciprocal plot. Furthermore, the product of the instrumental constant (ki) and the quantum efficiency of the complex (Qcomplex) can be assessed directly from the intercept. A modest and constant analytical concentration of the fluorescent probe (Cprobe) is utilized throughout these studies to maintain 1:1 complexation conditions. It is important to note that small contributions to the measured fluorescence intensity from the unbound fluorophore may introduce significant errors in the resulting complexation constant.18 Even in cases where the contribution of unbound fluorophore to the measured signal is less than 5%, a significant error is introduced because the equilibrium concentration of free fluorophore varies systematically with the analytical concentration of host species. That is, the concentration of unbound fluorophore is at a minimum at high βCD concentrations and at a maximum at the lowest βCD concentration. Thus, a systematic error is introduced in the slope of Benesi-Hildebrand plot, leading to an overestimation of the complexation constant. Using calibration curves, this error may be corrected by subtracting the fluorescence intensity attributable to the unbound probe from the overall measured intensity of the complex. In this way, all measured fluorescence intensities in the 2,6-ANS study were corrected for the intensity contribution of the unbound fluorophore. No contribution from the free fluorophore was measured in the 2,6MANS studies, due to the large binding constant and the low quantum yield of the free fluorophore. After iterative correction for the unbound fluorophore intensity, the Benesi-Hildebrand relationship was utilized to experimentally determine complexation constants from the measured fluorescence intensity as a function of the βCD concentration (eq 1). This method of data analysis yields not only the association constant but also the product of the instrumental constant and the quantum efficiency of the complex. In these studies, the effect of pressure on both the complexation constant and the complex quantum efficiency are evaluated at six pressures between 7 and 345 bar. From this information, the effects of uniform hydrostatic pressure on the equilibrium position of the inclusion reaction as well as on the fluorescence properties of the complex are determined. (17) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703-2707. (18) Hoenigman, S. M.; Evans, C. E. Anal. Chem. 1996, 68, 3274-3276.
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Experimental Design. In discerning the role of pressure on inclusion complexation, careful experimental design is required in order to isolate pressure from all other parameters. With this goal in mind, a capillary fluorescence cell is utilized to maintain constant temperature conditions by allowing rapid heat dissipation and simultaneously eliminating both inner and outer filter effects. The band-pass filters (fwhm ) 10 nm) are incorporated to ensure that the analytical signal will be unaffected by small frequency shifts in the excitation or emission maxima induced by elevated pressures (vide infra), while concurrently minimizing the contribution of unbound fluorophore intensity to the overall signal. Judicious choice of guest and host species also plays a significant role in the experimental design. Anilinonaphthalenesulfonate dyes (Figure 1) have been chosen as model solutes on the basis of their unique fluorescence properties. In the unbound state, these fluorophores are weakly fluorescent in aqueous solution, with quantum yields of 0.090 for 2,6-ANS and 0.006 for 2,6-MANS.19 Upon encapsulation within the hydrophobic βCD cavity, the excitation maxima are unchanged, the emission maxima are blue-shifted by 20 nm for 2,6-ANS and by 26 nm for 2,6-MANS, and the quantum yield is dramatically increased. The fluorescence intensity contributions of the unbound fluorophore are, thus, minimized or eliminated, and the measured analytical signal originates predominantly from the complexed species. In contrast with some ANS analogs,20 the 2,6-ANS‚βCD and 2,6-MANS‚βCD complexes show no dependence of the emission maxima on the βCD concentration. Under these conditions, association constants for complexation may be determined directly using the BenesiHildebrand approach (eq 1). In addition to pragmatic measurement considerations, these two ANS analogs have been chosen for their structural and chemical similarities. Both solutes contain no ionizable groups in the pH range from 2 to 11, eliminating pressure-induced ionization effects. Moreover, no solution buffering is required, which ensures that neither competing pressure-dependent equilibria nor quenching by buffer constituents will be a factor. Identical positional substitution on the naphthyl moiety of these probes allows both molecules to fit similarly inside the β-cyclodextrin cavity. Finally, the complex structure is such that the anilino nitrogen on the probe molecules is in proximity to the hydroxyls on the cyclodextrin rim.20,21 Substitution at the anilino nitrogen, therefore, is expected to yield insight into the effect of pressure on the site-specific rim interactions of these inclusion complexes. The host molecule, β-cyclodextrin, has been chosen for the well-defined cavity geometry that remains fixed over the pressure regime from 1 to 350 bar.1,10 In addition to its many analytical applications, this incompressible host is also commonly utilized to emulate hard-site binding in proteins. Consistent with the chosen probe molecules, the βCD host is not ionizable over pH 2-12. Furthermore, because βCD is nonfluorescent, it does not interfere with the analytical signal arising from the probecyclodextrin complex. Thus, this combination of analogous probe (19) Seliskar, C. J.; Brand, L. J. Am. Chem. Soc. 1971, 93, 5414-5420. (20) Nishijo, J.; Nagai, M.; Ysauda, M.; Ohno, E.; Ushiroda Y. J. Pharm. Sci. 1995, 84, 1420-1426. (21) Crescenzi, V.; Gamini, A.; Palleschi, A.; Rizzo, R. Gazz. Chim. Ital. 1986, 116, 435-440.
molecules and βCD provides a well-defined model system for the examination of pressure-induced perturbations on inclusion complexation. RESULTS AND DISCUSSION Pressure Dependence of Association Constants. The pressure dependence of equilibrium constants is described by the fundamental thermodynamic parameter, ∆V, and is based on volumetric considerations. For the complexation reaction,
probe + βCD S probe‚βCD
(2)
a shift in the equilibrium position is expected if a difference exists between the partial molar volumes of products and reactants.11 In solution, the partial molar volume depends upon the size of the molecules as well as the intermolecular forces that comprise the solvation environment. It is these interactions that define the solvation volume of the species involved in the reaction and thus the magnitude and directionality of ∆V. For the reactions under investigation here, the molar equilibrium constant, Kc, is defined as
Kc )
[probe‚βCD] [probe][βCD]
(3)
where [probe‚βCD], [probe], and [βCD] refer to the equilibrium molar concentrations of the complex, fluorophore, and β-cyclodextrin, respectively. To describe the expected pressure dependence of Kc, it is useful to proceed through Gibbs free energy, ∆G,
∆G ) -RT ln Kc
(4)
where R is the gas constant and T represents the absolute temperature. The pressure dependence of ∆G is based on the fundamental thermodynamic equation of state and is defined as
(∂∆G ∂P )
T
) ∆V
(5)
Under the modest pressure range of interest, the partial differential may be accurately expressed as a simple difference,11 and substitution of eq 4 into eq 5 yields
∆ ln Kc ) ∆V - ∆nRTκS ∆P
-RT
(6)
where the term ∆nRTκS accounts for any increase in molar concentration with pressure and relates directly to the bulk solvent compressibility, κS. The difference in the stoichiometric coefficients of products over reactants is ∆n, and the overall term is approximately 1.1 cm3/mol in these studies. For inclusion complexation reactions involving βCD, the ∆V is expected to be positive, because the host molecule is rigid and incompressible over 10 kbar.1,10 In contrast with many associated complexes, the βCD host molecule cannot change conformation to achieve minimum energy solvation of the guest, which would serve to decrease the volume of the complex. Of the limited studies investigating the effect of pressure on inclusion complexation equilibria, several hypotheses regarding the observed ∆V have been proposed. Torgerson et al.1 suggest that, in subjecting an incompressible host such as β-cyclodextrin to elevated pressure, the observed ∆V may be approximated as the difference in the compressibilities of the guest molecule and the volume of water occupying the unassociated cyclodextrin cavity. Because the aromatic guest molecules are more compressible than water,
Table 1. Association Constantsa (M-1) of ANS‚βCD Complexes at Various Pressures pressure (bar)
2,6-ANS‚βCDb
2,6-MANS‚βCDc
6.89 68.9 138 207 276 345 ∆V (cm3/mol)d
2160 ( 60 2130 ( 60 2070 ( 50 1970 ( 60 1910 ( 30 1830 ( 40 10.5 ( 2.0
16 500 ( 300 16 700 ( 400 16 100 ( 500 16 100 ( 300 16 000 ( 300 15 500 ( 300 1.2 ( 2.4
a Association constants are reported for a single representative trial. The uncertainty associated with each binding constant was calculated using the variances determined from a least-squares fit of the BenesiHildebrand plot for each pressure. b Experiment conducted at 292.5 K. c Experiment conducted at 292.3 K. d The overall ∆V was determined from three separate measurements of ∆V for each complex.
complex dissociation is favored with pressure, and this behavior is independent of the absolute value of the association constant. In other studies,5,10 ∆V is approximated using the sum of the individual volume changes occurring during solute complexation, including desolvation of the probe molecule, desolvation of the βCD cavity, solute inclusion inside the cavity, and any conformational changes in the βCD upon complexation. In summing these contributions, the magnitude of the increase in ∆V predicted for the desolvation processes is expected to dominate the negative ∆V accompanying the inclusion process. Since βCD is structurally rigid, no conformational changes upon complexation are expected to contribute to the overall ∆V. Thus, both of these approaches predict a positive change in molar volume upon complexation and the concomitant decrease in the complexation constant with pressure. The few experimental studies in the literature appear to be in general agreement with these predictions. High-pressure spectroscopic studies of 2-naphthyl acetate inclusion into βCD yielded a ∆V of +10 ( 2 cm3/mol.5 Studies with poly-β-cyclodextrin (polyβCD) and the 1,8-substituted ANS probe resulted in a measured ∆V of +9.3 cm3/mol.1 Based on the comparability in probe structure, the latter value may be used to predict the expected change in the current study. For the pressure range of 1-345 bar and a temperature of 292.5 K, this molar volume change corresponds to a 14% decrease in association constant. Experimentally measured Kc values under elevated pressure conditions are shown in Table 1, with representative doublereciprocal plots illustrated in Figure 3. The association constant determined for 2,6-ANS‚βCD at 7 bar is 2160 M-1 and is in general agreement with earlier studies conducted at atmospheric pressure.18,22 Literature values for the 2,6-MANS‚βCD complex formation constant at ambient pressure vary from 20 000 (ref 23) to 7360 M-1 (ref 22), while the value in this study was found to be 16 500 ( 300 M-1. Although the 2,6-ANS and 2,6-MANS fluorophores are almost structurally identical, the association constants at standard temperature and pressure are dramatically different. The fluorophores differ only by the presence of a methyl group on the anilino nitrogen atom, and this portion of the probe molecules is situated near the wider rim of the β-cyclodextrin cavity.20,21 A larger association constant for the 2,6-MANS‚βCD complex thus suggests that specific rim interactions play a key (22) Catena, G. C.; Bright, F. V. Anal. Chem. 1989, 61, 905-909. (23) Seliskar, C. J.; Brand, L. Science 1971, 171, 799-800.
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Figure 3. Benesi-Hildebrand data for the 2,6-ANS‚βCD (top) and 2,6-MANS‚βCD (bottom) complexes.
role in differentiating the complexation constants of these structurally similar analogs. As the hydrostatic pressure is increased, both complexes are well behaved, with double-reciprocal plots showing excellent linearity at all pressures (Figure 3). The resultant association constants as a function of pressure determined from these plots are shown in Table 1. For the 2,6-ANS‚βCD complex, a 14% decrease in the association constant is measured for an accompanying pressure increase of 338 bar. This result is in good qualitative agreement with theoretical predictions for the pressure dependence of inclusion complexes based on the complexation of 1,8-ANS with an incompressible host like poly-β-cyclodextrin. While the association constants for these two isomers are significantly different (K1,8-ANS‚poly-βCD ) 50 M-1;1 K2,6-ANS‚βCD ) 2160 M-1), the pressure-dependent behavior appears to be nearly identical over this pressure range. Because the volumetric considerations are essentially identical for the ANS‚βCD complexes, and because βCD cannot contract about the guest, the solvated complex occupies a greater volume than the dissociated species solvated by water. The measured ∆V for the 2,6-ANS‚βCD complex is +10.5 ( 2.0 cm3/mol, which is fully consistent with previous studies of 1,8-ANS‚poly-βCD complexation,1 where the ∆V was measured to be +9.3 cm3/mol for this analogous complex. This general agreement with earlier investigations suggests that 2140
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the pressure perturbation of the complexation equilibrium position is not strongly dependent on complexation geometry. For the positional isomers, 2,6-ANS is completely encapsulated inside the cyclodextrin cavity, while 1,8-ANS exhibits only modest entry into the cavity.21,24,25 In accordance with previous studies, the pressuredependent behavior would appear to be indifferent to the thermodynamic impetus that led to complex formation.1,5,10 In direct contrast with the behavior of the 2,6-ANS analog, the association constant of the 2,6-MANS‚βCD complex exhibits no significant pressure dependence (Table 1). As evidenced by a ∆V of +1.2 ( 2.4 cm3/mol, the expected dissociation of this structurally analogous complex with pressure is not realized. This lack of pressure dependence does not appear to be in agreement with predictions based on simple volumetric considerations. The nearly identical structures of the 2,6-ANS‚βCD and 2,6-MANS‚βCD complexes, together with the analogous structures of the unbound probe molecules (Figure 1), lead to the expectation of comparable ∆V values based on eq 6. Thus, the assumption of complex molar volume comparability based exclusively on analogous structural considerations appears to be insufficient. That is, volume changes associated with the solute desolvation are expected to be similar for these analogs, and the desolvation of the βCD cavity is clearly identical in both cases. Based on the previous model, the disparity in measured ∆V between these analogous solutes may be attributed to a difference in the volume upon inclusion. These analogs differ only in anilino substituents that have been directly implicated in hydrogen bonding with the rim of the βCD. As a result, the apparent difference in the molar volume change upon inclusion for these two molecules is consistent with a greater influence of local site interactions on the molar volume than previously appreciated. High-pressure NMR studies are presently underway to evaluate this local site perturbation with pressure for these analogous inclusion complexes. Furthermore, in the above models, the volume change associated with the inclusion process often considers the resultant complex as a single structure. However, previous observations of a distribution of emitting states for 2,6-ANS‚βCD and 1,8-ANS‚βCD1,26-28 may indicate the presence of a range of complex structures. That is, the complex may be comprised of a range of structures with varying probe positions inside the cavity. Since these complexes may no longer be discrete structures, the range of inclusion complex geometries and, therefore, complex molar volumes may differ for 2,6-ANS and 2,6-MANS. Unfortunately, evaluation of this contribution to the change in molar volume upon inclusion is not presently feasible, as no studies to our knowledge have assessed the role of pressure on the distribution of complex structures. Thus, local solvation and the detailed distribution of the probe within the cyclodextrin cavity may play a larger role in dictating the pressure dependent behavior for inclusion complexation than previously appreciated. Because the ∆V of a complexation reaction measures thermodynamic properties, detailed mechanistic information is not gained from these studies. The ∆V corresponds to perturbations in the initial and final states of the system, which is important to (24) Schneider, H.-J.; Blatter, T.; Simova, S. J. Am. Chem. Soc. 1991, 113, 19962000. (25) Nishijo, J.; Nagai, M. J. Pharm. Sci. 1991, 80, 58-62. (26) Bright, F. V.; Catena, G. C.; Huang, J. J. Am. Chem. Soc. 1990, 112, 13431346. (27) Huang, J.; Bright, F. V. J. Phys. Chem. 1990, 94, 8457-8463. (28) Nakamura, A.; Saitoh, K.; Toda, F. Chem. Phys. Lett. 1991, 187, 110-115.
Table 2. Qcomplexkia over 338 bar for ANS‚βCD Complexes pressure (bar)
2,6-ANS‚βCD
2,6-MANS‚βCD
6.89 68.9 138 207 276 345
532 ( 10 521 ( 10 513 ( 9 508 ( 10 498 ( 6 493 ( 8
226 ( 1 221 ( 1 218 ( 1 213 ( 1 209 ( 1 205 ( 1
% decrease
7.33
9.37 ( 0.25b
aQ 3 b complexki values were divided by 10 . The overall percent decrease is averaged from three trials.
be an accurate measure of Qcomplexki, the contribution of the unbound fluorophore must be eliminated (vide supra), and the quantum efficiency must be distinguished from fluorescence intensity decrease arising from the dissociation of the complex. Clearly, the fluorescence intensity decreases as the complex dissociates with pressure, thus increasing the number of unbound fluorophore molecules. The total fluorescence intensity is given by the sum of the intensities of the emitting species,
Ftotal ) Fcomplex + Fprobe
(7)
The fluorescence of each component is given by
Fcomplex ) Qcomplexki[complex]
(8)
Fprobe ) Qprobeki[probe]
(9)
and understanding general solvation changes induced by pressure. Since the fluorescent probes chosen for these studies are very sensitive to their solvation environment, further insight can be gained into the role of local solvation. The study undertaken here provides a unique opportunity to investigate any pressure-induced change in the fluorophore quantum efficiency. Pressure Dependence of Complex Quantum Efficiency. Solvent-sensitive probe molecules have been widely used in chemistry and biological studies. Fluorescence properties of emission frequency and quantum efficiency are both commonly utilized to assess perturbations in the solvation environment.29,30 For the probes of interest in these studies, significant blue shifts are observed on complexation; however, the bound and unbound 2,6-ANS exhibit no pressure-induced change in the emission frequency. This result is fully consistent with previous studies involving the positional isomer 1,8-ANS.31,32 As a result, no significant shifts in the fluorescence spectra of the free and complexed fluorophores are anticipated in the modest pressure regime. Moreover, the use of 10-nm band-pass filters for excitation and emission ensures that the analytical signal will be unaffected by any small frequency shifts induced by elevated pressure. The quantum efficiencies of the unbound and complexed solutes, however, may exhibit a pressure dependence. Using the present experimental configuration (Figure 2), the unbound solutes show no statistically significant change in fluorescence intensity over the pressure range of interest here. Pressureinduced perturbations on the quantum efficiency of the complexed solutes are assessed using the inverse of the intercept (kiQcomplex) for the Benesi-Hildebrand plot (eq 1). Measurements of the intercept as a function of pressure for 2,6-ANS‚βCD and 2,6MANS‚βCD are shown in Table 2. Since the instrumental constant is independent of pressure, any pressure-induced changes in this parameter provide a direct measure of the pressure dependence of the complex quantum efficiency. As shown in Table 2, the 2,6-ANS‚βCD and 2,6-MANS‚βCD complexes exhibit a decrease of 7.3% and 9.4% in the quantum efficiency over 338 bar, respectively. To confirm the validity of these determinations, it is important to isolate pressure-induced perturbations in the quantum efficiency of the complex from other possible factors. For the intercept to (29) Turner, D. C.; Brand, L. Biochemistry 1968, 10, 3381-3390. (30) Stryer, L. Science 1968, 162, 526-533. (31) Rollinson, A. M.; Drickamer, H. G. J. Chem. Phys. 1980, 73, 5981-5996. (32) Bismuto, E.; Sirangelo, I.; Irace, G.; Gratton, E. Biochemistry 1996, 35, 1173-1178.
Because a correction for any contribution of unbound fluorophore to the total intensity has been applied,18 the intensity used to determine the association constants and quantum yield is attributable solely to that of the complex. Using the Benesi-Hildebrand approach, the association constant is effectively isolated from the complex quantum efficiency (eq 1). As a result, pressure-induced effects in the quantum efficiency are determined independent of any pressure perturbation in the complexation constant. Thus, in these experiments, the overall decrease in fluorescence intensity observed with pressure is the result of both the decrease in the equilibrium concentration of the complex and the decrease in the complex quantum efficiency. Based on these observations, it is clear that a solvation environment perturbation occurs for both complexes, which is manifest in the decrease in quantum efficiency. The 2,6-ANS‚βCD complex shows a substantial decrease in both the quantum efficiency and the equilibrium constant, which suggests that a significant change in solvation has occurred over 338 bar. For the 2,6-MANS‚βCD complex, a similar decrease in quantum efficiency is not accompanied by a shift in the equilibrium position of the complexation reaction. Clearly, a pressure-induced perturbation in the fluorescence properties of the complex can be independent of the ∆V, so that not all of the solvation information is contained exclusively in the ∆V of a reaction. The steady-state fluorescence measurements, thus, are useful to distinguish between two different pressure-induced solvation perturbations, although it is not clear if the mechanism by which the quantum efficiency decreases is the same for both complexes, or if this mechanism is directly correlated to the ∆V of the 2,6-ANS‚βCD complex. Unfortunately, the specific mechanism by which the complex quantum efficiency decreases with pressure cannot be deduced from steady-state fluorescence measurements. Positing mechanisms is further impeded by the lack of detailed characterization of the excited states of the anilinonaphthalenesulfonic acids in neat solvents,33,34 much less in a mixed solvation environment where the naphthyl moiety is encapsulated in the βCD cavity. Thus, although it is possible to detect a clear, pressure-induced effect on the solvation environment, the exact origin of this perturbation requires more detailed mechanistic studies. Studies are presently underway in our laboratory to determine the localized perturbations in solvation structure induced in this (33) Robinson, G. W.; Robbins, R. J.; Fleming, G. R.; Morris, J. M.; Knight, A. E. W.; Morrison, R. J. S. J. Am. Chem. Soc. 1978, 100, 7145-7150. (34) Kosower, E. M.; Dodiuk, H. J. Phys. Chem. 1978, 82, 2012-2015.
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modest pressure regime. Among analytical techniques, NMR is well suited to provide the localized environment information required to discern pressure-induced changes within the unbound guest and host as well as the associated species. Previous studies with high-pressure NMR have demonstrated its capabilities in monitoring perturbations for a wide range of molecules and environments.35-37 Controlled-pressure NMR applied to this important class of inclusion complexes is expected to yield insight into those site-specific changes in solvation that have a direct impact on binding interactions. Moreover, mechanistic information regarding the role of the hydrophilic rim interactions may be more clearly delineated. CONCLUSIONS Commonly ignored in polar condensed phases, pressure is demonstrated here to have a significant impact on the host-guest interactions that form the basis for many analytical techniques. For two structurally analogous host-guest pairs, significantly differing pressure-dependent behaviors are observed. The 2,6ANS‚βCD complex shows a 14% decrease in association constant for a concomitant pressure increase from 7 to 345 bar, whereas no measurable effect on the complexation equilibrium is observed for the 2,6-MANS‚βCD complex. This result is somewhat surprising, based on theoretical predictions of pressure-induced dissociation when β-cyclodextrin is utilized as the host molecule. Simple volumetric additivity is insufficient to describe the pressuredependent behavior of these structurally analogous probe mol(35) Jonas, J.; Jonas, A. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 287-318. (36) Wallen, S. L.; Palmer, B. J.; Garrett, B. C.; Yonker, C. R. J. Phys. Chem. 1996, 100, 3959-3964. (37) Yonker, C. R.; Wallen, S. L.; Linehan, J. C. J. Supercrit. Fluids 1995, 8, 250-254. (38) Ringo, M. C.; Evans, C. E. J. Phys. Chem., submitted.
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ecules. However, the location of the positional substitution in these analogous probe molecules is consistent with pressureinduced perturbation in site-specific interactions with the hydrophilic rim of the cyclodextrin cavity. High-pressure NMR studies are presently underway to investigate the role of local site solvation on this pressure dependence of association. In addition to fundamental considerations, this pressuredependent behavior has a pragmatic impact on many measurements of analytical importance. As demonstrated here, pressure can act nonuniformly on inclusion complexation, yielding pressureinduced changes in interaction selectivity. Such pressure perturbations in selectivity are expected to have a direct effect on liquid chromatographic separations using inclusion complexation mechanisms.14 This important class of separations has pressure as an inherent parameter utilized to drive flow through the chromatographic column. In this case, solute capacity factor and selectivity may be affected by pressure-induced changes in solute complexation. Thus, it would seem prudent to begin to monitor and control pressure more carefully under these separation conditions. Although demonstrated here for positionally substituted solutes, studies are underway in our laboratory to assess the effect of pressure on chiral separations performed by inclusion complexation.38 ACKNOWLEDGMENT We gratefully acknowledge the General Electric Co. and the Eli Lilly Corp. for partial support of this research. Received for review December 16, 1996. Accepted March 19, 1997.X AC961271O X
Abstract published in Advance ACS Abstracts, May 1, 1997.
