J . Phys. Chem. 1992, 96, 5495-5501
5495
Solute-Solvated Cyclodextrin-Bonded Phase Interactions As Studied by the Spin Probe Technique A. J. Hooper, J. Heindl, P. Wright, M. P. Eastman,+and R. G.Kooser* Department of Chemistry, Knox College, Galesburg, Illinois 61 401 (Received: December 26, 1991)
The interaction of a series of spin probes with two solvated silica-bound cyclodextrin phases involving a and (3 cyclodextrins (Cyclobond 111 and I) have been investigated using electron paramagnetic resonance techniques. The results show that the interactions of the spin probes with the a cyclodextrin surface (Cyclobond 111) do not involve insertion into the cyclodextrin cavity but rather result from polar/nonpolar interactions with the solvated surface. With surface-immobilized p cyclodextrins (Cyclobond I), there is a bimodal behavior depending on the polar nature of the solute molecule. For probes with a nonpolar character, at least partial insertion into the cyclodextrin cavity is the major means of solute-surface interaction. However, when the probes become polar in nature, the probesurface interaction centers on the interactions between the solute molecule and the hydroxyl region surrounding the rims of the bound cyclodextrin. In all cases, the evidence supports the view that surface immobilized cyclodextrins are not simply a tethered version of free cyclodextrin molecules.
Introduction
Cyclodextrin-bonded silicas have found extensive use as stationary phases in liquid chromatography for the separation of chiral, diastereoisomeric, and routine compounds since their introduction by Armstrong and co-workers.',2 These systems have been studied by chromatographic3-'' and theoreticalI2J3methods which have demonstrated the powerful separating ability of these unusual stationary phases. On the other hand spectroscopic techniques (including fluorescence, infrared (IR), and nuclear magnetic resonance (NMR)) have been used extensively to characterize alkyl-modified silicasI4to determine how the structure and dynamics of the bonded phase correlates to chromatographic behavior and the microscopic mechanisms of separations. Recently, electron paramagnetic resonance (EPR) methods have been also used to aid in the understanding of alkyl modified While spectroscopic techniques have proven useful for understanding alkyl-modified silica surfaces, they have not been used in the study of cyclodextrin-bonded phases, but a variety of spectroscopic methods including fluorescence,'"22 NMR,23-30and EPR3'-42 have been applied to free cyclodextrins in solution. Electrochemical means have also been used to characterize association constants43of small paramagnetic molecules with cyclodextrins. These investigations have shown that certain probes actually insert into the cyclodextrin cavity while others form weaker associations with the cyclodextrin molecules. EPR techniques have proven to be a robust means for the investigation of the microscopic properties of many materials.One of these techniques is the spin probe method where paramagnetic molecules are allowed to intercalate into the material of interest. The spectroscopic properties of the probe hold information about the character of the host material. It is our objective to use the spin probe method to characterize the surface properties of solvated cyclodextrins bonded to silica (Cyclobonds) in order to understand the physical and chemical properties of the surface and to investigate the mechanism of chromatographic retention in these unusual compounds. We report here the first use of EPR to study a cyclodextrin (hexaamylose) and 8 cyclodextrin (heptaamylose) bonded to silica surfaces and one of the first studies to use spectroscopic techniques to understand the surface science of immobilized cyclodextrins. Experimental Section The spin probes of the 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO) family including TEMPO, TEMPOL (4-01), TEMPAMINE (Camino), and TEMPONE (4-carboxy) and di-tertbutyl nitroxide (DTBN) were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were used as received. The non-surface-immobilized cyclodextrins (a,@-cyclodextrins) were also 'Department of Chemistry, Northern Arizona University, Flagstaff, AZ.
obtained from Aldrich. All organic solvents were reagent grade and were purchased from Fisher Scientific (Chicago, IL). Doubly deionized water was used. Before use, the solvents were filtered through 0.2-pm membrane filters (Alltech Associates, Deerfield, IL). All mixed solvents were made on a volume/volume basis and were thoroughly degassed with either nitrogen or helium to excluded oxygen before EPR examination. The cyclodextrin phases Cyclobond I (P-cyclodextrin) and Cyclobond I11 (acyclodextrin) were used as purchased from ChromHelp (Boonton, NJ). The silica with just the spacer arm for the cyclodextrin attachment was a kind gift from D. W. Armstrong (University of Missouri-Rolla) and was washed with 0.01 M H2S04and rinsed with methanol and water to react the terminal epoxide group to a diol. EPR spectra were taken on a modified Varian V-4500 EPR spectrometer with a 64x1. magnet equipped with a MicroNow (Chicago, IL) modified bridge and a Stanford Research Systems (Palo Alto, CA) SR-565 lock-in amplifier and data acquisition software running on a Gateway 2000 386 computer. Data reduction was done using LabCalc (Galactic Enterprises, Salem, NH) software. The magnetic field modulation frequency was 100 kHz and the usual precautions were taken to avoid microwave saturation and field modulation distortions. The magnetic field sweep was calibrated using the known coupling constants of either pbenzosemiquinone ion in alkaline ethanol solution (0.2368 m P 7 ) or TEMPO in CCl, (1.535 mT48). All data reported here were the average of at least three separate runs. All experiments with the spin probes in contact with the Cyclobonds were done in a dynamic steady state mode. A column consisting of '/8-in.-o.d. Tefzel tubing (Upchurch, Oak Harbor, WA) held rigid by a l/,-in.-i.d. glass tube was connected on either end with unions (Upchurch model P-614) to 1/,6-in.PEEK tubing (Upchurch); one end of the PEEK tubing was attached to a Beckman llOB solvent delivery pump. The column was dry packed with the stationary phase which was held in the column by a frit ('/I6 in., Alltech, Model 720005) in the exit end of the column. The column was inserted into the cavity and held in place with aqueous flat cell holders on the Varian TE102 cavity. All experiments were done by pumping the degassed mobile phase of given composition containing the spin probe (concentration of 0.1 mM or less) through the column in the spectrometer and observing the probe signal when the column was under flow conditions. The entire solvent delivery tubing was encased in 1/2-in.-i.d.-diameter plastic tubing with a flow of nitrogen gas to prevent reincorporation of oxygen into the solvent through the permeable Teflon solvent delivery tubing. All columns were conditioned with the mobile phase/probe solution for 30 min before observation. Flow rates ranged from 0.1 to 0.5 mL/min. EPR paramaters for probes in the absence of the bonded cyclcdextrins were determined by degassing a probe solution with
0022-3654/92/2096-5495%03.00/00 1992 American Chemical Society
5496 The Journal of Physical Chemistry, Vol. 96, No. 13, 1992
nitrogen and transferring the solution to a 1.5" capillary tube in a nitrogen atmosphere in a glovebox. Rotational correlation time analysis was done using the fast tumbling theory of Freed and ~ e w o r k e r s . In ~ ~the , ~fast ~ tumbling regime, the line width can be expressed as a quadratic function of the nitrogen hyperfine index number, m:
6 = A 4- Bm -k Cm2
(1)
The values of A, B, and C were taken from line widths determined by measuring the amplitudes of all three spectral lines and the width of the central one. The widths were corrected for inhomogeneous broadening using the method of Bales.51 The calculation of the rotational properties required the knowledge of the g and hyperfine tensor components. Studies on TEMPONE5*have demonstrated that the g and hyperfine components are linear functions of the solvent polarity parameter, ET. The measured coupling constant allowed the establishment of an effective E T value from literature variation5* of the coupling constant with solvent polarity and then this effective ETvalue established the magnetic properties for the simulation for TEMPONE. Values for the g tensor for TEMPOL,53 and TEMPAMME54 (as the trimethylamine cation) were taken from the literature. The hyperfine tensors for TEMPO, TEMPAMINE, and TEMPOL were calculated from the effective ET values for the sovlent system and the linear fit to the uz values in water and cC14.48 Measurements of the retention factors of the spin probes on Cyclobond I were done using a 100 mm X 4.6 mm Rainin (Woburn, MA) AST-40010 5-pm particle size Cyclobond I column. An SSI (State College, PA) Model 300 pump and 210 damper, a Rheodyne injection valve, and an ISCO (Lincoln, NE) Model UA-5 detector operating at 254 nm constituted the chromatographic system. The probe solutes were made up at a concentration approximately 10 ppt. The dead time at a given flow was determined by the injection of a solution containing iodine.
Results and Discussion Before we examine the results, certain characteristics of the Cyclobond phase should be recognized. 1,2,56 These phases are synthesized by chemically modifying silica with spacer arm molecules bonded to the surface by a siloxane linkage. The spacer arm is an alkyl ether with terminal epoxide group. The epoxide reacts with the hydroxide groups around the rim of the cyclodextrin (CD) molecule to bond the moiety to the surface. Since both the bottom (C6 carbons) and the top (C2 and C3 carbons) of the cyclodextrin molecule have hydroxyl groups, various orientations and spacer arm configurations are possible. On the average each cyclodextrin molecule is bonded by two spacer arm chains with the C6 hydroxyls more reactive but less numerous than the C2 and 3 hydroxyls. Thus, there is some heterogeneity in orientation of the cyclobond cavity with respect to the silica surface as well as in the number of attached spacer arms to each cyclodextrin molecule.56 It should be noted that both the a- and &bonded cyclodextrins studied here are on identical silicas, spacer chains, and coverage.56 The percent surface coverage in the Cyclobonds is about the same as a non-end-capped C 18 silicas6such has been used in previous studies.l7*I8 Prior work on alkyl-modified s ~ r f a c e s ~with ' ~ ' ~the spin probes used here have shown that these are reasonable models for small chromatographic solutes containing the same functionality as the probes have a t the 4 position on the piperidine ring. Baaded a-Cyclodexbin (Cycloboad IU). Studies have been done on the four TEMPO family probes in contact with the Cyclobond I11 phase (CB 111), 0.010 M a-cyclodextrins in 50/50 methanol/water, and probes free in solution without CDs. The coupling constant results are displayed in Table I, and typical spectra are shown in Figure 1. Various studies'7J8*48,52 have shown that the coupling constant for nitroxides is an increasing function of the polarity of the solvent media in the near neighborhood of the nitroxide group so that the spectral shifts from the values of the probe free to those in contact with cyclodextrins (CD) argue clearly for an association between the probes and the cyclodextrins. The
Hooper et al. TABLE I: Coupling Constants for Spin Probes Interacting with a-Cyclodextrin in Solution and Cyclobond 111 in SO/SO Methnnol/Water Mobile Phase mT a-CD CB-111
ON."
probe
free
OCH3 TEMPO
1.676
1.723
1.690
TEMPONE
1.581
1.577
1.600
TEMPOL
1.650
1.699
1.673
TEMPAMINE
1.632
1.692
1.653
CHI
H3C H3C
0
A
G
"Sample deviation *0.008 mT.
Figure 1. EPR spectra of TEMPAMINE (A), TEMPOL (B), and TEMPO (C) in contact with Cyclobond I11 bonded phase solvated with 50/50 v/v methanol/water. Notice the broader lies in the TEMPAMINE and TEMPOL cases, indicating greater interaction with the bonded cyclodextrin moiety.
coupling constant increase over the free state for TEMPO, TEMPOL, and TEMPAMINE in both the a-CD and CB I11 cases shows the influence of the C D hydroxyls in the formation of a nitroxide-CD association. On the other hand, TEMPONE coupling constants in the free and a-CD cases are identical within experimental error; in fact the spectra are nearly superimpcwable, suggesting that TEMPONE does not associate with the nonbound CD. Interestingly, there is a decrease in the polarity of the nitroxide environment upon surface immobilization for the three interacting probes, reflecting a decrease in the importance of hydroxyl interactions and underscoring the observation that Cyclobond phases are not just silica-bonded analogs of cyclodextrins, and further demonstrating that the surface exerts a profound influence on the probe-CD interaction. This is clearly the case in TEMPONE-CB 111, where we know the interaction is minimal with the CD in solution, but the introduction of the bonded surface does cause a significant upward shift in the coupling constant. More subtle information can be gathered from the line widths and line intensities of the probes as seen in Table 11. A general
The Journal of Physical Chemistry, Vol. 96, No. 13, 1992 5491
Cyclodextrin-Bonded Phase Interactions
TABLE 11: EPR Spectral Features of Spin Probes Interacting with a-Cyclodextrin Solutions and Cyclobond I11 in 50/50 MethnnoVWater Mobile Phase line amplitude relative to center line low field high field
center width,O mT probe TEMPO TEMPONE TEMPOL TEMPAMINE
" Uncorrected
free
0.101 0.0490 0.167 0.171
a-CD 0.146 0.050 0.172 0.180
CB 111 0.124 0.0561 0.168 0.198
a-CD 0.999 0.992 0.991 0.998
free 1 .oo 1 .oo 1 .oo 1 .oo
CB 111 0.912 0.982 0.993 0.976
free
0.966 0.871 0.958 0.919
a-CD 0.979 0.838 0.951 0.934
CB I11 0.816 0.828 0.884 0.665
for inhomogeneous broadening; i4.0% error.
TABLE 111: Line Widths and Rotational Correlation Times for Spin Probes Interacting with Cyclobond 111 and Free a-Cyclodextrins Compared to Probes Free in 50/50 Methanol/Water Solution line widths" probe B, mT X 10 C, mT X 10 rB, s X 10" rC,s X 10" Tc/TB x 10" TEMPO free 0.0006 0.0006 0.4 0.4 1. 0.4 a-CD -0.0066 0.0073 0.4 0.8 2. 0.6 CBIII -0.019 0.064 1.3 6.3 4.8 2.9 TEMPOL free -0.015 0.015 1.1 1.1 1 .o 1.1 a-CD -0.015 0.021 1 .o 2.4 2.4 1.5 CBIII -0.026 0.051 2.2 5.1 2.3 3.3 TEMPAMINE free -0.029 0.029 3.2 3.2 1 .o 3.2 a-CD -0.025 0.027 2.5 2.8 1.2 2.6 CBIII -0.125 0.146 13. 15. 1.1 14. TEMPONE free -0.015 0.015 1 .o 1 .o 1 .o 1.o a-CD CBIII -0.021 0.026 1.7 2.6 1.5 2.1 "Errors are fl in the last significant place. bGeometric mean of rC and rB.
TABLE IV: Spectral Variations of TEMPOL as a Function of Solvent Composition Free in Solution Compared to Interactions with CB I11 Phase line amplitude re1 to center line aN/ mT center width: mT low field high field mobile phase free CB I11 free CB 111 free CB 111 free CB 111 100% H20 1.713 1.708 0.180 0.166 1 .oo 0.901 1 .oo 0.820 0.171 1 .oo 0.966 0.988 0.855 1.678 1.688 0.170 75% H20/25% MeOH 1 .oo 0.884 0.993 0.958 1.650 1.673 0.167 0.168 50% H20/50% MeOH 1 .oo 0.935 1 .oo 0.955 1.598 1.622 0.171 0.168 100% MeOH
" iO.008mT.
Uncorrected for inhomogeneous broadening.
increase in the line width is seen when the TEMPO and TEMPAMINE probes are in contact with cyclodextrin containing systems, indicating a decrease of rotational mobility as would be expected for a complex formation. This is most marked in TEMPAMINE when shifting from free CD to CB 111. However, the width shifts and changes in line amplitudes are not great in moving from probes free in solution to those interacting with the CD phases. It would be expected and has been observed3'-33*35*40 that insertion into the CD cavity causes a large increase in the central linewidth and large amplitude decrease in the high and low field lines. The lack of this kind of spectral change shows that insertion is not the mode of complexation in either a-CD or CB 111. Analysis of the line widths leads to the determination of rotational correlation times from the line-width dependence given in eq 1 from either the B or C term if the motion is isotropic. However, when the motion is anisotropic, T~ and T~ are unequal and indicate which is the faster molecular rotation axis; T~ > T~ it is the x axis (parallel to the N - O bond in the nitroxide) and T~ > rB it is either the z (in the plane of N O ?r bond) or the y axis.5s It should be noted that in the case of anisotropic motion, neither T~ nor T~ is the true rotational correlation time; however, simulations based on the line-width theory of Freed49show that (1) the experimental values of T B or TC are closer than an order of magnitude to the true correlation time and (2) the ratio of T,-/T~ depends only on the anisotropy of the rotation and not on the mean rotational rate. Line-width values, corrected for inhomogeneous broadening,51 and correlation times appear in Table I11 for the case of 50/SO methanol/water solvent. All probes rotate isotropically free in
solution as demonstrated by the equality of T~ and rC as is expected for small, approximately spherical molecules in isotropic solv e n t ~ . ~When ~ - ~ *the probes, except for TEMPONE, interact with CD phases, there is a restriction of the rotational motion demonstrated by an increase in T~ and/or T~ and concomitant increase in the ratio T C / T B . Since the coupling constant evidence indicates a probe-CD complex, this restriction is to be expected and is consistent with complex formation. When the CB I11 phase is compared to the a-CD in solution for TEMPO, there is a dramatic shift to slower motion and a greater increase in the ratio. It is likely that this is the result of the immobilization of the C D molecules and the consequent decrease in rotational freedom of the CD-probe complex. The largest shift in rotational restriction comes from the probe TEMPAMINE. In octadecyl-modified surfaces1618 it has been shown that the amine group of the probe interacts strongly with surface immobilized hydroxyls, yielding a bimodal spectrum of a fairly mobile probe with a strongly immobilized signal from hydroxyl interaction typical of slow tumbling nitroxides. While the TEMPAMINE-CB I11 system shows decreased rotational mobility, it is not as immobilized as the octadecyl case indicating that in CB 111the interaction is at the C D hydroxyls rather than the surface. Chromatographic s t u d i e have ~ ~ ~indicated ~ ~ ~ that surface silanols are not important in retention in CDs and our results with TEMPAMINE confirm that observation. If we examine T~ as the correlation time characteristic of the motion,s7we find the fastest rotational motion for TEMPO with TEMPOL and TEMPONE slightly slower, a result of the fact that TEMPO has no polar group in the 4-position while TEMPAMINE is an order of magnitude slower. The effects seen here are certainly due to interactions exterior to the CD cavity
Hooper et al.
5498 The Journal of Physical Chemistry, Vol. 96, No. 13, 1992
TABLE V Coupling Constants for Various Spin Probes Interacting with Cyclobond I in Water/Methanol Solvents Compared to Probes Free in Solution solvt UN (av)? mT composition ('3% methanol) 0 25 50 100 TEMPO free 1.732 1.710 1.676 1.636 CB I 1.746 1.726 1.713 1.676 Au,? 8.1 9.3 22.0 24.0 TEMPONE free 1.615 1.596c 1.581 1.526 CB I 1.626 1.606 1.601 1.536 AureI 6.8 6.3 13 6.5 TEMPOL free 1.713 1.678 1.650 1.598 1.685 1.676 1.689 1.626 CB I Aurel -16 -1.2 24 17 TEMPAMINE free 1.694 1.666' 1.632 1.607 CB I 1.719 1.693 1.692 1.636 Aarer 15 16 37 18 a f0.008 mT. * Au,,, = 1000[uN(CBI) - uN(free)]/uN(free). Extrapolated from linear fit to other three values.
rather than insertion, confirming the results of the coupling constant comparisons. To see what effects solvent composition might have, a study was done on TEMPOL in a variety of methanol/water mobile phases and the results are shown in Table IV. Coupling constants of this probe free in solution are linear functions of the solvent composition for the methanol/water In contact with CB 111, TEMPOL has coupling constants that are uniformly higher than the bulk phase except for the case of pure water, indicating probe interaction in the polar region around the cyclodextrin rims. Using the linear variation of the coupling constant for free probes and hyperfine values for the probes in contact with CB 111, it is possible to establish an effective solvation composition. For pure water, 25% MeOH, 50% MeOH, and pure MeOH, the effective solvation compositions in percent methanol are 1%, 19%, 32%, and 77%, respectively. The pure solvent cases clearly demonstrate the mediating effect of the cyclodextrin hydroxyls on the solvation character of the CB 111, maintaining some organic character in pure water but some aqueous character in pure methanol. Table IV also contains motional information about TEMPOL as a function of solvent composition. The probe motion in the absence of CB I11 is nearly isotropic (as demonstrated by the nearly equal line heights) and shows little line-width variation, indicating that nothing much is changing with regard to rotational motion as a function of solvent composition. In the case of CB 111, this same uniformity is also apparent in the widths, but the heights of the outer lines undergo a significant decrease as the water content increases, indicating an increase in the restriction of rotation. This change in rotational behavior is consistent with a strong probe-CD interaction in media with high water content and a decrease in this interaction as the percent organic modifier increases. Retention s t u d i e ~ have ~ ~ . demonstrated ~~ the bonded CD phases show a switch from reversed to normal phase behavior when the mobile phase changes from high to low aqueous content. TEMPOL behavior confirms this because the switch to normal phase behavior at high methanol decreases the organic probesurface interaction. Bonded 8-Cyclodextrin(Cyclobond I). In buffered aqueous solution, /3-cyclodextrins can completely insert small spin probe molecules. The most well-characterized inclusion complex formation has been demonstrated by TEMP0,33*35.36*43 which inserts with the N O group pointing out of the cyclodextrin basket and DTBN35,38and related molecule^"^^^ which insert with the N O group sideways. While other spin probes do associate with 8CD,33-35*43 evidence for actual inclusion complex formation of TEMPONE, TEMPOL, and TEMPAMINE is weak except for possibly TEMPONE at high CD concentration^.^^ The question to be answered in this study is whether inclusion is the mechanism for the interaction of these probes with the CB I phase. The answer has relevance for the understanding of the retention mechanism in chromatographic applications of CB I and
n
I \
Figure 2. EPR spectra of TEMPO interacting with Cyclobond I phase solvated with pure water (A), 50/50 v/v methanol/water (B), and pure methanol (C). Note the satellite lines in A from probe dimer formation.
of chemical behavior modification with surface immobilization. Table V contains the study of spin probe coupling constants as the average of the high- and low-field values for the solutes in association with CB I and free in solution. The latter all exhibit the expected linear variation of the coupling constant with solvent comp~sition,~'J~ whereas the probes in association with the bonded cyclodextrin show considerable variety in their response to solvent variation. Perhaps the easiest way to examine the character of the shifts is by defining a relative coupling constant shift, Pam,: Parel = 1000[aN(CBI) - aN(ffee)] /aN(fffX)
(2)
where aN(CBI) and aN(free) are the coupling constants of the probe in contact with CB I and free in solution, respectively. Positive values of Aamiindicate that the probe is in a more polar environment than the corresponding solution without the CB I; negative values indicate a less polar environment. Two general kinds of behavior in PareIcan be observed in Table V; TEMPO stands in a class by itself with a monotonic increase with increasing methanol, while the others do not. Such behavior is explained as a matter of course if the TEMPO molecule inserts with the nitroxide group facing out of the cavity, exposing the N O group to the polar solvated region around the rim of the cyclodextrin molecule; however, the NO moiety, not directly interacting with the rim hydroxyls, would be expected to track in a linear way the changes in the bulk solvent polarity as its composition changes. TEMPONE and TEMPAMINE probes demonstrate a behavior that shows approximately a constant coupling constant, and hence constant solvent composition in the mixed solvent region, changing dramatically only in pure water and methanol. This is explainable if the nitroxide group was pointed toward the CD cavity or into the hydrocarbon spacer chain region. In fact, TEMPOL actually has an Aamithat is negative in the high water solvents, indicating perhaps that the nitroxide group has been inserted into the hydrophobic interior of the cyclodextrin cavity or into the hydrocarbon spacer chain region. The variation of Pa,, for the three probes with polar groups at the 4-position indicates the greatest shift in polarity from the bulk solvent at about 50/SO methanol/water. The shift toward coupling constants more characteristic of the bulk solvent at high methanol indicates a decreased interaction between the spin probes and the CB I surface which arises from methanol itself becoming competitive with the probes for inclusion and hydroxyl interaction, decreasing probe interactions. This is likely since it is well known that aliphatic alcohols can include and/or modify inclusion behavior of other compounds.-* It should be mentioned that coupling constant evidence does not show inclusion as the mechanism for surface interaction for TEMPOL, TEMPAMINE, and TEMPONE. In the case of TEMPO and DTBN at high water content (75% and 100% water) satellite lines appear on either side of the well-known three-line nitroxide spectrum arising from dimer formation63d5 (see Figure 2). In this case the pair formation
The Journal of Physical Chemistry, Vol. 96, NO. 13, 1992 5499
Cyclodextrin-Bonded Phase Interactions
do interact with the spacer chain surface and undergo a substantial inhibition of rotation. When the CD unit is attached to the surface, the probe is actually freer to rotate than with just the spacer arm present; however, the line amplitude trends suggest that the rotation is more anisotropic. The likely molecular picture is that the rather disordered spacer chains cause considerable inhibition of rotation when the probes intercalate; however, when the CDs are present the surface order increases but the interaction between the probe and bonded surface is more directional. This study reveals that the spacer arms can play a role in solute-surface interaction but that the CD basket is a major player in the surface properties of Cyclobonds with the CD hydroxyl groups being important modifiers of surface solvation and/or probe interaction. Another item to note is the dramatic change in line amplitude shift seen in TEMPAMINE in moving from the spacer surface to the CB I. This is further proof that the rotational inhibition shown by TEMPAMINE is due the presence of the CD basket and not unmodified hydroxyls on the silica surface. The probe line widths also contain information about the modified silica surface and are given in Table VII; also included here are the rBand rC values and the ratio of the rC/rB. The TEMPO probe shows distinctive behavior in that its rotation is among the fastest (see rB),yet it is the most anisotropic ( T C / T B ) , a circumstance that is explainable if the molecule partially inserts into the CD cavity. Among the other probes what is fairly remarkable is the lack of much variation as the concentration of the solvent changes, except in pure water. Both TEMPOL and TEMPAMINE show significant rotational slow down at all concentrations, indicative of the strong association with CB I hydroxyls that was inferred from the coupling constant data. Since all probes possess a nitroxide moiety, but only TEMPOL and TEMPAMINE have 4-position functional groups capable of hydrogen bonding, the rotational immobility shown by the two must be due to 4-position interaction with the chemically modified surface. TEMPONE in the mixed-solvent region shows faster rotational motion than TEMPOL and TEMPAMINE and shifts to isotropic rotational motion in pure methanol indicating that the probe undergoes weak interactions in the mixed solvent region which disappear all together in pure methanol. In pure water as a solvent, significant rotational slow down is observed for all probes indicating that the antagonistic behavior of the probes toward the highly polar mobile phase forces a tighter interaction between the bonded phase and the probes. Microscopic explanations must square with macroscopic behavior. To see if the molecular pictures coming from EPR data have relevance for the chromatographic behavior of CB I and to test the predictions about surface-probe interaction, a series of chromatographic retention studies was done to determine the capacity factor, k', for these probes on a commercial CB I column. The results are displayed in Table VI11 where the capacity factors for the four TEMPO family probes are given along with the values
TABLE VI: EPR Coupling COM~SII~S, Line Widths, and Relative Line Amplitudes for TEMPO Family Probes Interacting with Spacer Onin Only Modified Silica in So/!%) Meth.nol/Water aN: mT bo? mT h-l f ha h+llhQ probe surface 0.101 1.00 1.676 0.966 TEMPO free in solution 0.162 0.986 0.962 1.688 spacer chain 0.123 0.986 0.943 1.713 CB-I 0.167 0.991 0.958 TEMPOL free in solution 1.650 0.191 1.01 1.666 0.902 spacer chain 1.689 0.651 0.166 1.00 CB-I 1.632 0.9 19 0.171 1.00 TEMPAMINE free in solution 1.656 spacer chain 0.191 0.975 0.930 1.692 CB-I 0.183 0.950 0.837 Free solution and CB-I values repeated from Table V for convenience; h0.008 mT error. b*4.0% error.
is caused by probe antagonism for the aqueous solvent environment which creates high surface loading, enough to create dimers. Reduction of the probe concentration in the mobile phase to 1 X M caused the disappearance of the dimer spectra. To test the effect of the spacer chain that anchors the CD to the silica surface, silica modified with just the spacer chain and no CD was investigated. The terminal epoxide group had been hydrolyzed before use so the two end carbons each had a hydroxyl attached to them. DTBN in pure water gave dimer spectra in the cyclodextrin-modifiedsurface, but the dimers were absent from the spacer chain-modified silica. Comparison of spacer chain DTBN spectra with DTBN free in solution showed remarkable similarity while those from CB I showed definite rotational hinderance. The coupling constant also shifted down by 0.034 mT with the spacer chain as compared to cyclodextrin-modified surface, illustrating that most of the effective polarity of the CB I surface comes from the presence of the cyclodextrin molecules. A second series of spacer chain experiments was done using the four TEMPO family probes in 50/50 methanol/water solution. Table VI contains the data from the experiments, noting that the TEMPONE results were omitted because asymmetric line shapes precluded accurate measurement of the EPR parameters. In all cases, the coupling constant trend is for an increase from free in solution to spacer chain surface to CB I. The increase in going from free solution to spacer surface is due to the nitroxide interaction with the spacer chain hydroxyls or water enriched solvation layer near the bonded surface brought about by the chain hydroxyls. The next leap in going to the CB I surface is the result of the rim hydroxyls on the immobilized CDs and demonstrates the importance of the cyclodextrin hydroxyl groups in determining solute behavior on the surface. All probes show similar line-shape behavior with an increase in line widths in going from free in solution to the spacer surface followed by a decrease when the CB I phase is present accompanied by a uniform decrease in the high and low field line amplitudes. This indicates that the probes
TABLE VII: Line Widths and R o t P t i o ~ lCorrelation Times for Spin Probes Interactinn with Cvclobond I in Various Methawl/Water Solutions line widths" .- ____ ... ..
probe TEMPO
TEMPOL
% water 100 75 50 0 100 75 50 ~~~
0
TEMPAMINE
100 15
TEMPONE
50 0 100 75 50 0
B, mT X 10 b
b -0.01 1 -0,006 -0.063 -0.057 -0.054 -0.039 -0.17 -0.10 -0.10 -0.14 -0,040 -0.010 -0,007 0.0
C, mT X 10
T*,
s
x 10"
Tc,
s
x 10"
TCITB
h
b 0.019 0.019 0.194 0.091 0.054 0.039 0.28 0.12 0.14 0.17 0.11 0.012 0.008 0.0
Error i 1 in the last significant place. bDimer formation precludes measurement.
0.64 0.37 5.9 5.5 2.8 3.8 21.0 12.0 12.0 18.0 2.8 0.66 0.46
2.0 2.1 20.0 9.7 5.4 4.1 27.0 12.0 14.0 19.0 9.7 1.3 0.91
3.1 5.8 3.4 1.8 1.9 1.1 1.3 1.o
1.2 1.o 3.5 2.0 2.0
5500 The Journal of Physical Chemistry, Vol. 96, No. 13, 1992 TABLE VIII: Capacity Factor for a Series of Probes and Molecules on a Cyclobond I Column as a Function of Solvent Composition capacity factor k'" 100% 50/50 10/90 probe MeOH MeOH/H,O MeOH/H,O TEMPO 0.18 1.6 15 TEMPOL 0.14 0.29 1.3 TEMPAMINE 0.13 0.06 1.3 TEMPONE 0.09 0.31 1.6 DTBN 0.15 1.9 17 @-naphthol 0.12 1.69 not eluted a
Less than 2% error over four trials.
for DTBN (a known inserter into the CD cavity in free /3 CD in aqueous solutions) and /3 naphthol which has a strong affinity for the stationary phase.66 The larger the k'value, the longer the solute remains on the column and, by inference, the greater the interaction between the solute and the stationary phase. Under conditions of 100%methanol, all the solutes show nearly uniform behavior with little retention, a confirmation of the situation inferred from coupling constant data that showed methanol is competing with the solutes for the CD moiety. In the mixed solvents, TEMPO, DTBN, and &naphthol are strongly retained, while TEMPOL, TEMPONE, and TEMPAMINE show an order of magnitude less retention. EPR evidence argued for partial insertion of TEMPO into the CD cavity and the results here are consistent with that inference. The interactions of TEMPONE, TEMPOL, and TEMPAMINE are of a different character and are consistent with solute-surface interactions that involve the hydroxyls on the CD rims, either through direct probe-hydroxyl interaction or hydrogen bonding with solvent molecules associated with the rim hydroxyls.
Conclusions Evidence in this paper shows that the Cyclobond phases are not just surface-immobilized cyclodextrins. Rather there is a synergy among the silica surface, the spacer chains, and the CDs that modifies the interactions between small solute molecules and the CD moiety. Cyclobond IIIspin probe EPR data suggest strongly that these molecules do not form inclusion complexes. Coupling constant evidence in the presence of the a-cyclodextrins and the spacer chains alone show that the probes interact with the polar, solvated hydroxyls about the CD rim. Line-width studies indicate that this surface interaction slows down the rotational motion of the probes and creates a much more anisotropic, directional interaction with the surface. The interaction with CB I11 is strongest for the TEMPAMINE and TEMPO although the mode of interaction for the two are different. TEMPAMINE has a hydrogen bonding interaction through the amine group and TEMPOS association is through nonpolar means. The changes that occur in TEMPOL spectra when the organic modifier increases show a change in the probe-cyclodextrin interaction characteristic of a switch from reversed phase to normal p h a ~ e . Since ~ ~ . these ~ ~ molecules differ in the group at the 4-position of the piperidine ring, the spectral differences come about because of these functionalities, making these spin probes useful models for small solute molecules that are alcohols, hydrocarbons, amines, and ketones. In Cyclobond I, the larger cyclodextrin cavity makes it more likely that inclusion complex formation may play a role in the interaction of the probes with the surface. Coupling constant and line-width evidence in the case of TEMPO would argue for inclusion with the nitroxide group out. Dimer formation in both TEMPO and DTBN indicate a strong association with the modified surface and set the two probes apart from TEMPONE, TEMPOL, and TEMPAMINE, confirming the importance of inclusion in their interactions with the surface, a fact supported by the retention measurements. Those probes that have a polar group in the 4-position of the ring clearly interact with the surface, but do not include into the CD basket. Because of the extent of rotational immobilization shown by TEMPOL and TEMPAMINE, it is likely that the predominate interaction of these
Hooper et al. probes is through the hydrogen bonding groups at the 4-position of the ring with the hydroxyls on the cyclodextrin ring or with solvent molecules that are interacting with the rim hydroxyls. TEMPONE, not capable of hydrogen bonding, has a weak CDprobe interaction. The probe studies show that mechanisms other than inclusion exist for the interaction of the probe with the bonded CD and demonstrates the importance of the hydroxyl groups in the rim of the CD molecule in determining surface-probe interaction. Acknowledgment. We acknowledge support for this work from NSF Grant CHE-891987, the Educational Committee of the E. I. du Pont de Nemours Co., and Keck Foundation. Acknowledgment is also made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this work. Special thanks goes to D. W. Armstrong of the University of Missouri-Rolla for his kindness in lending materials and for his review of the manuscript and to Knox students Diane Suzuki and Talib Ali, who ran certain critical experiments. Registry No. TEMPO, 2564-83-2; TEMPOL, 2226-96-2; TEMPAMINE, 14691-88-4; TEMPONE, 2896-70-0; DTBN, 2406-25-9; CHSOH, 67-56-1.
References and Notes (1) Armstrong, D. W. J . Liq. Chromatogr. Suppl. 1984, 2, 353. (2) Armstrong, D. W.; Demond, W. J. Chromatogr. Sci. 1984, 22,411. (3) Armstrong, D. W.; DeMond, W.; Alak, A.; Hinze, W. L.; Riehl, T. E.; Bui, K. H. Anal. Chem. 1985, 57, 234. (4) Tanaka, M.; Shono, T.; Zhu, D.; Kawaguchi, Y. J . Chromatogr. 1989, 469, 429-33. ( 5 ) Han, S. M.; Han, Y. I.; Armstrong, D. W. J . Chromatogr. 1988, 441, 376-81. (6) Chang, C. A.; Wu, Q. J . Liq. Chromatogr. 1987, 10, 1359-68. (7) Issaq, H. J.;Glennon, M. L.; Weiss, D. E.; Fox, S. D. ACSSymp. Ser: Ordered Media, 1987, 342, 260. (8) Armstrong, D. W.; Yang, X.; Han, Soon M.; Menges, R. A. Anal. Chem. 1987, 59, 2594-6. (9) Pirkle, W. H.; Pochapsky, T. C. J . Chromatogr. 1986, 369, 175. (10) Lipkowitz, K.; Landwere, J. M.;Darden, T. Anal. Chem. 1986, 56, 1611. (1 1) Topiol, S.; Sabio, M.;Moroz, J.; Caldwell, W. 8. J . Am. Chem. SOC. 1988, 110, 8367. (12) Boehm, R. E.; Martirre, D. E.; Armstrong, D. W. Anal. Chem. 1988, 60, 522. (13) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beesley, T. E. Science 1986, 232, 1132. (14) Gilpin, R. K. Anal. Chem. 1985, 57, 1465A. (15) Gilpin, R. K.; Kasturi, A.; Gelerinter, E. Anal. Chem. 1987,59, 1177. (16) Malcom, T.; Gorse, J.; Kooser, R. G. J . High Resolut. Chromatogr. Chromatogr. Commun. 1988, 11, 416. (17) Miller, C.; Dadoo, R.; Kooser, R. G.; Gorse, J. J . Chromatogr. 1988, 458, 255. (18) Miller, C.; Joo, C.; Roh, S.; Gorse, J.; Kooser, R. G. In Chemically Modified Oxide Surfaces, Chemically Modified Surfaces; Leyden, D. E., Collins, W. T., Eds.; Gordon & Breach: New York; 1990; Vol. 3, p 251. (19) Nelson, G.; Patonay, G.; Warner, I. M. Anal. Chem. 1988,60, 274. (20) Nelson, G.; Patonay, G.; Warner, I. M. J . Inclusion Phenom. 1988,
-. (21) Hamai, S.J . Phys. Chem. 1990, 94, 2595.
6. -217.
(22) McGown, L. B.; Warner, I. M. Anal. Chem. 1990, 62, 255R. (23) Beregon, R. J.; Chaning, M. A.; McGovern, K. J. J . Am. Chem. SOC. 1978. 100. 2878. (24) Ohara, M.; Kobatashi, G. Nippon Kagaku Kaishi 1983, 2, 300. (25) Inoue, Y.; Hoshi, H.; Sakuri, M.; Chujo, R. J. J. Am. Chem. SOC. 1985, 107, 2319. (26) Eastman, M. P.; Brainard, J. R.; Stewart, D.; Anderson, G.; Lloyd, W. D. Macromolecules 1989, 22, 3888. (27) Lacelle, S.; Gerstain, B. C. Carbohydr. Chem. 1989, 8, 29. (28) Murakami, T.; Harata, K.; Morimoto, S. Chem. Express 1989, 4, 645. (29) Ripmeester, J. A.; Ratcliffe, C. I.; Mameron, I. G. Carbohydr. Res. 1989, 192, 69. (30) Yoshida, N.; Seiyama, A,; Fujimoto, M. J . Phys. Chem. 1990, 94, 4254. (31) Paton, R. M.; Kaiser, E. T. J . Am. Chem. SOC.1970, 92, 4723. (32) Birrell, G. B.; Van, S. P.; Griffith, 0. H. J . Am. Chem. SOC.1973, 95, 2451. (33) Martinie, J.; Michon, J.; Rassat, A. J . Am. Chem. SOC.1975, 97. 1818. (34) Flohr, K.; Paton, R. M.; Kaiser, E. T. J. Am. Chem. SOC.1975, 97, 1209. (35) Okazaki, M.; Kuwata, K. J . Phys. Chem. 1985.89, 4437.
J . Phys. Chem. 1992,96, 5501-5505 (36) Harten, B.; Darcy, R.; Lyons, R. New J. Chem. 1986, I O , 569. (37) Kubozono, Y.; Ata, M.; Aoyagi, M.; Gondo, Y. Chem. Phys. Letr. 1987, 137,467. (38) Eastman, M. P.; Freiha, B.; Hsu, C. C.; Lum, K. C.; Chang, C. A. J. Phys. Chem. 1987, 91, 1953. (39) Eastman, M. P.; Freiha, B.; Hsu, C. C.; Chang, C. A. J. Phys. Chem. 1988, 92, 1682. (40) Kotake, Y . ;Janzen, E. G. J. Am. Chem. SOC.1988, 110, 3699. (41) Kotake, Y.; Janzen, E. G. J. Am. Chem. SOC.1989, 1 1 1 , 7319. (42) Sueishi, Y.; Nishimura, N.; Hirata, K.; Kuwata, K. Chem. Express 1989, 4, 567. (43) Saint-Aman, E.; Serve, D. New J. Chem. 1989, 13, 121. (44) Spin Labeling. Theory and Amplications; Berliner, L. J., Ed.; Academic: New York, 1976. (45) Spin hbeling II; Berliner, L. J., Ed.; Academic: New York, 1979. (46) Biological Magnetic Resonance: Spin Labeling, Theory & Applications, Berliner, L. J., Reubuen, J., Eds.; Plenum: New York, 1989. (47) Venkataraman, B.; Fraenkel, G. K. J. Chem. Phys. 1955, 23, 588. (48) Windle, J. J. J. Magn. Reson. 1981, 45, 432. (49) Freed, J. H. J. Chem. Phys. 1964,41, 2077. (50) Goldman, S.A.; Bruno, G. V.; Polnaszek, C. F.; Freed, J . H. J . Chem. Phys. 1972, 56, 716. (51) Bales, B. L. In Biological Magnetic Resonance: Spin Labeling, Theory and Applications; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1989; Chapter 2.
5501
(52) Zager, S. A.; Freed, J. H. J. Chem. Phys. 1982, 77, 3344. (53) Reference 44, Appendix 11. (54) Romanelli, M.; Ottaviani, M. F.; Martini, G. J. Colloid Interface Sci. 1983, 96, 373. (55) Polnaszek, C. F.; Scheirer, S.;Butler, K. W.; Smith, I. C. P. J . Am. Chem. SOC.1978, 100, 8223. (56) Armstrong, D. W. University of Missouri-Rolla, private communications, July 1990, Mar 1992. (57) In the case of anisotropic but axially symmetric rotations, there are two characteristic correlation times, parallel and perpendicular to the symmetry axis (2). See ref 50. The assumption of axial symmetry in the hyperfine tensor means that there is only one measurable correlationtime, perpendicular to r; this infers that tg is best estimate of motion (see ref 55). (58) Armstrong, D. W.; Li, W. Chromatography 1988, 2, 43. (59) Armstrong, D. W.; Jin, L.H. J. Chromatogr. 1989, 462, 219, (60) Fujiwara, H.; Arakawa, H.; Murata, S.;Sasaki, Y. Bull. Chem. Soc. Jpn. 1987, 60, 3891. (61) Nelson, G.; Patonay, G.; Warner, I. M. Anal. Chem. 1988,60,274. (62) Lukovits, I. J. Mol.Struct. 1988, 170, 249. (63) Veloso, D. P.; Rassat, A. J. Chem. Res. Symp. 1979, 168. (64) Kooser, R. G. Macromolecules 1987, 20, 435. (65) Eaton, S.S.;Kundalika, K. M.; Sawant, B. M.; Eaton, G. R. J. Am. Chem. SOC.1983, 105, 6560. (66) Cyclobond Handbook; Advanced Separation Technologies: Whip pany, NJ.
Measurement of the Charge In a Double Layer at a Solid/Liquld Interface. Use of a Conducting Polymer Pierre Chartier, Laboratoire d’Electrochimie et de Chimie-Physique du Corps Solide, Universite Louis Pasteur, URA au CNRS No. 405, 4, rue Blaise Pascal, 67000 Strasbourg, France
Benjamin Mattes, and Howard Reiss* Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90024 (Received: January 16, 1992; In Final Form: March 5. 1992)
A method is described for measuring the equilibrium double layer charge at the interface between an aqueous solution of HCI and NaCl and the emeraldine form of polyaniline. The method involves the analysis of the measured change in double layer potential that attends the transfer of a polyaniline electrode from a reference solution to one having a different pH. Both experiment and theory are presented. Although double layer charge at liquid/liquid interfaces has been measured previously (using electrocapillarity methods), its measurement at a solid/liquid interface does not appear to have been accomplished previously. The present method may have some generality.
I. Introduction Although the capacitance of a double layer can frequently be measured, the actual total charge stored on the capacitance presents a more difficult problem although it has been measured effectively using mercury electrodes and electrocapillarity theory.’ In addition, in a very original study Reid, Melroy, and BuckZhave measured the double layer charge a t the liquid/liquid interface between nitrobenzene and water using electrocapillarity methods while a t the same time performing theoretical analyses by means of Gouy-Chapman-Verwey-Niessen the0ry3q4for comparison with their experimental result. Agreement was in fact satisfactory. In the present paper we describe a method that makes possible the measurement of the double layer charge a t a solid/liquid interface. The method promises to be of some generality and has grown out of some recent studies of a modified Donnan phenomenon involving the conducting polymer, polyaniline (in its semioxidized form, i.e., emeraldine). Work on this modified Donnan phenomenon has already been p~blished.~ Briefly, in that work, emeraldine base was immersed in aqueous solutions containing HCl and NaCl. Protons are strongly absorbed by the imine nitrogen atoms in the polymer and are followed by C1- ions as counterions. Nevertheless, the polymer develops a positive charge relative to the solution after equilibrium
is achieved. The absorbed protons play the role offixed ions in an ion exchange membrane so that the proton-doped emeraldine behaves as an anion selective membrane. Since the protons are not infinitely strongly bound, their concentration in the polymer phase (Le., the concentration of thefixed ions) is affected by the composition of the bathing solution. Since in the usual Donnan equilibrium this is not the case, the equilibrium has been termed a modified Donnan phenomenon. In the studies of ref 5 , the uptake of protons was determined by the measurement of the conductivity of the dried emeraldine film, using earlier calibrations6 of conductivity as a function of doping. In this way it was possible to measure the change in uptake (at constant pH) induced by a change of NaCl concentration in the bathing solution. Comparison of these measurements with the theory for the modified Donnan equilibrium, for the case in which NaCl is absent, allowed the determination of the equilibrium constant K for distribution of protons between free and bound states within the polymer. This value could then be used to predict the effect (via the modified Donnan equilibrium) of NaCl on the proton uptake. Results were in semiquantitative agreement with the predictions of theory. It should be mentioned that the concentration of imine nitrogens in the swollen immersed polymer was estimated to be 2 X loz1
0022-3654/92/2096-5501$03.00/00 1992 American Chemical Society
