Murray B. McBride
196
Nitroxide Spin Probes on Smectite Surfaces. Temperature and Solvation Effects on the Mobility of Exchange Cations Murray B. McBride' Department of Land Resource Science, Unlversity of Guelph, Guelph, Ontario, Canada (Received June 30, 1975)
Electron spin resonance (ESR) studies of the paramagnetic probe cation, protonated 4-amino-2,2,6,6-tetramethylpiperidine N-oxide, exchanged on the surfaces of a smectite (layer silicate) have revealed important properties of the silicate surface. The probe cation is motionally restricted by its association with the external exchange surfaces on fully solvated smectites, and is restricted and oriented to an even greater degree in interlamellar regions, especially as the distance between silicate sheets is decreased. The type of interlamellar solvent alters the molecular orientation, with water apparently allowing the hydrocarbon fraction of the probe ion to associate closely with the silicate surfaces. In addition, the species of exchangeable metal ion present on the surfaces has an effect on the probe's mobility and orientation. The mobility of the probe is greatly reduced on the hydrated smectite surfaces a t temperatures below OOC, a fact attributed to the migration of water from between the silicate sheets and the reduction of thermal motion. Similar lowtemperature ESR studies of smectite exchanged with Cu2+ demonstrate the transition from a solution-like to a solid-like environment between the silicate plates in the -25O to -5OOC temperature range. The ESR spectra are supported by x-ray diffraction data which allow the spacings between smectite plates to be determined.
Introduction The use of electron spin probes in membrane biophysics has recently gained impetus because of the availability of stable nitroxide radicals as probe molecules. The anisotropic g tensors and hyperfine splitting values ( A ) of the nitroxide probes, as well as the solvent dependence of g and A , permit considerable information to be obtained from electron spin resonance (ESR) spectra of the probes when doped into membrane systems.2J Effective viscosities of the local environments of the spin probes can be estimated from the degree to which anisotropies in the ESR spectrum are averaged by molecular tumbling. Paramagnetic cations such as Mn2+ and Cu2+have been utilized as spin probes to characterize the environment of exchangeable cations adsorbed on smectite surface^.^,^ These ions exist as Mn(H20h2+ or C u ( H 2 0 ) ~ ~ complexes + in fully hydrated smectites, and demonstrate solution-like tumbling mobility in interlamellar regions. However, lowered relative humidities produce rigidly oriented hydration complexes of these ions as the interlamellar regions collapse until only about one to two monolayers of water remain between the silicate sheets. In the present study, an attempt is made to relate the mobility of exchange cations on smectites to the extent of dehydration of the smectite. Dehydration is achieved both by lowering the relative humidity, and by freezing the clay a t various temperatures between 0 and -5OOC. A protonated nitroxide spin probe is doped into Na+-, H+-, and Mg2+-saturatedsmectite at about the 5% level of exchange, and used to estimate the interlamellar mobility. In addition, the rotational mobility of the spin probe on fully solvated smectites is determined. Materials and Methods A California hectorite ( < 2 - particle ~ size), with chemical formula and exchange capacity previously reported,5 was saturated with Na+ and Mg2+ exchange ions using excess The Journal of Physical Chemistry, Vol. 80, No. 2, 1976
quantities of aqueous chloride salt solutions. Excess salt was then washed from the clay suspensions by centrifuging and discarding the supernatant until a negative AgN03 test for chloride was obtained. The H+-saturated hectorite was prepared by passing Na+ hectorite through an acid resin column; however, partial decomposition of this acid hectorite probably resulted in some Mg2+ moving from the structure into exchange positions. An aqueous solution of Nthe spin probe (4-amino-2,2,6,6-tetramethylpiperidine oxide), referred to here as Tempo, was titrated with HC1 past the equivalence point (as determined by a pH meter) in order to protonate the amine group of the probe. Known quantities of this cationic form of the probe were added to the Mg2+- and Na+-saturated hectorites to produce clays doped a t about the 5% level of exchange capacity. The clays were then washed several times in distilled water to remove chloride and neutral probe molecules. Aqueous suspensions of the doped hectorites were dried on polyethylene sheets to produce oriented, self-supporting films to be used for ESR studies. The ESR spectra were recorded for oriented films of hectorite in quartz tubes using an X-band Varian E-12 spectrometer. Sample temperature was controlled to & l 0 C between 20 and -1OOOC by flowing nitrogen gas cooled by liquid nitrogen through the sample cavity and using a variable heating element. Hectorite suspensions dried on glass slides produced well-oriented films for x-ray diffraction. A GE XRD-6 diffractometer with Ni-filtered Cu radiation was used to determine the basal spacings of these films under various conditions of solvation. The temperature of the clay films could be controlled by drying the clays on aluminum foil and mounting these foil-supported films on the sample holder. The holder was then placed in contact with an apparatus that varied the temperature using a reservoir of liquid nitrogen and a variable heating coil. A thermocouple attached to the sample holder allowed the temperature a t the sample to be determined to k l 0 C . Thus, the sample temperature could be varied from +20 to
197
Nitroxide Spin Probes on Smectite Surfaces 1
-5OOC while a slight excess of water was maintained on the clay films during the cooling process by covering the films with cellophane.
I
20 GAUSS
H .
Results and Discussion
Nitroxide S p i n Probes on Smectites under Various Conditions of Solvation. The nitroxide spin probes are observed to tumble rapidly enough in aqueous solution at 25OC to completely average anisotropies in g and hyperfine splitting ( A ) values. As a result, a three-line ESR spectrum is obtained centered on go (average g) and with splitting value A. (average A ) . The correlation time, rC,for the tumbling of Tempo1 has been calculated to be 0.1 X sec in water at 250Cfi A similar correlation time is expected for Tempo+, the protonated form of Tempo, and is verified by the observation of a symmetrical three-line spectrum resulting from rapid molecular tumbling (Figure 1).However, the protonated probe can occupy exchange sites on hectorite surfaces and may demonstrate reduced mobility in the adsorbed state. The spectra of Figures 2 and 3 for Tempo+-doped Na+ and Mg2+ hectorite demonstrate the loss of rotational mobility of Tempo+ adsorbed on silicate surfaces that are solvated with HzO or 95% ethanol (EtOH). Orientation of the hectorite films with the plane of the silicate sheets parallel (11) and perpendicular (I) to the magnetic field, H,produces considerably different line positions and values of A for the hectorites equilibrated a t 100% relative humidity (Figure Z), an effect that is much less evident for the hectorites solvated in 95% EtOH (Figure 3). This anisotropic behavior is more pronounced as the relative humidity of equilibration is reduced, a result demonstrated by the spectra of Na+ and Mg2+ hectorite at 93%, 75%, and 33% relative humidities (Figures 4 and 5). Closer analysis of the ESR spectra of Figures 2 and 3 reveals that the three sharp resonance lines are superimposed on a broad spectrum representing immobilized Tempo+. The actual intensity of this broad spectrum is greater than is apparent from the spectra because of its greater line widths relative to the widths of the sharper resonance lines. The intensity of the broad spectrum is lowest when the hectorites are fully solvated in water or 95% EtOH (lowfield and high-field shoulders of Figures 2 and 3) but increases as the relative humidity is decreased (Figures 4 and 5). Apparently, two distinct types of Tempo+ are present on the hectorite surfaces: probe ions that are essentially immobilized and probe ions that are much less motionally restricted. Analysis of the narrow-line spectra of Tempo+ on fully solvated hectorites allows calculation of correlation times from the following equatiomfi 71 72
-
=
Figure 1. ESR spectrum at 2OoC of the nitroxide spin probe, Tempo+, in aqueous solution acidified to pH 2.4. The vertical line on this spectrum (and following spectra) represents the position of g = 2.00.
n a
Figure 2. ESR spectra at 2OoC of the nitroxide spin probe, Tempo+, doped at the -5% exchange level into (a) Na+ hectorite equilibrated at 100% relative humidity; (b) Mg2+ hectorite equilibrated at 100% relative humidity.
-2211 WOR-
Ho
= 0.65 Wo(R+- 2)
where 71 and 7 2 are two estimates of r c ,the correlation time in nanoseconds, Wo is the line width of the central peak (G),and Ho is the magnetic field ( G ) .R+ = ( / ~ ~ / h + ~ ) 1i/ 2 (h~/h--d” where ~ ho,+l,-l = height of the middle, low, and high field peaks, respectively. The calculated values of 7‘ are given in Table I for Na+, H + , and Mg2+ hectorites solvated in water and 95% EtOH. Because of the anisotropy of the spectra, the T~ values do not always agree for the perpendicular and parallel orientations of the silicate sheets.
Figure 3. ESR spectra at 2OoC of the nitroxide spin probe, Tempo’, doped at the -5% exchange level into (a) Na+ hectorite solvated in excess 95% ethanol: (b) Mg2+ hectorite solvated in excess 95% ethanol.
In general, the values of ,TC are in the range of 1-5 X 10-9 sec, meaning that the tumbling mobility of the nonimmobilized Tempo+ is reduced by a factor of 100-500 when compared with the probe in aqueous solution. However, a number of trends are apparent from Table I. The correlation time is always greater for the hydrated systems than for the The Journal of Physical Chemistry, Vof. 80, No. 2, 1976
Murray B. McBride
198
TABLE I: Calculated Rotational Correlation Times (nsec) of Tempo+ Doped into Solvated Na+, H+, and Mg2+ Hectorit@
A‘ 75%
Figure 4. ESR spectra at 2OoC of the nitroxide spin probe, Tempo+, doped at the -5% exchange level into Na+ hectorite equilibrated at 93, 75, and 33% relative humidity.
IJFigure 5. ESR spectra at 2OoC of the nitroxide spin probe, Tempo+, doped at the -5% exchange level into Mg2+ hectorite equilibrated at 93, 75, and 33% relative humidity. solvated (EtOH) systems despite the greater distances between silicate sheets for hectorites (Na+, Mg2+,H+) wetted The Journal of Physical Chemistry, Vol. 80, No. 2, 1976
Exchange form of hectorite
Correlation time, T~ of Tempo+ ( X l o 9 sec) Wetting solvent
7,
1
7,
II
J.
II
Na+ 1.7 2.6 3.9 3.8 HZO 95% EtOH Na+ 1.4 2.0 2.3 2.4 H+ 2.9 1.4 5.2 2.9 H2O H+ 95% EtOH 1.2 1.3 1.8 1.5 Mgz+ 3.3 1.5 5.9 3.4 H*O Mgz+ 95% EtOH 0.9 1.o 1.6 1.7 a 1 and 11 represent the orientation of the hectorite films to the magnetic field, H. in water than for those solvated in 95% EtOH (Table 11). The basal spacings of the hectorites show that the interlamellar distance is m7.5 A in 95% EtOH, and a t least 10.5 A in water. If Tempo+ occupies interlamellar positions, steric hindrance of tumbling would be expected for the EtOHsolvated hectorites especially, since the Tempo+ molecule is about 8 A in length. Because the probe tumbles more rapidly on the EtOH-solvated than on the water-solvated hectorite, it is likely that the spectra of the mobile probe represent Tempo+ on external surfaces rather than in interlamellar regions while the broad spectra (immobilized probe) represent Tempo+ sterically restricted from tumbling in the interlamellar spaces. The immobile fraction of Tempo+ increases relative to the mobile fraction as the relative humidity is lowered from 100 to 93% (Figures 2, 4, and 5) as evidenced by the increase in the intensity of the broad “rigid glass” spectrum.2 Lowering the relative humidity further to 75 and 33% essentially eliminates all evidence of mobile Tempo+ (Figures 4 and 5). Essentially all of the probe at 33% relative humidity is restricted, either in interlayers as the silicate sheets collapse together (thereby eliminating most of the external surfaces that were present in fully solvated hectorites), or on partially dehydrated external surfaces. Another trend apparent from Table I is the dependence of correlation times upon the exchangeable cation that largely occupies the hectorite surface. Thus, for EtOH-solvated hectorite, T~ decreases in the order Na+ > H+ 2 Mg2+ for both perpendicular and parallel orientations of the hectorite. For hydrated hectorite, T~ follows the order Mg2+ 1 H+ > Na+ for the perpendicular orientation and the order Na+ > Mg2+ 2 H + for the parallel orientation. In general, the H+ and Mg2+ hectorite seem to behave similarly (similar T~ values) while Na+ hectorite has different behavior, probably because of the weak energy of solvation Therefore, the correlation of Na+ relative to Mg2+ (or H+). time of the probe on EtOH-solvated Na+ hectorite may be lengthened as a result of the ability of Tempo+ ions to closely approach and be adsorbed to the weakly solvated silicate surface. In contrast, the strong solvation of Mg2+ and H+ ions could more effectively inhibit probe-surface interactions by producing a layer of solvent molecules strongly attracted to the surface exchange cations through ion-dipole forces. (The similarity between the T~ of Mg2+ and H+ hectorite may be a t least partly due to acid decomposition of H+ hectorite to produce a (Mg2+, H+) hectorite.) The T~ values of the hydrated hectorites depend on the orientation of the silicate sheets relative to H as well as the exchangeable cation (Table I). The orientation effect
1QQ
Nitroxide Spin Probes on Smectite Surfaces on T~ is a result of preferred alignment of the probe ion on the hydrated silicate surfaces, an effect that is much reduced on the EtOH-solvated surfaces. From the spectra recorded in Figures 2, 3, 4, and 5, it is obvious that orientation of the hectorite films with the plane of the silicate sheets parallel and perpendicular to the external magnetic field, H, produces spectra with different hyperfine splitting values (A). Since the largest value of A, A,,, is expected when H is parallel to the nitroxide z axis of the spin probe,3 and is observed with H perpendicular to the folly hydrated hectorite films (Figure 2), the N-0 bond axis of Tempo+ is demonstrating a tendency to orient parallel to the hydrated silicate surfaces. If the hyperfine splitting value, A , is given as the distance in gauss between the low field line and the central line of the narrow spectrum, then A 1 and A 1 are defined as the values of A for the two principal orientations of the hectorite films relative to H. The difference, A l - Ail, is denoted as AA, and is directly related to the degree of order of the nitroxide probe.7 Thus, for perfect alignment of the z axis of the nitroxide normal to the silicate surface with no molecular motion, AA = A,, - A,, = 31.8 - 7.6 = 24.2 G, assuming that A,, Ayy. The principal values of A (A,,, A,, and Azz) were determined for di-tert- butyl nitroxide oriented in a host c r y ~ t a l . ~ The values of AA for fully-hydrated Na+, H+, and Mg2+ hectorite are 5.1, 3.4, and 3.2 G, respectively. Molecular tumbling and imperfect orientation result in AA values much below the maximum possible. As for correlation times, H+ and Mg2+ hectorite have similar values of AA, while Na+ hectorite has a higher value. This result indicates that Tempo+ is better aligned on the Na+ hectorite film, suggesting that the probe ion is spending a fraction of its time adsorbed directly on the silicate surfaces, allowing the longest dimensions of the probe to align with the surfaces. Again, the strongly hydrated surfaces of H + and Mgz+ hectorite may deter such direct adsorption. The values of AA for H+, Mg2+, and Na+ hectorite solvated in EtOH (-0.5, -0.6, and -1.3 G, respectively) are much smaller than those for the hydrated hectorites and indicate near-random tumbliqg of the mobile fraction of adsorbed Tempo+. Since All > A 1 for these systems (the reverse of hydrated systems), AA has a negative sign, and the probe has a slight preference for a n orientation with the N-0 axis of the nitroxide normal to the solvated silicate surfaces. As with the hydrated hectorites, the larger AA for the Na+ hectorite is evidence for more direct probe-surface contact allowed by the weak solvation energy of Na+. The broad ESR Spectrum representing immobilized Tempo+ is observed even in the fully solvated systems (Figures 2 and 3) as the low- and high-field shoulders of the spectrum are partially separated from the more narrow spectrum of mobile Tempo+. With the hydrated systems these shoulders are more intense for the perpendicular orientation, but the reverse is true with the EtOH-solvated systems. Thus, immobilized Tempo+ has A I > A 11 on hy9 A I on EtOH-solvated hectodrated hectorites and All ( rites. This orientation dependence, observed even on fully solvated hectorites, is further evidence that the immobilized probes are restricted in interlamellar regions. Using the positions of the low-field and high-field resonances of the broad spectrum, one can determine A,, to be 34 G for both EtOH-solvated and hydrated systems, a value agreeing closely with the expected 32 G. Apparently, even the fully solvated hectorites immobilize part of the probe ions,
-
but the type of solvent determines the alignment of these ions with the surface as indicated by the opposite dependence of A on orientation of hydrated and EtOH-solvated systems. Since the alignment relative to the silicate sheets of both the mobile and immobile fractions of Tempo+ is reversed when the solvent is changed from water to EtOH, it is evident that the probe-surface interaction is greatly influenced by the solvent. The hydrophobic nature of Tempo+ is demonstrated by fully saturating the exchange sites on hectorite with the probe ion. The resulting basal spacing (Table 11) for dry hectorite shows that the probe ions hold the clay plates about 5.7 A apart, suggesting that the Tempo+ molecules form a monolayer and are oriented in a way that minimizes the basal spacing. The Tempo+saturated hectorite does qot expand appreciably in water, but does swell in 95% EtOH (Table 11), indicating the hydrophobic nature of the adsorbed Tempo+ arising from the hydrocarbon nature of the molecule.8 Therefore, water molecules cannot compete as readily with Tempo+ for adsorption sites on the silicate as can ethanol molecules. The hydrocarbon nature of ethanol may reduce the preference of the methyl groups of Tempo+ to approach the silicate surfaces, thereby causing the molecule to assume an “upright” position on the external surfaces that permits considerable freedom of motion. The positively charged end of the Tempo+ molecule is electrostatically attracted to the surface so that totally random motion is not achieved. In contrast, the hydrophobic portion of Tempo+ associates closely with external silicate surfaces in the presence of water because of the lack of hydrocarbon-water attraction and the probe tends to lie “flat” on the surfaces. This model of interaction explains the large positive values of AA for the mobile fraction of Tempo+ on hydrated systems, and the small negative value of AA on EtOH-solvated systems. The immobilized Tempo+ on the solvated hectorites is oriented in the same general sense as the mobile Tempo+ on the same systems, but the alignment is much more rigid and pronounced. Steric hindrance of molecular tumbling combined with maximized probe-surface interaction can account for the orientation of the immobilized probes if it is assumed that these probe ions are in interlamellar regions. In the EtOH-solvated H+, Na+, and Mg2+ hectorites, the N-0 bond axis of immobilized Tempo+ tends to orient normal to the silicate sheets. Since the interlamellar dis(Table 11), the -8.0-A long probe could tance is -7.5 bridge across interlamellar space with the -NH:3+ end of the molecule “keyed” into an hexagonal cavity of one surfaceg and the other end adsorbed to the adjacent surface by the attraction between the silicate oxygen atoms and methyl groups. In the hydrated H+, Na+, and Mg2+ hectorites, the N-0 axis of immobilized Tempo+ tends to orient parallel to the silicate sheets. The interlamellar distance of -10.5 A could allow restricted molecular tumbling since the interlamellar region has a viscosity similar to that of water;5 however, the maximum probe-surface interaction is achieved when the molecule lies “flat” on an interlamellar surface with the -NH3+ group “keyed” into the surface. The rigid glass spectrum of immobilized Tempo+ indicates that molecular motion is absent or very slow on the ESR time scale, Le., T ~
